Application of miR-140 in preparation of medicine for inhibiting breast cancer proliferation and migration
阅读说明：本技术 miR-140在制备抑制乳腺癌增殖和迁移药物中的用途 (Application of miR-140 in preparation of medicine for inhibiting breast cancer proliferation and migration ) 是由 郭志刚 胡志刚 鲁潇 于 2019-08-06 设计创作，主要内容包括：本发明公开了miR-140在制备抑制乳腺癌增殖和迁移药物中的用途。miR-140可以通过直接结合到FEN1的3′UTR来抑制FEN1的蛋白表达,从而导致DNA修复受损,并使肿瘤细胞的增殖和迁移受到抑制。在裸鼠移植瘤模型中进一步发现过表达miR-140可以增加乳腺癌细胞对化疗药物的敏感性,其中,与阿霉素的联合作用效果最佳。该发明为在临床上利用miR-140来诊断和治疗乳腺癌,以及提供药物靶点方面提供了应用价值。(The invention discloses application of miR-140 in preparation of a medicine for inhibiting breast cancer proliferation and migration. miR-140 can inhibit protein expression of FEN1 by binding directly to the 3' UTR of FEN1, resulting in impaired DNA repair and inhibition of tumor cell proliferation and migration. The miR-140 overexpression is further found in a nude mouse transplantation tumor model, so that the sensitivity of the breast cancer cells to chemotherapeutic drugs can be increased, and the combined effect of the miR-140 overexpression and adriamycin is optimal. The invention provides application value for clinically diagnosing and treating breast cancer by using miR-140 and providing a drug target.)
Use of miR-140 or an expression vector containing miR-140 in preparation of drugs for inhibiting breast cancer.
2. Use according to claim 1, characterized in that: the medicament is a medicament for inhibiting proliferation and migration of breast cancer.
3. Use according to claim 1, characterized in that: the miR-140 sequence is shown as SEQ ID NO: 1 is shown.
4. Use according to claim 1, characterized in that: the preparation form of the medicine is liquid preparation, granular preparation, tablet or capsule.
5. Use according to claim 1, characterized in that: the medicament also comprises a pharmaceutically acceptable carrier.
6. Use according to claim 1, characterized in that: the miR-140 promoter is combined with a transcription factor or an inhibitor Ying Yang1, and the Ying Yang1 sequence is shown as SEQ ID NO: 2, respectively.
7. Use according to claim 6, characterized in that: the binding site is site4, and the sequence of site4 is shown as SEQ ID NO: 3, respectively.
8. Use according to claim 1, characterized in that: the miR-140 is overexpressed.
Technical Field
The invention relates to the technical field of biology, in particular to application of miR-140 in preparation of a medicine for inhibiting breast cancer proliferation and migration.
Background
Breast cancer is of various types and properties, and different modern drugs are used for treating breast cancer, and antiestrogens such as raloxifene or tamoxifen can be used for drug treatment of breast cancer, and chemotherapy drugs including 5-fluorouracil, paclitaxel, platinum drugs (cisplatin, carboplatin), adriamycin, cyclophosphamide and the like can also be used for treatment according to the condition of a patient. The principle of chemotherapy is that the mechanism of interfering cell division is usually used to inhibit the growth of cancer cells or accumulate DNA damage of tumor cells to induce apoptosis of tumor cells, and most chemotherapy drugs have no specificity, so that normal tissue cells undergoing cell division are killed at the same time. Chemotherapy is usually the combination of two or more drugs, known as "combination chemotherapy," and most cancer patients are treated with chemotherapy in this manner. The development of drug resistance in tumors is an important factor in the failure of tumor therapy during chemotherapy. Reversing the drug resistance of the tumor cells and improving the sensitivity of the tumor cells to drugs are popular in the research field of tumor treatment. Chemotherapeutic drugs can act to kill tumor cells by exacerbating DNA damage to the tumor cells. The improvement of the DNA damage repair capability is related to the generation of drug resistance in the tumor chemotherapy process.
Targeted therapy has achieved significant efficacy in treating certain types of cancer starting late in the 90 s, as effectively as chemotherapy, but with much less side effects than chemotherapy. The most common targeted therapeutic for breast cancer is herceptin, which is indicated for metastatic breast cancer with over-expression of
MicroRNA (miRNA) is a group of small-molecule non-coding RNAs with the length of about 18-25nt, and can regulate and control related biological processes by regulating downstream target genes. Based on the characteristic that one miRNA can regulate and control various downstream target genes, the miRNA has the diversity of functions. mirnas are involved in regulation in cells mainly by degradation of mRNA of a target gene through cleavage or inhibition of translation of the target gene. A large number of researches show that miRNA can be used as oncogene or cancer suppressor gene to participate in the generation and development of tumor.
FEN1 is used as a structure-specific metal multifunctional endonuclease and participates in two ways of DNA replication and DNA damage repair. In DNA replication, FEN1 was involved in the maturation of okazaki fragments; while FEN1 is involved in the process of long-fragment Base Excision Repair (BER). In addition, FEN1 also has the function of maintaining genome stability and telomere stability.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide application of miR-140 or an expression vector containing miR-140 in preparation of a medicine for inhibiting breast cancer, and aims to solve the problems of proliferation and migration of breast cancer in the prior art.
The technical scheme is as follows: the invention provides application of miR-140 or an expression vector containing miR-140 in preparation of a drug for inhibiting breast cancer.
Further, the medicament is a medicament for inhibiting proliferation and migration of breast cancer.
Further, the sequence of the miR-140 is shown as SEQ ID NO: 1 is shown.
SEQ ID NO：1：5’-cagugguuuuacccuaugguag-3’
Further, the preparation form of the medicine is liquid preparation, granular preparation, tablet or capsule.
Further, the medicine also comprises a pharmaceutically acceptable carrier.
Further, the promoter of the miR-140 is combined with a transcription factor or an inhibitor Ying Yang1, and the sequence of Ying Yang1 is shown in SEQ ID NO: 2, respectively.
Further, the binding site is site4, and the sequence of site4 is shown in SEQ ID NO: 3, respectively.
SEQ ID NO：3：
The entire sequence is the sequence recognized by the primer of site4 chip experiment Qpcr, wherein the capital part is the- (235-225) sequence).
Further, the miR-140 is overexpressed.
FEN1 was highly expressed in many different tumors and was less expressed in normal cells. The proliferation capacity of tumor cells can be reduced by inhibiting FEN1 expression, and the sensitivity of the tumor cells to chemotherapeutic drugs can be enhanced, so FEN1 can be used as a potential target point for tumor treatment.
The primers of FEN1 for PCR amplification are:
FEN1-F：5’-cggggtaccaatgggaattcaaggcctggc-3’-F(SEQ ID NO：4)
FEN1-R：5’-tgctctagattattttccccttttaaacttcc-3’-R(SEQ ID NO：5)
miR-140 can inhibit protein expression of FEN1 by binding directly to the 3' UTR of FEN1, resulting in impaired DNA repair and inhibition of tumor cell proliferation and migration. The miR-140 overexpression is further found in a nude mouse transplantation tumor model, so that the sensitivity of the breast cancer cells to chemotherapeutic drugs can be increased, and the combined effect of the miR-140 overexpression and adriamycin is optimal. By using the adriamycin resistant cell strain for breast cancer, the drug resistance of the breast cancer can be reversed by over-expressing miR-140, so that the drug resistant cells for the breast cancer are sensitive to the adriamycin again. In addition, the over-expression of FEN1 can reduce the accumulation of DNA damage of breast cancer cells caused by miR-140 and reduce the sensitivity of the breast cancer cells to chemotherapeutic drug adriamycin, thereby proving that the accumulation of the damage of miR-140 to the breast cancer cells and the influence on the chemotherapeutic drug play a role by down-regulating
In addition, bioinformatics analysis and experimental verification prove that the transcription factor/inhibition factor Ying Yangl (YY1) is directly combined with the miR-140 promoter to activate miR-140 expression, and an optimal binding site4 is found. YY1 can be directly combined with a promoter of FEN1 to inhibit the expression of FEN1, and the invention proves that YY1 can also indirectly inhibit the expression of FEN1 by activating expression miR-140, wherein miR-140 is low in expression and YY1 is high in expression in breast cancer adriamycin-resistant cells. Under the condition of adriamycin resistance, the capacity of YY1 for binding the miR-140 promoter is obviously reduced.
Has the advantages that: the research result of the invention shows that miR-140 inhibits DNA damage repair by inhibiting FEN1, thereby inhibiting proliferation and migration of breast cancer cells, enhancing the sensitivity of the breast cancer cells to chemotherapeutic drugs and reducing drug resistance of the breast cancer cells. miR-140 can be used as a novel anti-tumor factor for adjuvant therapy of breast cancer, and provides a new target and a new idea for designing and screening medicaments for treating breast cancer.
Drawings
FIG. 1 shows that miR-140 directly affects FEN1 expression, (A) qPCR detects the influence of miR-140 overexpression on FEN1 mRNA level in four breast cancer cells, (B) Weston Blotting detects the influence of miR-140 overexpression on FEN1 protein level in four breast cancer cells, (C) miR-140 and FEN 13 ' UTR binding sequence, and in a rectangular frame area, Wild Type (WT) or Mutant (MT) FEN1 overexpression 3 ' UTR luciferase reporter plasmid and miR-140 or negative reference nucleus are arranged in FEN 13 ' UTR and miR-140 binding site mutation base (D) HEK-293 for luciferase activity analysis;
FIG. 2 shows the tumor expression detection and prognosis relationship between FEN1 and miR-140, (A) and (B) are the relative expression difference between FEN1 and miR-140 in the TCGA database in breast cancer and para-cancer tissues, respectively, (C) and (D) are the expression relationship between FEN1 and miR-140 and patient prognosis, respectively, (ns, no significant difference, P < 0.01;,. P < 0.001);
FIG. 3 shows the effect of miR-140 on cell proliferation, (A) qPCR measures the expression of miR-140 in stable transgenic cell lines, (B) continuous cell counts, measuring the proliferation rate of cells, (C) comparing the number of clones formed after crystal violet staining, (D) is (C) a statistical analysis of the number of clones (ns, no significant difference,. about.P.P.0.01;. about.P.0.001);
FIG. 4 shows the migration of MCF-7 cells after stabilizing miR-140, (A) the cell scratch culture is analyzed by photographing for 24h, (B) the migration efficiency statistical chart of scratches, (C) the cell culture channel Transwell chamber is taken for 48h, and the cell culture channel is photographed after crystal violet staining, (D) the number statistical after crystal violet staining, (ns, no significant difference,. about.P, P < 0.01;. about.P, P < 0.001);
FIG. 5 shows the effect of miR-140 on cell cycle, (A) shows that cell immunofluorescence experiment detects the expression of Ki67 in stable miR-140 and its negative control cell; the scale in the graph is 20 μ M, (B) is a quantitative plot from plot (a), and (C) and (E) are flow cytometry analyses of the distribution of transiently transfected miR-140 and its negative control cells in sub-G1, G1, S or G2-M phases, (D) and (F) histograms represent the results of one typical experiment in (C) and (E), respectively, (ns, no significant difference, ×, P < 0.01;, ×, P < 0.001);
FIG. 6 shows the observation of tumor models of nude mice over-expressing miR-140, (A) subcutaneous tumor volumes of different groups of miR-140 nude mice, (B) subcutaneous tumor mass maps of nude mice from different drug-treated groups, (C) tumor mass weight records, (D) staining of tumor sections H & E, FEN1, Ki67, and cleared-
FIG. 7 shows the sensitivity of miR-140 over-expressed in cells to different chemotherapeutic drugs, (A-D) survival of MCF-7 cells after miR-140 over-expression by MTT treatment with different concentrations of 5FU, Paclitaxel, Cisplatin, Camptothecin and other chemotherapeutic drugs, (E) and (F) survival of MCF-7 cells after miR-140 over-expression by MB231 treatment with different concentrations of ADR (ns, no significant difference, P < 0.01;,;. P < 0.001);
fig. 8 is the effect of miR-140 overexpression on apoptosis, (a) cytomorphological analysis of MCF-7 overexpression miR-140 addition to ADR for cell status, (B) typical flow cytogram showing the co-staining of MCF-7 apoptotic cells with annexin v and PI under different transfection, (C) histogram showing the statistics for (B) plot, (D) typical flow cytogram showing the co-staining of MB231 apoptotic cells with annexin v and PI under different transfection, (E) histogram showing the statistics for (D) plot, (ns, no significant difference, P < 0.01;, P < 0.001);
fig. 9 shows the effect of miR-140 and FEN1 on drug resistance in drug-resistant cells, (B) whether MCF-7-ADR-140 was stable over MCF-7-ADR-140 was determined by qPCR, (a) cell morphology analysis, cell status when miR-140 and FEN1 were treated against MCF-7-ADR, and (C) cell viability assay, cell viability was determined using MTT, (ns, no significant difference, P < 0.01;, P < 0.001).
Detailed Description
The sequences of the substrates and primers involved in the present invention are shown in Table 1.
TABLE 1 substrate and primer sequences related to the invention
1. The experimental method comprises the following steps:
1.1 recovery, passage and cryopreservation of cells
The source of the cells and the media components, etc., are listed in Table 2 below (the following discussion is for 10cm dish cells for example):
TABLE 2 sources of cells and Medium types
All cells were cultured in medium before 10% serum and 1% penicillin and streptomycin were added. All cells were cultured at 37 ℃ in 5% CO2And a tray containing sterilized ultrapure water is placed at the bottom of the incubator to ensure that the incubator maintains proper humidity.
And (3) recovering the cells: cells were removed from the liquid nitrogen tank quickly and carefully, keeping in mind liquid nitrogen frostbite. And quickly placing the freezing tube into a 37 ℃ water bath to quickly melt, placing the melted freezing solution into a sterile centrifuge tube, adding 1mL of fresh culture medium, centrifuging at 1000rpm for 2min, sucking out the supernatant, gently blowing and suspending the cell sediment by using 1mL of fresh culture medium, and transferring the cell sediment into a cell culture dish with the culture medium added in advance for culture.
Passage of cells: when the cells grow to the density of about 80-90% in the dish, the cell culture medium in the dish is sucked off, PBS is slowly added along the inner wall of the cell culture dish, and the cells are blown off from the dish by force too violently in a cautious way. Washing cells for 2 times, adding 0.25% of pancreatin (containing 0.02% of EDTA) for cell digestion, observing cell morphology at any time under a microscope, sucking away the pancreatin timely after most cells become round, adding 2-3mL of fresh culture medium into a culture dish to stop digestion, then suspending the cells by blowing, and distributing the cells into 2-3 culture dishes for continuous culture (a dish of cells is passed for several times during passage depending on cell concentration and state).
Freezing and storing the cells: after digesting full and well-conditioned cells, blowing and suspending the cells by using a culture medium and transferring the cells into a centrifuge tube, centrifuging the cells at 1000rpm for 2min, removing supernatant, blowing and suspending the cells by using a prepared freezing solution, subpackaging the cells into freezing tubes, wherein each tube of the freezing solution is about 1-1.5 mL, the name of the cells is clearly marked on the freezing tubes, the freezing time and an operator are marked, and the freezing tubes are placed in a freezing box. The frozen storage box was placed in a-80 ℃ freezer. Taking out the mixture on the next day, and storing in liquid nitrogen tank for long term or temporarily storing in refrigerator at-80 deg.C for short term. The formula of the frozen stock solution is as follows: 50% medium + 40% fetal bovine serum + 10% DMSO.
1.2 preparation of drugs in the experiment
The chemotherapy drugs and sources used in this experiment are listed in table 3 below:
TABLE 3 chemotherapeutic drugs and sources used mainly in this experiment
Medicine name (English)
Medicine name (Chinese)
Sources of drugs
Medicine solvent
Paclitaxel
paclitaxel
Selleck
DMSO
Camptothecin
Camptothecin
Selleck
DMSO
Doxorubicin
Adriamycin
Selleck
DMSO
Cisplatin
Cis-platinum
DMSO
5FU
5 Fluorouracil
Selleck
DMSO
1.3 cell count
(1) Taking the cells out of the incubator, sucking the cell culture medium in a super clean bench, adding 2mL of PBS into a 10cm cell dish to clean the cells for 2 times, removing the PBS, adding 1mL of pancreatin, slowly shaking to enable the pancreatin to uniformly cover the whole cell dish, sucking away the pancreatin immediately when most of the cells are observed to be round under a microscope, adding 3mL of the culture medium to stop digestion, slightly blowing and suspending the cells, and repeatedly slightly blowing and beating the cells to enable the cells to be fully digested into single cells.
(2) And collecting the digested cells in a centrifuge tube in an ultra-clean bench, and gently blowing and stirring the cells in the centrifuge tube uniformly. Then, 10. mu.L of the cell suspension was aspirated from the centrifuge tube into a 200. mu.L centrifuge tube, and 10. mu.L of trypan blue was mixed with the cell suspension outside the ultraclean bench and blown uniformly. The entire 20 μ of the mixed solution was slowly added to the cell counting plate, which was inserted in parallel into the cell counter. Cells were counted using a matched cell counter. To ensure the reliability of the data, the knob of the cell counter was turned, the number of cells in different fields of view was read, the average was taken, and the counting process was repeated 3 times for each sample. After trypan blue is added, the whole counting process preferably does not exceed 5 min. If the concentration of the cell sample to be counted is too high, it can be diluted in an appropriate ratio and then measured. Too high or too low a concentration of the cell sample may result in inaccurate measurements.
1.4 transient transfection of cells into lentiviruses infection and identification
(1) The cells in good condition were sorted into 12-well plates or 6-well plates, and 12-well plates were used as an example here. Firstly, cells are digested, the specific process refers to the previous operation steps, the cells of the digested cells are transferred into a centrifuge tube or directly operated in a cell culture dish, and the individual habit can be followed. Approximately 2 × 105 cells were transferred to 12-well plates. Transfection of a plasmid typically establishes three parallel wells as an intragroup repeat. Negative control, namely transfection of unloaded plasmids and the like, is required for each transfection, 1mL of culture medium is added into each hole in the 12-hole plate, each hole is blown by a pipette, attention is paid to precaution of pollution, then the 12-hole plate is taken out of a super clean bench, and the 12-hole plate is shaken left and right and back and forth on a flat table, so that cells are uniformly distributed in the 12-hole plate. The 12-well plate is stood for 2min, and then is gently moved under a microscope to observe whether the settled cells are uniformly distributed in the 12-well plate. Transfection efficiency was affected if cells were packed together, and the 12-well plate was shaken again if the distribution was not uniform. The density of the cells was observed under a microscope the next day. Cells can be transfected at cell densities around 70%.
(2) Before transfection, the old medium in the 12-well plate was removed, fresh serum-free and double-antibody-free medium was added, and the cells were starved for about 2 hours in advance in a culture incubator.
(3) An appropriate amount of 1.5mL centrifuge tube was added to 300. mu. LOpti-MEM per tube and labeled. Transfection of one plasmid corresponds to two centrifuge tubes, and liposomes (2. mu.L per well, 6. mu.L if three wells are transfected) are added to one centrifuge tube and allowed to stand for 5min to allow the liposomes to diffuse well. miR-140 RNAimimic or target plasmid (5ng per well, the quantity of the added mimic is calculated according to the actual number of transfection wells) is added into the corresponding centrifuge tube, and the negative control group is also the same, and only the plasmid is changed into the corresponding no-load plasmid or nonsense plasmid. The contents of the two centrifuge tubes were mixed. The mixture was allowed to stand for 20 min. The mixture was added to a 12-well plate at 200. mu.L per well, and the medium in the plate was removed after 6 hours (the time can be appropriately extended or shortened depending on the type of transfected cells). Adding fresh culture medium containing serum and double antibody, and culturing for 48h to obtain cell sample.
The miR-140RNAmimic is a double chain of a mature body sequence, and the sequence is as follows:
5’-cagugguuuuacccuaugguag-3’
3’-gtcaccaaaatgggataccatc-5’。
1.5 Lentiviral infection and identification
The miR-140 overexpression lentivirus used in this experiment was purchased by the company and previously titre-determined.
(1) The slow virus infection experiment needs to be operated in a biological safety cabinet, safety protection is well done before the experiment, and the virus infection experiment is carried out when the cell density in the well-inoculated six-hole plate reaches about 40-50%.
(2) Before virus infects cells, the original culture medium is discarded, 1-2mL of fresh culture medium is added, 2 μ L of polybrene is added according to the proportion of 1: 1000, then proper amount of slow virus suspension is added uniformly, and the culture is continued for 48 h.
(3) And after 48 hours, carefully observing the cells after virus infection under a fluorescence microscope, observing whether the cells have fluorescence or not and the intensity and the density of the fluorescence, if the fluorescence intensity and the density are moderate, indicating that the virus infection is successful, replacing fresh culture solution, and adding a proper amount of specific medicine for medicine screening, wherein the experiment is to add puromycin medicine screening, MCF7 and MB231 cells according to the ratio of 1: 1000.
(4) And (3) screening the medicine for 2-3 days, gradually increasing the fluorescence intensity in the virus-added cells after the negative cells without the virus are completely dead, finishing screening the medicine, establishing a stable cell strain, and performing amplification culture and freezing and storing the cell.
(5) WesternBlot or qPCR methods were used to identify the effect of viral infection. As the experimental lentivirus over-expresses miR-140, a qPCR method is used for detection.
1.6 drug susceptibility test
(1) An appropriate amount of 96-well plates were prepared, and 300. mu.L of sterilized PBS or sterilized ddH was added to the edge row of each 96-well plate2O。
(2) The cells were counted according to the procedure of 1.3 and diluted so that the number of cells per well was 4000 and the total amount of the medium per well was 200. mu.L, and the cells were uniformly dispersed in the culture plate by shaking gently. After the cells attached for 24h, a gradient of diluted drugs was added. Wherein the concentration gradient of paclitaxel is 0, 0.1, 0.5, 1, 5, 10 μ M; the concentration gradient of the adriamycin is 0, 0.05, 0.1, 0.5, 1, 5 mu M; the concentration of camptothecin, 5FU and cisplatin is set as follows: 0, 5, 10, 50, 100, 200. mu.M.
(3) Adding medicine for 48 hr, sucking off the culture medium containing medicine, washing with PBS for 2-3 times, adding 200 μ L of fresh culture medium and 10 μ L of cellTMThe Countingkit-8(CCK8) reagent was incubated for 3-4h, and the corresponding OD at 450nm was read and the inhibition of cell growth by the drug was calculated using the solvent well reading as 100%.
1.7 clone formation experiments
(1) Old medium in the dish was gently aspirated in a cell ultraclean bench with a pipette and washed 2 times with sterile 1 × PBS and the cells were digested.
(2) Digestion was stopped with medium, gently blown with a gun, and then aspirated into a 15mL cell centrifuge tube, centrifuged at about 1000rpm for 2min at room temperature.
(3) After the supernatant is sucked away and then the corresponding culture medium is added for blowing, 20 mu L of the supernatant is taken and added with trypan blue with the same volume, and if the cells needing to be inoculated are few, a plurality of new culture mediums can be added for diluting the cells by using a cell counting instrument so as to improve the accuracy.
(4) According to the counting result, about 300-500 cells are inoculated to each hole of the six-hole plate, and about 2mL of fresh culture medium is added for shaking.
(5) At 37
(6) Until the clone cell clusters with proper size appear in the six-hole plate, dyeing for 10-15min by using crystal violet dye solution, washing for 2-3 times by using double distilled water until the cells are cleaned, and photographing for storage.
1.8 cell cycle assays
(1) The cell culture medium was discarded from the well-grown cells, and the cells were washed 2-3 times with precooled PBS and digested with pancreatin.
(2) The cells were suspended by blowing with 1-2mL of the medium and transferred to a centrifuge tube and centrifuged at 1000rpm for 2 min.
(3) The cells were then resuspended in 1ml PBS and centrifuged, and the washing of the cells was repeated 3 times.
(4) After the final centrifugation, the supernatant was removed, 1mL of pbs was added to suspend the cells by blowing and 3mL of glacial ethanol was added slowly, the cells were blown evenly by gentle blowing with a pipette, and the centrifuge tube was left at-20 ℃ overnight to perforate.
(5) The next day, the tube containing the cell suspension was removed, the cells were washed 2 times with ice PBS, 0.5ml of PI/RNase stain was aspirated to gently suspend about 1X 106 cells until uniform, and the cells were incubated at room temperature for 30 min.
(6) And (3) placing a filter membrane of 200 meshes above the flow-type tube, sucking cells by a pipette, blowing before sucking to prevent the cells from agglomerating, abutting the pipette head on the filter membrane to filter cell agglomerates, and detecting the collected cell suspension by using a flow cytometer.
1.9 apoptosis assay
(1) The cell culture medium was pipetted into a clean 15mL centrifuge tube, the cells were washed with
(2) Centrifugation at 1000rpm for 3min at room temperature removed the supernatant, washing of the cells with ice PBS was continued 2 times and the supernatant was removed.
(3) Resuspend cells with 1 × biningbuffer in the apoptosis kit for BD (100 μ L with approximately 1 × 105Individual cells), the cells to which the solvent only is added will be used as the negative control of the experiment, the experimental group is divided into three parts, single staining is respectively carried out by annexin V-FITC or Propidium Iodide (PI), the other samples are added with annexin V-FITC and Propidium Iodide (PI) staining solution for double staining, and the incubation staining is carried out for 15min at room temperature in the dark.
(4) Before filtration, 400 μ L of 1 × biningbuffer was added to each 1.5mL of LEP tube, the tube was flushed with a pipette several times to prevent cell clumping, a 200-mesh filter was placed over the flow tube, the pipette was used to aspirate cells, the pipette was pressed against the filter to filter out cell clumps, and the cell suspension was rapidly assayed for apoptosis using a flow cytometer within 1 h.
1.10 Total RNA extraction and miRNA purification of cells
(1) Trizol lysed cells: using a six well plate as an example,
(2) And (3) separating RNA by using chloroform: mu.L of chloroform was added to the tube at a ratio of VTrizol to V of 5: 1, vortexed, mixed, and then allowed to stand at room temperature for 10min, followed by centrifugation at 12000rpm at 4 ℃ for 15 min. After the centrifugation is finished, the liquid in the tube is divided into three layers, and the uppermost layer of water phase is taken and transferred into a new RNaseFree centrifugal tube.
(3) Isopropanol precipitation of RNA: according to the ratio of V Trizol to V isopropanol of 2: 1, 250 μ L of isopropanol is added into a tube, the mixture is evenly mixed by inversion, and then the mixture is kept stand for 20min at room temperature and centrifuged for 10min at 12000rpm and 4 ℃. After the centrifugation was completed, the supernatant was discarded, and the bottom RNA precipitate was observed.
(4) Ethanol cleaning of RNA: add 500. mu.L of 75% ethanol solution (RNaseFreedH) into the tube2O preparation), bounce the bottom sediment, centrifuge at 7500rpm for 10min at 4 ℃, discard the supernatant, and clean again.
(5)RNaseFreeddH2O dissolving RNA: drying the RNA precipitate at room temperature for 10min, adding 20-30 μ LRNaseFreedH2O dissolution, and the concentration and purity of the extracted RNA are determined using a micro-spectrophotometer and recorded. After extraction, RNA can be stored at-80 ℃ to avoid repeated freeze thawing. When used again, the RNA concentration needs to be determined again.
1.11 fluorescent Real-time quantitative PCR (Real-time PCR) for detecting the relative expression of mRNA and miRNA of cells (the primers for carrying out fluorescent quantitative detection on related target genes or miRNA are shown in the serial number 4-11 in Table 1)
(1) Reverse transcription: mRNA was reverse-transcribed (10. mu.L system) with 1. mu.g of RNA according to the following system; 5 × RTmix, 1 μ L; RNaseFreedH2O, to 10 μ L; reverse transcription procedure: 42 ℃ for 15 min; at 85 ℃ for 5 s; the cDNA was stored at-20 ℃ at 12 ℃ with a concentration of 100nM after the infinity reverse transcription.
Reverse transcription of miRNA was performed according to the following system (10. mu.L system, reverse transcriptase does not contain Oligo-dT)
First-step unwinding: RNA, 1. mu.g;
RNaseFreeddH2O to 4.4. mu.L of RNA and
miRNARTPrimer unwinding procedure 70 c,
10min, ice bath, 2 min.
And a second step of reverse transcription, wherein the following reagents are added into a first step unwinding system: dNTP0.8. mu.L; RTenzyme, 0.2. mu.L; RNaseFreedH2O to 5.6 μ L;
reverse transcription procedure: 42 ℃ for 60 min; at 85 ℃, 5s12 ℃ and infinity,
the product can be stored at-80 deg.C.
(2) Fluorescent real-time quantitative PCR:
whole transcriptome cDNA transcribed from mRNA Real-timePCR (20. mu.L system) was performed according to the following system:
Primer-F(10nM)、0.4μL；Primer-R(10nM)、0.4μL
cDNA、4μL；SYBGreen、10μL；ddH2o, to 20 μ L;
the whole transcriptome cDNA Real-time PCR reaction program transcribed from mRNA is as follows
miRNA-cDNA transcribed from mRNA Real-timePCR was performed according to the following system (20. mu.L system)
Primer-F(10nM)、0.8μL；Primer-R(10nM)、0.8μL；
miRNA-cDNA、4μL；SYBGreen、10μL；RNaseFreeddH2O, to 20 μ L;
the miRNA-cDNA Real-time PCR reaction program transcribed by mRNA is as follows
The detection temperature of the melting curve is 70 ℃ to 95 ℃, the heating rate is 0.4 ℃/time, and the constant temperature time is 1 s/time. The resulting Ct values were used for statistical analysis.
1.12 cellular immunofluorescence
(1) A cell slide with a diameter of about 1cm was soaked in 75% ethanol in advance, and the slide was briefly subjected to a grill sterilization with an alcohol burner before use and placed in a 12-well plate. (2) Digesting the cells in a good state by using pancreatin, blowing and suspending the cells by using a culture medium, inoculating a proper amount of cells into each hole, uniformly shaking, and carrying out overnight adherent culture in an incubator. (3) When the cell density is suitable for the experiment, the main H of the medicine used in the experiment is added according to the experiment requirement2O2And doxorubicin. After the cell treatment was completed, the cells were washed 3 times with PBS and the PBS was removed. (4) About 1mL of 4% paraformaldehyde was added to each well to allow the fluid-covered slide to soak at room temperature for 30min, and the cells were fixed. (5) Excess liquid was aspirated, and the cell slide was washed 3 times with PBS at room temperature, 10min each time with PBS aspirated. (6) Adding 0.5% Triton-X100 solution prepared in advance, immersing the cell slide at room temperature for 15min, and perforating the cells. (7) Excess liquid was aspirated, washed 3 times with PBS, 10min each time, and PBS was removed. (8) In 12-well plates, approximately 1mL of 5% BSA was added per well, the cell slide was submerged, incubated at room temperature for 1h, and blocked. (9) Absorbing the sealing liquid, completely absorbing the liquid around the climbing sheet by absorbent paper, adding the primary antibodies prepared in advance, wherein the primary antibodies mainly used in the experiment are gamma H2AX and 53BP1, and reference description of the required dilution ratio of the antibodiesBook, 4 ℃ refrigerator overnight. (10) The following day the cell slide was rinsed 3 times with PBST (containing 0.1% Tween20), 15min each time and the PBST was removed. (11) And (3) dropwise adding a prepared fluorescent secondary antibody on the cell slide, referring to the specification for the dilution ratio of the secondary antibody, and incubating for 1-2h at 37 ℃ in a dark place. (12) PBST (containing 0.1% Tween20) rinse
1.13 detection of protein expression in cells
1.13.1 Total cell protein extraction Using 12-well plate as an example
(1) When the growth density of the cells in the culture dish reaches 80-90%, the optimal period for extracting the total protein of the cells is provided. The culture broth in the dish was aspirated in a clean bench, and the cells were washed 2 times with sterilized PBS, and the PBS was aspirated. (2) 1mL of protein lysate was taken and 5. mu.L of 200 XPSF and 10. mu.L of 100 XCKTail were added. The prepared protein lysate is added to 12-well plates, approximately 100. mu.L per well, and if the cell amount is large, the amount of the cell lysate is increased appropriately. Gently shake to allow the cell lysate to cover the well plate evenly. The cells were placed on ice and lysed for about 30 min. (3) The lysed cells were scraped off completely with a cell scraper and all cell lysates were transferred to a new 1.5ml lep tube at 12000rpm, 4 ℃ and centrifuged for 10 min. (4) The centrifuged supernatant was carefully pipetted into a new 1.5ml lep tube, which was the protein sample from the cells.
1.13.2 Western immunoblot (Blot)
(1) Adding 6 Xloadingbuffer into the cell lysate, mixing uniformly, and boiling for about 5 min. Taking out, centrifuging for a short time by using a centrifuge, and centrifuging the liquid drops evaporated and solidified on the tube wall to the bottom of the tube. According to the size of the protein to be detected in the experiment, SDS-PAGE gel with corresponding concentration is prepared, and the higher the concentration of the separation gel is, the more favorable the separation of small protein is. All proteins referred to herein can be tested with 12% separation gel.
(2) Placing the prepared rubber plate in a vertical electrophoresis tank, adding 1 Xrunningbuffer into the electrophoresis tank, vertically and carefully pulling out a comb on the rubber plate, taking 10 mu L of each sample, regulating voltage and current, and performing electrophoresis at a constant voltage of 80V for about half an hour; after the sample runs out of the concentrated gel, the marker starts to separate, the voltage is adjusted to 120V to continue to run the separation gel, and the specific time length of gel running is adjusted according to the size of the target protein. If the target protein is large, the voltage can be increased according to actual conditions, the electrophoresis time can be properly prolonged, and the electrophoresis can be stopped when the bromophenol blue is about to run out of the bottom of the gel plate under general conditions.
(3) After electrophoresis is finished, the rubber plate is taken out, the foam and the buffer on the rubber plate are washed away, the PAGE glue is stripped, care needs to be taken when the PAGE glue is stripped, the glue is prevented from being torn, and the glue is flatly laid on the film rotating clamp. And (3) covering the PVDF membrane which is activated by the methanol with glue, carefully driving off bubbles, loading the PVDF membrane into a membrane conversion electrophoresis tank, and adding a precooled 1 × transbuffer into the electrophoresis tank in advance for 60min of membrane conversion at a constant pressure of 100V. The voltage of the transmembrane and the duration of the transmembrane can be adjusted according to the size of the protein.
(4) And (3) after the membrane is transferred, quickly taking out the membrane, putting the membrane into 5% skimmed milk powder or 5% BSA solution prepared in advance for blocking, preparing blocking liquid by PBS, and incubating for 1.5h by a shaking table at normal temperature.
(5) After blocking, the membrane was removed from the blocking solution, washed three times with PBS, and then the PVDF membrane was carefully cut according to the size of the target protein, taking care to set the internal control protein. The cut membrane was soaked in the corresponding antibody and the primary antibody was incubated overnight at 4 ℃ with the dilution ratio of the primary antibody being as described in the specification.
(6) The next day, primary antibody was recovered by aspiration and membranes were washed 3 times with PBST (containing 0.1% Tween20) for 15min each time.
(7) Incubating the secondary antibody at normal temperature, generally 1-2h, washing the membrane for 3 times by PBST (containing 0.1% Tween20), and recovering the secondary antibody 15min each time.
(8) And taking the film out, uniformly dropwise adding the prepared ECL developing solution with the right side facing upwards, putting the film into a dark box of an imager for exposure when the developing solution is used as it is, and storing the image.
1.13.3 Bradford method for determining protein concentration
(1) A previously prepared BSA standard protein solution (1.47mg/mL) was diluted with 1 XPBS at 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5mg/mL to prepare a calibration curve. (2) The standard protein samples were sequentially added to a 96-well plate at 10. mu.L per well, and the samples were repeatedly loaded 3 times, taking care to avoid air bubbles and making the measurement error larger. (3) Firstly, a sample to be measured is diluted by 10 times by PBS, so that the problem that the concentration of a protein sample is too high and exceeds the maximum concentration of a standard curve, which causes inaccurate measurement is mainly prevented. The diluted samples were then added sequentially to a 96-well plate and the loading was repeated 3 times. (4) The Bradford dye liquor is diluted by 5 times by using double distilled water, and the dye liquor needs to be prepared immediately when used. 200 μ L of diluted dye solution was added to each sample well and allowed to stand at room temperature for 1-2 min. (5) And measuring the OD value by using an enzyme-labeling instrument, wherein the used wavelength is 595nm, drawing a standard curve, and calculating the protein concentration of the sample to be measured.
1.14 immunohistochemistry
(1) The tissue mass or tumor mass peeled from the skin of the nude mouse was transferred to paraffin sections prepared by google bio-corporation. (2) And (3) baking the paraffin sections in an oven at 60 ℃, and taking out the paraffin sections after 4-6 h. (3) Dewaxing the dried slices in xylene for 2 times, 10min each time; then sequentially transferring to 100%, 90%, 80%, 70%, 50% ethanol solution with different concentrations for dehydration twice, each for 5min, and finally using ddH2O5min, washing twice. Excess water on the periphery of the section was carefully blotted with absorbent paper. (4) Selecting a proper tissue area, carefully circling a closed area along the outer edge of the tissue by using a water-blocking pen, and soaking the slice; soaking in 3% H2O2And (4) incubating at room temperature for about 10-15min in a dark place. (5) Sections were rinsed 3 times with PBS for about 5min each. (6) Sucking off excessive water from the slices with absorbent paper, and dripping the zone in the ring with a repellentThe original repair solution was allowed to cover the entire tissue area at 37 deg.C for 15 min. (7) Sections were carefully rinsed 3 times with 1 × PBS for about 5min each. (8) The sections were soaked in a previously prepared 3% BSA blocking solution and blocked by shaking slowly in a shaker at room temperature for 1 h. (9) Excess liquid on the sections was aspirated off and carefully blotted dry with absorbent paper, primary antibody (diluted with 3% BSA blocking solution, dilution scale instructions) was added drop-wise over the tissue sample to cover the sample and incubated overnight at 4 ℃. This step allows the setting of a negative control, i.e. a control in which only an equal amount of BSA solution is incubated above the negative sample. (10) The next day, the antibody cassette was removed and the slides were rewarmed for about 45min and washed 3 times with PBST (containing 0.1% Tween20) for about 10min each time. (11) The water on the slide is sucked dry by absorbent paper, the corresponding secondary antibody is dripped on the tissue, the incubation is carried out for about 1-2h at 37 ℃, and the corresponding secondary antibody is also dripped on the negative control. Slides were washed with PBST, as described above. (12) And (3) dropwise adding the prepared DAB (toxic, attention is paid to experimental specifications during operation) to the tissue sample, and covering, and dyeing in a dark place for about 2min (the dyeing condition of the tissue can be observed under a microscope, and the reaction is stopped in time). (13) By ddH2And O, washing the slices on the reverse side, and stopping the reaction. (14) Draining off water on the section, counterstaining with hematoxylin for about 2min, and adding 1% hydrochloric acid methanol differentiation solution when observing the color of the tissue under microscope, wherein the differentiation time is about 5-20s, and is determined according to the situation. (15) Using ddH2And O stops the reaction. (16) The slides were dehydrated by soaking in 50%, 70%, 90%, 100% ethanol in sequence, 2 times each for about 3min and finally the sections were placed in xylene in a fume hood for 2 times each for about 3 min. (17 in the fume hood naturally dry the slide, and drop the appropriate amount of neutral resin cover slip on the tissue, care to avoid air bubbles as much as possible) (18) observe and take pictures under the microscope.
1.15 plasmid construction
(1) Designing a primer, designing and synthesizing a primer suitable for a target gene by searching CDS (coding sequence coding) region data of a related target gene sequence, (2) amplifying the target gene, designing PCR amplification conditions with a proper annealing temperature and an extension time according to the primer of the target gene and the size of the target gene, (3) carrying out PCR amplification, (3) adding a part of a PCR product into loadingbuffer for spotting, adding the spotted PCR product into agarose gel which is prepared in advance, 100V and 30min, determining the size of an amplified band according to the size indicated by a marker, (if no band appears, the optimal amplification conditions can be changed, and carrying out re-amplification), (4) purifying the target gene, (5) carrying out purification of the PCR product by using an axygen purification kit, (6) carrying out enzyme digestion, carrying out enzyme digestion on the purified target gene and a vector to be connected in a water bath at 37 ℃ by using the same enzyme, (6) carrying out gel recovery, (6) adding all spots of the digested product into a nucleic acid gel, carrying out gel digestion, carrying out gel recovery on the digested product, carrying out gel digestion, carrying out gel recovery, carrying out gel amplification, carrying out gel recovery, carrying out gel amplification, and carrying out single-amplification, carrying out gel recovery, and carrying out gel recovery, (3) carrying out gel recovery, carrying out amplification on a gel recovery, a.
1.16 Dual luciferase reporter assay
(1) Cell lysis: after the reporter gene cell lysate is fully and uniformly mixed, the reporter gene cell lysate is added in the following mode to fully lyse the cells.
a. For adherent cells: after the cell culture fluid is aspirated, a proper amount of reporter gene cell lysate is added with reference to the following table; for suspension cells: after centrifugation to remove the supernatant, appropriate amounts of reporter cell lysates were added as referenced in the table below.
Ware type 96-well plate 48-well plate 24-well plate 12-well plate 6-well plate reporter gene cell lysate (microliter/well) 100150200300500
Note: if the luciferase expression level is low, it may be attempted to use less lysate, e.g.a minimum of 100. mu.l per well in a 6-well plate.
b. After sufficient lysis, 10,000-15,000g were centrifuged for 3-5 minutes and the supernatant was taken for assay.
Note: the luciferase may be assayed immediately after cell lysis or may be frozen and assayed later. The frozen samples need to be thawed and measured after reaching room temperature.
(2) And (3) melting the firefly luciferase detection reagent and the renilla luciferase detection buffer solution, and reaching the room temperature. Renilla luciferase assay substrate (100X) was placed on an ice bath or ice box for use.
(3) According to the amount of 100 microliters required by each sample, a proper amount of renilla luciferase detection buffer solution is taken, and a renilla luciferase detection substrate (100X) is added according to the ratio of 1: 100 to prepare renilla luciferase detection working solution. For example, 10. mu.l of Renilla luciferase assay substrate (100X) is added to 1ml of Renilla luciferase assay buffer and mixed well to prepare about 1ml of Renilla luciferase assay working solution.
(4) According to the instrument operation instruction, the chemiluminescence instrument or the multifunctional microplate reader with the function of detecting chemiluminescence is started, the measurement interval is set as 2 seconds, and the measurement time is set as 10 seconds.
(5) For each sample, 20-100. mu.l of sample is taken (if the sample amount is sufficient, 100. mu.l is added; if the sample amount is insufficient, the amount can be reduced properly, but the amount of the sample in the same batch is kept consistent).
(6) Add 100. mu.l firefly luciferase assay reagent, gun-mix or mix well by other suitable means and measure RLU (relative light unit). Reporter cell lysates were used as blank controls.
(7) After completion of the above-mentioned firefly luciferase assay procedure, 100. mu.l of Renilla luciferase assay working solution was added, and RLU (relative light unit) was assayed after beating with a gun or mixing by another appropriate means.
(8) In the case of Renilla luciferase as an internal control, the RLU value obtained by firefly luciferase assay was divided by the RLU value obtained by Renilla luciferase assay. And comparing the activation degree of the target reporter gene among different samples according to the obtained ratio. Similar calculations can be made if firefly luciferase is used as an internal control.
1.17 animal experiments
(1) BALB/c nude mice purchased about 4 weeks from south university model one week in advance were allowed to adapt to the breeding environment in advance. (2) Simultaneously culturing cell lines of which MCF-7 and MB231 are stably transformed into NC and miR-140 in a large quantity, carrying out expanded culture on the cells, and when the cells grow to be enough for experimental dosage and have good state, using pancreatinThe cells were digested and the same cells were collected in a 15mL centrifuge tube. (3) 10 μ L of the cell suspension was removed and counted using a cell counter. (4) Taking out the matrix glue subpackaged in advance, melting the matrix glue on ice, mixing the matrix glue and the cell suspension in a volume of 1: 1, and injecting 250 ten thousand cells into each mouse subcutaneously, wherein the injection volume is 100 mu L. (5) Note that the growth of subcutaneous tumors in mice was observed, and when the tumors grew to 50m3, the nude mice were randomly divided into 2 groups. (6) Simultaneously measuring and recording the length, width and weight of the mouse tumor, and calculating the volume of the tumor, wherein the calculation formula is as follows: length x width2/2. (7) One mouse was randomly taken out of each
1.18 cell invasion assay
(1) Preparing a crystal violet dye solution: dissolving 0.05g of crystal violet in methanol to prepare 0.5 percent of crystal violet solution, and using PBS solution at a ratio of 1: 5-8 to prepare 0.1 percent of crystal violet dye solution. (2) Preparation of transwell invasion well: BD matrigel and serum-free medium were diluted 1: 8, and 80. mu.l was pipetted into the upper chamber of a transwell and placed in an incubator for at least 30 minutes. (3) Cell centrifugation: cells were digested, centrifuged, resuspended without serum, and diluted to a cell suspension of
1.19 cell viability assay
(1) Digesting and inoculating the cells: single cell suspensions were prepared from culture medium containing 10% fetal calf serum and seeded into 96-well plates at 2000 cells per well, 200ul per well volume. (2) Culturing the cells: the culture was carried out for 2 days (the culture time can be determined according to the purpose and requirements of the test) under the same general culture conditions. (3) Color generation: after 2 days of incubation, 20ul of MTT solution (5mg/ml in PBS) was added to each well. Incubation was continued for 4 hours, the culture was terminated, and the culture supernatant in the wells was carefully aspirated, after centrifugation was required for suspension cells, and the culture supernatant in the wells was aspirated. Add 150ul DMSO/well and shake for 10 minutes to fully melt the crystals. (4) Color comparison: selecting 490nm wavelength, measuring the light absorption value of each well on an enzyme linked immunosorbent assay, recording the result, and drawing a cell growth curve by taking time as an abscissa and the light absorption value as an ordinate.
1.20 data processing and statistics
The experimental data are collated by Excel, statistical analysis is carried out by Origin8 or Graphprism software, the difference analysis between two groups of data uses t test of sample average comparison, and the difference analysis of multiple groups of data adopts one-factor analysis of variance. P < 0.05 indicated significant difference, p < 0.01 indicated very significant difference, and p < 0.001 indicated very significant difference.
The Comgusyn software is used for the analysis of the effect of the combination of the two drugs, and the final judgment is made by an isobologram method.
2. Results of the experiment
2.1miR-140 targets and downregulates the expression of FEN1
Whether the miR-140 can regulate the expression of FEN1 is verified through experiments. The Sharp Bio Inc. of Guangzhou was entrusted with the synthesis of mimic of miR-140 and a negative control for miRNA.
The mimic of the miR-140 is transfected into different types of breast cancer cell lines including MCF-7, MB231, T47D and Bcap37 by transiently transfecting the mimic of the miR-140 and a negative control, and the influence of the miR-140 on the levels of FEN1 protein and mRNA is detected. The data show that miR-140 overexpression in all four cell lines can significantly down-regulate the FEN1 protein level without affecting the mRNA level of FEN1 (fig. 1A, B). This indicates that the regulation effect of miR-140 on FEN1 is to regulate the expression of FEN1 by inhibiting the translation of target genes. Next, it was further investigated whether the effect of miR-140 on FEN1 expression is related to the direct binding of miR-140 to the 3' UTR of FEN1 predicted by Targetscan. A3 'UTR sequence containing FEN1 bound to miR-140 was constructed, and three bases were mutated at the site sequence where FEN1 and miR-140 are perfectly complementarily paired (FIG. 1C FEN1MT 33' UTR right side AAU). Namely, a dual-luciferase reporter system expression plasmid of pMIR-FEN1-3 'UTR-WT and pMIR-FEN 1-3' UTR-MUT is constructed. Two plasmids are transfected in HEK-293 cells respectively, and miR-140 or negative reference and Renilla are co-transfected. We then performed luciferase reporter assays and mutation assays. The results were recorded as the light signal generated by firefly luciferase, RL1, and the signal generated by Renilla luciferase,
Wherein, the primer sequence of pMIR-FEN 1-3' UTR-WT is as follows:
pMIR-FEN1-3′UTR-WT-F：5’-gctaactagtgctcaggaaaatatgt-3’-F(SEQ ID NO：：6)
pMIR-FEN1-3′UTR-WT-R：5’-gctaaagcttctccctctcttctcc-3’-R(SEQ ID NO：7)
the primer sequence of pMIR-FEN 1-3' UTR-MUT is:
pMIR-FEN1-3′UTR-MUT-F：5’-cattaaactgcctgagaagatttttgagcctgacatattttcctgagctaaaacagg-3’-F(SEQ ID NO：8)
pMIR-FEN1-3′UTR-MUT-R：
5’-cctgttttagctcaggaaaatatgtcaggctcaaaaatcttctcaggcagtttaatg-3’-R(SEQID NO：9)
2.2 expression level and prognosis relationship between FEN1 and miR-140 in breast cancer
mRNA levels of FEN1 were found to be highly expressed in breast cancer, while miR-140 was low expressed in breast cancer by database (TCGA) analysis (fig. 2A, B). Survival analysis of breast cancer patients by Kaplan Meier showed a better prognosis for patients with low-expression FEN1, while the prognosis for high-expression miR-140 in patients was better than for miR-140 under-expression (FIG. 2C, D).
2.3miR-140 inhibits cell proliferation and clone formation of MCF-7 and MB231
Preliminary experiments have demonstrated that miR-140 can inhibit the expression of FEN1, and therefore miR-140 is suspected to exist as an oncosuppressor in breast cancer cells. Since one of the main features of tumor cells is rapid proliferation. The stronger the proliferation capacity of the tumor cells, the higher the degree of malignancy and the higher the risk of recurrent metastasis in the patient. The proliferation ability of tumor cells is an important index of the malignancy of tumors. Thus, miR-140 is over-expressed to detect migration and proliferation of tumor cells and the like. MCF-7 and MB231 cells were first infected with a lentivirus of miR-140 and a negative control, and a stable cell line was selected. Four stable transfectant cell lines, namely MCF-7-NC, MCF-7-140, MB31-NC and MB231-140, are obtained. First, qPCR was used for validation to confirm that miR-140-stable cell line construction was successful (FIG. 3A). In order to further explore the influence of miR-140 on the proliferation capacity of breast cancer cells, two miR-140-stable-overexpression breast cancer cell lines MCF-7-140 and MB231-140 and negative control cells corresponding to the breast cancer cell lines are compared in cell proliferation capacity. It was found that the proliferation rate of both cell lines overexpressing miR-140 was significantly lower than their corresponding negative control cell lines (FIG. 3B). After counting four cells, dividing the counted cells into six-well plates with 1000 cells per well, and continuously culturing for 10 days, the clone formation experiment also shows that the number of clones formed by the cells after over-expressing miR-140 is obviously reduced (FIG. 3C, D). The above studies indicate that over-expression of miR-140 reduces the proliferation rate of breast cancer cells.
2.4 Effect of miR-140 on cell migration of MCF-7
Cell scratch (figure 4A, B) and Transwell cell invasion experiments (figure 4C, D) are carried out on MCF-7-NC and MCF-7-140 cells, and the experimental results show that the migration capacity of the MCF-7 cells stably transforming miR-140 is obviously lower than that of the MCF-7-NC cells.
2.5 Effect of overexpression of miR-140 on cell cycle
Preliminary experiments prove that the over-expression of miR-140 inhibits the proliferation rate of breast cancer cells. Thus, the influence of miR-140 on the cell cycle is further detected. Ki67 is expressed actively during cell growth, but is silenced when cell growth is stopped, and thus can be used as a marker of cell growth status. Ki67 fluorescent staining of breast cancer cells also demonstrated that cell proliferation was very significantly inhibited after miR-140 was overexpressed (FIG. 5A, B). The effect of miR-140 on the cell cycle was further analyzed by flow cytometry. To prevent the interference of the flow cytometric analysis technique by the green fluorescence carried by the stably transfected cell lines, miR-140 was transiently transfected in MCF-7 and MB231 cells using transient transfection. The cell cycle change of two breast cancer cells after transient miR-140 transfection is detected by a cell flow technology. For example, (FIG. 5C, E) the classical flow histogram simulates the image of each cell cycle stage, (FIG. 5D, F) is the statistical result of the distribution ratio of the cells in MCF-7 and MB231 cells at each stage
2.6 role of overexpression of miR-140 in nude mouse tumor model
In the above-described in vitro cell experiments, it has been found that the proliferation and migration of cells become slow after miR-140 is overexpressed. Therefore, whether the over-expressed miR-140 has the obvious inhibition effect on tumor tissues in vivo is further detected, and a nude mouse is used for carrying out in-vitro transplantation tumor experiment for verification. Approximately 5 weeks of nude mice were first randomized into groups and injected with approximately 2.5X 10 cells of MCF-7-140, MCF-7-NC, MB231-140, and MB231-NC, respectively6Injecting subcutaneously until the tumor grows to about 50mm3Thereafter, changes in tumor volume and body weight of nude mice were recorded every three days, and the survival of the mice was closely followed. FIG. 6A shows the change in the growth of subcutaneous tumor mass in nude mice. After the tumor mass had grown to volume, all mice were sacrificed and the tumor mass was photographed (fig. 6B) and weighed for statistical analysis (fig. 6C). These results indicate that miR-140 can inhibit the proliferation of tumor cells in mice. FIG. 6D shows H histological sections of tumors formed by subcutaneous injection of MCF-7-140 and MCF-7-NC cells in mice&E. FEN1, Ki67, and cleaned-
2.7 analysis of the sensitivity of miR-140 overexpression in cells to different chemotherapeutic drugs
Chemotherapy is the treatment of cancer with chemical drugs that kill cancer cells. Since cancer cells differ from normal cells in the greatest degree by rapid cell division and proliferation, the principle of action of chemotherapeutic drugs is usually to inhibit cancer cell proliferation by interfering with cell division mechanisms, such as inhibiting DNA replication or preventing chromosome segregation. Most chemotherapeutic drugs are not specific and kill tumor cells while killing normal tissue cells that undergo cell division, thereby often damaging healthy tissue that needs to divide to maintain normal function. Therefore, the sensitivity of the tumor cells to the chemotherapeutic drugs is improved, the effect of killing the tumor can be achieved by using the low-dose chemotherapeutic drugs, and the damage of the chemotherapeutic drugs to normal tissue cells can be reduced. To further explore that miR-140 does not affect the sensitivity of breast cancer cells to chemotherapeutic drugs, it was examined in MCF-7-NC and MCF-7-140 cells. Two MCF-7 cells were treated with 5FU, Paclitaxel (Paclitaxel), Cisplatin (cissplatin), Camptothecin (Camptothecin), and Adriamycin (ADR), and MTT was used to test the viability of the cells after culturing the two cells for 48h under different drugs and different drug concentrations. The results prove that the miR-140 overexpression in MCF-7 can increase the sensitivity of cells to adriamycin (FIG. 7E), and the sensitivity effect on other drugs is not obvious (FIGS. 7A-D). Adriamycin is an antitumor antibiotic widely used clinically, can inhibit the synthesis of RNA and DNA, has the strongest inhibition effect on RNA, has wider antitumor spectrum, has killing effect on various tumors, and belongs to a periodic non-specific medicament. Doxorubicin is a commonly used chemotherapeutic agent in the treatment of breast cancer. Then, the sensitizing effect of miR-140 on doxorubicin was further verified in MB231 cells (FIG. 7F), and the obtained results were consistent with the effects in MCF-7 cells.
2.8 overexpression of miR-140 increases Adriamycin-induced apoptosis
In view of previous research, after miR-140 is over-expressed, cell cycle is blocked, DNA damage degree of cells is also obviously increased, and ADR treatment of tumor cells is more sensitive, so that breast cancer over-expressing miR-140 is suspected to promote apoptosis of tumor cells after ADR treatment, so that the breast cancer is more sensitive to ADR. Then, through cytomorphology analysis, the cells over expressing miR-140 are obviously fewer in number and worse in cell morphology than the cells in the reference group after being added with ADR treatment. After FEN1 is over-expressed in MCF-7-140 and MCF-7-140, ADR is added for treatment, the resistance of breast cancer cells to ADR is obviously improved, and the cell state is better (figure 8A). Apoptosis was further detected by flow cytometry. The miR-140 over-expressing cell line used before is a virus-infected stable cell line with green fluorescence, and in order to avoid the influence of fluorescence, miR-140 is over-expressed through transient transfection, and miR-140 and FEN1 are over-expressed simultaneously and then treated with ADR for 24 h. Cells to be detected are doubly stained by annexin V-FITC and PI, a flow cytometer is used for detecting the apoptosis condition, the apoptosis rate of the cells added with ADR is obviously higher than that of cells of an NC group after miR-140 is over-expressed, FEN1 is over-expressed in the cells over-expressing miR-140, and the apoptosis rate is obviously reduced (figure 8B-E)
2.9 miR-140 reverses drug resistance of drug-resistant tumors by downregulating FEN1
To further explore the effect of miR-140 on ADR resistance of MCF-7 cells. MCF-7-ADR cells are infected by the lentivirus of miR-140, and a cell line, namely the MCF-7-ADR-140 cell line, of which the MCF-7-ADR stably over-expresses the miR-140 is obtained through screening (figure 9B). In addition, cell morphology analysis showed and cell viability assay both indicated that miR-140 can reverse drug resistance of MCF-7-ADR cells to doxorubicin while overexpression of FEN1 attenuated this effect of miR-140 after 48h of ADR treatment (FIG. 9A, C).
Therefore, the over-expression of miR-140 significantly inhibits the proliferation of MCF-7 cells and MB231 cells, and the transplantation of tumors in nude mice also demonstrates that the growth rate of miR-140-overexpressing breast cancer xenograft tumors is significantly lower than that of tumors produced by control cells. Furthermore, immunohistochemistry as such also indicated an impaired FEN1 protein content in breast cancer xenografts with the presence of miR-140. These results indicate that miR-140 can at least partially inhibit the occurrence and development of tumors by inhibiting FEN1 expression and inducing DNA damage.
The resistance of breast cancer cells to conventional chemotherapy remains a major obstacle in the treatment of breast cancer
The invention discovers that the MCF-7 is more sensitive to a plurality of chemotherapeutic drugs including adriamycin by over-expressing miR-140, wherein MCF-7 cells over-expressing miR-140 are most sensitive to the adriamycin. miR-140 promotes doxorubicin-induced apoptosis in MCF-7 and MB231 cells, whereas overexpression of FEN1 reverses apoptosis. That is, down-regulation of FEN1 enhanced chemotherapy-induced apoptosis, while over-expression of FEN1 inhibited some chemotherapy-induced apoptosis.
In addition, the study proves that miR-140 can overcome the drug resistance of breast cancer cells to adriamycin, and the property is further weakened by over-expression of
In addition, the research proves that YY1 is combined with the promoter of miR-140 to positively regulate the expression of miR-140, and further inhibit the expression of
In conclusion, the research proves that miR-140 plays a certain role in regulating and controlling DNA repair, genome stability, drug sensitivity and drug resistance of breast cancer cells by regulating and controlling FEN1 expression. The research result of the invention determines the correlation between the function of miR-140 and the occurrence, development and treatment of breast cancer, which has certain significance for the chemotherapy process of breast cancer. Therefore, the development and the use of the agonist of the breast cancer cell endogenous miR-140 are a brand new idea.
Sequence listing
<110> university of Nanjing university
Application of miR-140 in preparation of medicine for inhibiting breast cancer proliferation and migration
<160>9
<170>SIPOSequenceListing 1.0
<210>1
<211>22
<212>RNA
<213> human (Homo sapiens)
<400>1
cagugguuuu acccuauggu ag 22
<210>2
<211>1244
<212>DNA
<213> human (Homo sapiens)
<400>2
atggcctcgg gcgacaccct ctacatcgcc acggacggct cggagatgcc ggccgagatc 60
gtggagctgc acgagatcga ggtggagacc atcccggtgg agaccatcga gaccacagtg 120
gtgggcgagg aggaggagga ggacgacgac gacgaggacg gcggcggtgg cgaccacggc 180
ggcgggggcg gccacgggca cgccggccac caccaccacc accatcacca ccaccaccac 240
ccgcccatga tcgctctgca gccgctggtc accgacgacc cgacccaggt gcaccaccac 300
caggaggtga tcctggtgca gacgcgcgag gaggtggtgg gcggcgacga ctcggacggg 360
ctgcgcgccg aggacggctt cgaggatcag attctcatcc cggtgcccgc gccggccggc 420
ggcgacgacg actacattga acaaacgctg gtcaccgtgg cggcggccgg caagagcggg 480
gcggcggctc gtcgtcgtcg ggaggcggcc gcgtcaagaa gggcggcggc aagaagagcg 540
gcaagaagag ttacctcagc ggcggggccg gcgcggcggg cggcggcggc gccgacccgg 600
gcaacaagaa gtgggagcag aagcaggtgc agatcaagac cctggagggc gagttctcgg 660
tcaccatgtg gtcctcagat gaaaaaaaag atattgacca tgagacagtg gttgaagaac 720
agatcattgg agagaactca cctcctgatt attcagaata tatgacagga aagaaacttc 780
ctcctggagg aatacctggc attgacctct cagatcccaa acaactggca gaatttgcta 840
gaatgaagcc aagaaaaatt aaagaagatg atgctccaag aacaatagct tgccctcata 900
aaggctgcac aaagatgttc agggataact cggccatgag aaaacatctg cacacccacg 960
gtcccagagt ccacgtctgt gcagaatgtg gcaaagcttt tgttgagagt tcaaaactaa 1020
aacgacacca actggttcat actggagaga agccctttca gtgcacgttc gaaggctgtg 1080
ggaaacgctt ttcactggac ttcaatttgc gcacacatgt gcgaatccat accggagaca 1140
ggccctatgt gtgccccttc gatggttgta ataagaagtt tgctcagtca actaacctga 1200
aatctcacat cttaacacat gctaaggcca aaaacaacca gtga 1244
<210>3
<211>129
<212>DNA
<213> human (Homo sapiens)
<400>3
tgcctggcac ataataggca taaggttgcc aggaaataga aatacagata ctcaattcaa 60
tttgatcttc agataatcaa aaaatacttt ttttagtata aatatgtccc attctgtatt 120
tgggacata 129
<210>4
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
cggggtacca atgggaattc aaggcctggc 30
<210>5
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
tgctctagat tattttcccc ttttaaactt cc 32
<210>6
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
gctaactagt gctcaggaaa atatgt 26
<210>7
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
gctaaagctt ctccctctct tctcc 25
<210>8
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
cattaaactg cctgagaaga tttttgagcc tgacatattt tcctgagcta aaacagg 57
<210>9
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
cctgttttag ctcaggaaaa tatgtcaggc tcaaaaatct tctcaggcag tttaatg 57