阅读说明：本技术 一种长非编码rna在诊断和治疗乳腺癌中的应用 (Application of long non-coding RNA in diagnosis and treatment of breast cancer ) 是由 张智弘 狄世豪 于 2019-09-26 设计创作，主要内容包括：本发明属于基因工程领域,特别涉及长非编码RNA LCT-AS1在乳腺癌中作为发生和转移的分子标记物且成为乳腺癌治疗靶点的潜在作用。通过改变长非编码RNA LCT-AS1的表达对乳腺癌细胞的增殖、转移等产生影响,说明敲低长非编码RNA LCT-AS1的表达能够抑制乳腺癌细胞的增殖、转移和细胞周期进展,促进凋亡。(The invention belongs to the field of genetic engineering, and particularly relates to a potential effect of long non-coding RNA LCT-AS1 serving AS a molecular marker for generation and transfer in breast cancer and becoming a breast cancer treatment target. The influence of the expression of the long non-coding RNA LCT-AS1 on the proliferation, the metastasis and the like of the breast cancer cells is changed, so that the knocking down of the expression of the long non-coding RNA LCT-AS1 can inhibit the proliferation, the metastasis and the cell cycle progression of the breast cancer cells and promote the apoptosis.)

1. A long non-coding RNACT-AS 1 for determining the prognosis of breast cancer or AS the target of breast cancer therapy, the nucleotide sequence of the long non-coding RNA is shown in SEQ ID NO. 1.

2. Use of a marker for identifying the long non-coding RNA of claim 1 in the manufacture of a medicament for treating breast cancer and a product for determining prognosis, said marker including but not limited to:

(1) A primer/primer set that binds to the long non-coding RNA or a fluorescently labeled primer/primer set that binds to the long non-coding RNA;

(2) a small molecule compound that binds to the long non-coding RNA;

(3) Biological macromolecules that bind the long non-coding RNA include, but are not limited to: an antibody or functional fragment of an antibody, a fluorescently labeled antibody or functional fragment of an antibody, an RNA-binding protein or functional fragment thereof, a fluorescently labeled RNA-binding protein or functional fragment thereof.

3. The use according to claim 2, wherein the nucleotide sequence of the primer set or the fluorescently labeled primer set is shown as SEQ ID No.2 and SEQ ID No. 3.

4. A reagent or kit for determining the prognosis of breast cancer comprising the marker of claim 2 or 3.

5. use of the long non-coding RNA of claim 1 as a diagnostic agent for screening for prognosis of breast cancer.

6. the use of the long non-coding RNA of claim 1 as a screening agent for the treatment of breast cancer.

7. Use of the long non-coding RNA of claim 1 in the preparation of a medicament for the treatment of breast cancer.

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to application of long non-coding RNA LCT-AS1 in diagnosis/treatment of breast cancer.

Background

breast Cancer (BC) is one of the most common malignant tumors in China and is the leading cause of Cancer death of women all over the world, and with the development of comprehensive treatment means such as surgery, endocrine therapy, chemotherapy and targeted therapy, the death rate of Breast Cancer is on the decline, but still is one of the main causes of Cancer-related death of postmenopausal women, accounting for 23% of all Cancer deaths. Early-stage breast cancer can be cured by treatment, but most breast cancer patients are in the middle and late clinical stage in the initial diagnosis due to the female self-examination and the negligence of clinical examination of the breast, and even if the treatment means is comprehensively applied, the risk of recurrence and metastasis is high, and the prognosis is not optimistic.

Chip technology and complete transcriptome sequencing technology research show that RNA capable of coding protein in human genome only accounts for 2%, and the rest is non-coding RNA (ncRNA). Long non-coding RNA (LncRNA) is a RNA molecule with the length of more than 200nt and without protein coding capacity, and is widely involved in regulating and controlling various vital activities of cells at the transcription, transcription and epigenetic level, and the abnormal expression of the RNA molecule is closely related to the occurrence and development of malignant tumors. A large number of researches show that lncRNA plays an important role in the occurrence and development process of breast cancer. For example, an increase in the expression levels of LncRNA AFAP1-AS1, ARNLA, ZNF469, DANCR and a decrease in the expression levels of LncRNA H19, LncRNA GASS are associated with a poor prognosis of TNBC. For another example, LncRNA MALAT1 regulates a key pathway for the occurrence and progression of Triple Negative Breast Cancer (TNBC), and high-expression MALAT1 can up-regulate the expression levels of c-MET and SOX4 by competitively binding to targeted mRNAs such as miR-34a/c-5p and miR-449 a/b, thereby promoting the proliferation and metastasis of tumor cells.

Disclosure of Invention

The present invention finds that LncRNA LCT antisense RNA 1(LCT-AS1) is significantly up-regulated in breast cancer tissues compared to adjacent normal tissues. Researches find that the over-expression LncRNA LCT-AS1 can promote the proliferation and metastasis of breast cancer cells, is an important carcinogenic factor of the breast cancer, can be used AS a potential molecular marker for the occurrence and metastasis of the breast cancer and a target point for the treatment of the breast cancer, and therefore has good application prospect in the aspect of predicting and treating the breast cancer.

the LncRNA LCT-AS1 is an LncRNA with the length of 1862bp, and is RNA which is provided by the inventor for normal breast tissue and breast cancer tissue samples, is screened by TCGA database analysis and qPCR detection and is remarkably and highly expressed in the breast cancer tissue. The invention discovers the regulation and control function in the breast cancer for the first time and has novelty.

The invention aims to provide LncRNA LCT-AS1 for judging the prognosis condition of breast cancer or serving AS a breast cancer treatment target, the nucleotide sequence of which is SEQ ID NO:1,

The invention also relates to the application of the marker for identifying the long non-coding RNA in the preparation of a diagnostic product for judging the prognosis of breast cancer treatment, wherein the marker comprises but is not limited to:

(1) A primer/primer set that binds to the long non-coding RNA or a fluorescently labeled primer/primer set that binds to the long non-coding RNA;

(2) A small molecule compound that binds to the long non-coding RNA;

(3) Biological macromolecules that bind the long non-coding RNA include, but are not limited to: an antibody or functional fragment of an antibody, a fluorescently labeled antibody or functional fragment of an antibody, an RNA-binding protein or functional fragment thereof, a fluorescently labeled RNA-binding protein or functional fragment thereof.

The nucleotide sequences of the primer group or the fluorescence labeled primer group are shown as SEQ ID NO.2 and SEQ ID NO.3,

Primer F(SEQ ID NO：2)：

5’-CGGGAGGAAGATAAACGGGG-3’，

Primer R(SEQ ID NO：3)：

5’-TGACCACGGGAACACCTTCAG-3’。

the invention also relates to a reagent or a kit for judging the prognosis of breast cancer treatment, which comprises the marker for identifying the long non-coding RNA.

The invention also relates to the application of the marker for identifying the long non-coding RNA or the reagent or the kit containing the marker in judging the treatment prognosis condition of the breast cancer.

The invention also relates to the application of the long non-coding RNA in the preparation of medicaments for treating breast cancer;

The invention also relates to application of the long non-coding RNA in screening and judging a diagnosis reagent of breast cancer prognosis.

The invention also relates to application of the long non-coding RNA in screening of drugs for treating breast cancer.

Technical scheme

1. Tissue collection

56 samples of breast cancer and corresponding paracancerous tissue were obtained from patients who were operated at the first subsidiary hospital of the Nanjing medical university. All cases were confirmed to be breast cancer based on histopathological evaluation. These patients have not undergone local or systemic treatment prior to surgery. All collected tissue samples were immediately snap frozen in liquid nitrogen and stored at-80 ℃ until use. Our study was approved by the research ethics committee of the first subsidiary hospital of the university of medical, Nanjing.

2. Cell culture

two breast cancer cell lines (T-47D & MDA-MB-231) were purchased from Shanghai cell Bank, China, academy of sciences. The culture condition of the T-47D cells is RPMI 1640+ 10% fetal bovine serum +0.2Units/ml insulin; the MDA-MB-231 cell culture condition is L-15+ 10% fetal bovine serum. Complete medium containing 10% fetal bovine serum, 100U/ml penicillin and 100 mg/ml streptomycin was cultured at 37 ℃ in a 5% CO2 incubator.

3. RNA extraction and quantitative PCR analysis

Total RNA was isolated using Trizol reagent according to the instructions for use of the reagent. Reverse transcription was performed using TaKaRa Prime Script kit (TaKaRa, Dalian, China). The reverse transcription kit reverse-transcribes 1. mu.g of total RNA to a final volume of 20. mu.l. And (4) analyzing results: analyzing the specificity and the amplification efficiency of the primer, and judging the reaction specificity of the primer according to the dissolution curve. And (5) obtaining a Ct value according to the amplification curve, and analyzing the relative expression quantity of the target gene by adopting a relative quantity method and an internal reference GAPDH. The calculation formula is as follows: 2^ (-. DELTA.Ct), and [ Delta ] Ct is Ct gene-Ct control.

4. Plasmid construction

the full-length cDNA sequence of human LCT-AS1 was synthesized and inserted into the pcDNA vector. Ectopic overexpression of LncRNA-LCT-AS1 was obtained by transfection with pcDNA-LCT-AS1, against an empty pcDNA vector. qRT-PCR measured the expression level of LncRNA LCT-AS1 after 48 hours.

5. Cell transfection

All plasmid vectors used for transfection were extracted with a plasmid extraction kit (DNA miniprep kit, Qiagen) for endotoxin removal. The interference sequence and the random control (si-NC) of LncRNA LCT-AS1 were purchased from Invitrogen (Invitrogen, Calif., USA).

T-47D and MDA-MB-231 cells were plated at 2X 10 per well⁵Planting the cells on a 6-hole culture plate, and after the cells adhere to the wall, discarding the original culture medium 12h before transfection, and replacing the original culture medium with a double-antibody-free culture medium; diluting 10 μ l liposome in 250 μ l OPTI-MEM, gently pumping and mixing, and incubating at room temperature for 5 min; 100pmol siRNA, si-NC or 4ug pcDNA, pcDNA-LCT-AS1 were diluted in 250. mu.l OPTI-MEM, blown and mixed well, incubated at room temperature for 5 min; mixing the incubated liposome with siRNA or plasmid diluent, gently blowing, uniformly mixing, and continuously incubating for 20min at room temperature; uniformly dripping the mixture into a 6-hole culture plate added with 1.5mL of OPTI-MEM in advance, gently mixing, and continuously culturing in a 5% CO2 cell culture box at 37 ℃; culturing for 6h, removing the OPTI-MEM culture medium, replacing with complete culture medium, and culturing at 37 deg.C in 5% CO2 cell culture box; and collecting cells to extract total RNA or protein 24-48h after transfection, and performing qRT-PCR detection or western blot analysis.

Plasmid vectors (pcDNA3.1-LncRNA LCT-AS1 and empty pcDNA vector) were transfected with lipofectamine2000 transfection reagent according to the protocol. T-47D and MDA-MB-231 cells were seeded into six-well plates upon confluency and then manipulated as described. 48 hours after transfection, cells were harvested for qRT-PCR or immunoblot analysis.

6. CCK8 experiment T-47D and MDA-MB-231 cells 24h after transfection were seeded at 3000 cells per well in 96-well culture plates; after 80% of cells adhere to the wall, synchronizing the cells for 12h, and discarding the original culture medium. For each sample, 6 duplicate wells were set, with a total reaction volume of 200. mu.l per well. 20. mu.l of CCK8 reaction solution (5mg/ml in PBS) was added to each well, incubated at 37 ℃ for 2 hours in the absence of light, and the absorbance at 450nm was measured with a microplate reader. Five measurements were made, each at the same interval.

7. Clone formation experiments

digesting with 0.25% trypsin, blowing into single cell suspension, and inoculating to 6-well plate at appropriate cell density (500 cells) to disperse cells uniformly; placing into a cell culture box, changing the culture solution every 4 days, and culturing for 2 weeks. When macroscopic colonies appeared in the culture dish, the culture was terminated. Discarding the supernatant, and gently washing with PBS for 2 times; adding pure methanol or 1: 3 acetic acid/methanol 1ml, fixing for 15 minutes; the methanol fixing solution was discarded, 1ml of 0.1% crystal violet staining solution was added for 15 minutes, and then the staining solution was slowly washed away with PBS and air-dried. The 6-well plate was inverted and overlaid with a piece of transparent film with a grid, and the clones were counted directly with the naked eye or the number of clones larger than 10 cells was counted in a microscope (low power mirror). And finally calculating the clone formation rate.

8. flow cytometry

Preparation of cell samples: the cell culture fluid was carefully collected into a centrifuge tube for use. Digesting the cells with pancreatin until the cells can be blown down by a pipette or a gun head, adding the previously collected cell culture solution, blowing down all adherent cells, and blowing off the cells gently. Again collected in the centrifuge tube. Centrifuging at 1000rpm for 3-5 min to precipitate the cells. For a particular cell, if the cell pellet is insufficient, the centrifugation time may be appropriately prolonged or the centrifugation force may be slightly increased. The supernatant was carefully aspirated, and about 50. mu.l of the culture medium was left to avoid aspiration of the cells. Approximately 1mL of ice-cooled PBS was added, the cells were resuspended, the pelleted cells were again centrifuged, and the supernatant carefully aspirated. Add again 1mL ice-cooled PBS and resuspend the cells.

cell fixation: 4ml of ice-bath precooled 95% ethanol is taken, 1ml of cell suspension is added by quotient (operated on water) while low-speed vortex application is required, and after uniform mixing, the cell suspension is fixed for 2 hours or longer at 4 ℃. The effect may be better when the fixation is carried out for 12-24 hours. Centrifuging at 1000rpm for 3-5 min to precipitate the cells. For a particular cell, if the cell pellet is insufficient, the centrifugation time may be appropriately prolonged or the centrifugation force may be slightly increased. The supernatant was carefully aspirated, and about 50. mu.l of ethanol remained to avoid aspiration of the cells. About 5ml of ice-cooled PBS was added to resuspend the cells, re-pellet the cells by centrifugation, carefully aspirate the supernatant, and about 50. mu.l of PBS may remain to avoid aspiration of the cells. Gently flick the bottom of the centrifuge tube to properly disperse the cells and avoid cell clumping.

Preparation of an propidium iodide staining solution: with reference to the following table, appropriate amounts of propidium iodide staining solution were prepared according to the number of samples to be tested:

Note: the prepared propidium iodide staining solution can be stored at 4 ℃ in a short time, and is suitable for use on the same day.

Dyeing: 0.4mL of propidium iodide staining solution was added to each tube of cell sample, and the cell pellet was slowly and thoroughly resuspended and incubated at 37 ℃ in the dark for 30 minutes. Subsequently, the cells can be stored at 4 ℃ or in an ice bath protected from light. After the staining is finished, the flow detection is preferably finished within 24 hours, and the flow detection can be preferably finished on the same day.

Flow detection and analysis: the red fluorescence was detected with a flow cytometer at the 488nm excitation wavelength, with the light scattering detected. Cellular DNA content analysis and light scattering analysis were performed using appropriate analysis software.

9. The transfected cells were digested by the Transwell assay, the medium was discarded by centrifugation after termination of the digestion, washed 1-2 times with PBS and resuspended in serum-free medium containing BSA. Adjusting cell density to 1-10X 10⁴. Cell suspension 300. mu.l was added to the Transwell chamber. 700. mu.l of a medium containing 20% FBS or a chemokine is added to the lower chamber of the 24-well plate, and the mixture is placed into an incubator for conventional culture for 12-48 h. The cells in the upper chamber were wiped off with a cotton swab from the chamber, and the stained cells on the outer bottom surface of the chamber were stained with 0.1% crystal violet, and the stained cells attached to the lower chamber side of the basement membrane of the Transwell chamber were photographed and counted using an inverted microscope.

10. Data processing

The experimental data were analyzed using the SPSS17.0 software, expressed as mean ± standard error of the experiment, and the differences between groups were tested using the two-tailed Student's T test, the rank-sum test, and the chi-square test.

Drawings

FIG. 1 upregulation of LncRNA LCT-AS1 in breast cancer tissue

1A: detecting that the expression of LncRNA LCT-AS1 in the breast cancer tissue is up-regulated compared with that in the normal tissue by a QRT-PCR method;

1B: the expression level of LncRNA LCT-AS1 in breast cancer tissues was divided into two groups.

FIG. 2 expression level of LncRNA LCT-AS1 in breast cancer cells

LncRNA LCT-AS1 is up-regulated in breast cancer cells compared with normal cells;

2B: detecting the interference efficiency of transfected si-LCT-AS1 in breast cancer cells by a QRT-PCR method;

2C: LncRNACT-AS 1 was overexpressed in breast cancer cells AS above.

FIG. 3 LncRNA LCT-AS1 promoting proliferation of breast cancer cells

3A: CCK8 experiment shows that after the LncRNA LCT-AS1 is interfered, the proliferation of the breast cancer cells is inhibited;

3B: the clone formation experiment shows that after the LncRNA LCT-AS1 is interfered, the proliferation of the breast cancer cells is inhibited;

3C: CCK8 experiment shows that the LncRNA LCT-AS1 is over-expressed to promote the proliferation of breast cancer cells;

3D: the cloning experiments show that the LncRNA LCT-AS1 is over-expressed to promote the proliferation of the breast cancer cells.

FIG. 4 Effect of LncRNA LCT-AS1 on apoptosis and metastasis of breast cancer cells

4A, 4B: flow cytometry analysis finds that the interference LncRNA LCT-AS1 can promote apoptosis of breast cancer cells;

4C: the Transwell experiment indicates that the interference LncRNA LCT-AS1 can inhibit the breast cancer cell metastasis;

4D: the LncRNA LCT-AS1 is overexpressed to promote the metastasis of the breast cancer cells.

Detailed Description

The invention is further illustrated by the following examples, without restricting the invention thereto.

general description: