Application of UPK1A-AS1 inhibitor in preparation of antitumor drugs
阅读说明：本技术 Upk1a-as1抑制剂在制备抗肿瘤药物中的应用 (Application of UPK1A-AS1 inhibitor in preparation of antitumor drugs ) 是由 张冬艳 吴德华 刘莉 邹雪晶 宋旸 张雅璇 于 2021-07-13 设计创作，主要内容包括：本发明属于药物技术领域,公开了UPK1A-AS1抑制剂在制备抗肿瘤药物中的应用。本发明首次公开了UPK1A-AS1抑制剂或其药学上可接受的盐在制备抗肿瘤药物中的应用,这是基于发明人发现抑制UPK1A-AS1表达和/或活性可以抑制肿瘤细胞及肿瘤组织的增殖,并且对给药动物的体重无显著影响,同时,给药期间,未观察到药物治疗副作用；表明：UPK1A-AS1抑制剂或其药学上可接受的盐可以抑制肿瘤组织的增殖,且无副作用,可用于制备抗肿瘤药物。(The invention belongs to the technical field of medicines, and discloses application of a UPK1A-AS1 inhibitor in preparation of an antitumor medicine. The invention discloses the application of a UPK1A-AS1 inhibitor or pharmaceutically acceptable salts thereof in preparing antitumor drugs for the first time, which is based on the discovery that the inhibition of the expression and/or activity of UPK1A-AS1 can inhibit the proliferation of tumor cells and tumor tissues, and has no significant influence on the body weight of an animal to which the drug is administered, and meanwhile, no side effect of the drug treatment is observed during the administration period; shows that: the UPK1A-AS1 inhibitor or its pharmaceutically acceptable salt can inhibit proliferation of tumor tissue, has no side effect, and can be used for preparing antitumor drugs.)
An application of UPK1A-AS1 inhibitor or pharmaceutically acceptable salt thereof in preparing antineoplastic drugs.
2. Use according to claim 1, characterized in that: the UPK1A-AS1 inhibitor is an agent for inhibiting UPK1A-AS1 expression and/or reducing UPK1A-AS1 activity;
preferably, the inhibitor of UPK1A-AS1 is at least one of an antisense oligonucleotide targeting UPK1A-AS1, an interfering RNA targeting UPK1A-AS1, a CRISPR targeting UPK1A-AS1, a TALEN targeting UPK1A-AS1, and a zinc finger nuclease targeting UPK1A-AS 1.
3. Use according to claim 2, characterized in that:
the interfering RNA targeting UPK1A-AS1 comprises dsRNA, siRNA and shRNA targeting UPK1A-AS 1.
4. Use according to claim 2, characterized in that:
the antisense oligonucleotide targeting UPK1A-AS1 contains at least one modified nucleotide group;
preferably, the modified nucleotide group is at least one of (1) to (3):
(1) a phosphate group-modified nucleotide group;
(2) a ribose group-modified nucleotide group;
(3) a base-modified nucleotide group.
5. Use according to claim 4, characterized in that:
the phosphate group modification is to modify oxygen in a phosphate group, and comprises sulfo modification and boration modification;
preferably, the ribose group modification is the modification of 2 ' -hydroxyl in the ribose group, and comprises 2 ' -fluorine modification, 2 ' -methoxy modification, 2 ' -methoxyethyl modification, 2 ' -2, 4-dinitrophenol modification, locked nucleic acid modification, 2 ' -amino modification and 2 ' -deoxy modification;
further preferably, the antisense oligonucleotide targeting UPK1A-AS1 is a locked nucleotide targeting UPK1A-AS 1.
6. Use according to claim 5, characterized in that:
the locked nucleotide of the targeting UPK1A-AS1 is at least one of (4) to (6);
(4) a locked nucleotide of sequence AGCAGACCTTCCTAAC (SEQ ID No. 1);
(5) a locked nucleotide of sequence AACAGCACTGTCAAGG (SEQ ID NO. 2);
(6) a locked nucleotide of sequence TCTTTGCCCACTTTAC (SEQ ID NO. 3);
preferably, the locked nucleotide targeting UPK1A-AS1 is at least one of (5) to (6).
7. Use according to claim 6, characterized in that:
the anti-tumor medicament also comprises a pharmaceutically acceptable carrier or diluent;
preferably, the pharmaceutically acceptable carrier is a colloidal dispersion system, a macromolecular complex, a nanocapsule, a nanoparticle, a microsphere, a bead, an oil-in-water emulsion, a micelle, a mixed micelle, or a liposome;
preferably, the diluent is PBS.
8. Use according to claim 6, characterized in that:
the dosage form of the anti-tumor medicament is at least one of a solid preparation, a liquid preparation and a semi-solid preparation;
preferably, the solid formulation includes tablets, granules, powders and capsules;
preferably, the liquid formulation comprises an injection;
preferably, the semi-solid formulation comprises an ointment and a cream.
9. Use according to any one of claims 1 to 8, wherein:
the tumor is at least one of liver cancer, ovarian cancer, lung cancer, gastric cancer, colorectal cancer, esophageal cancer, breast cancer, thyroid cancer, cervical cancer, cerebroma and pancreatic cancer.
10. A medicament, comprising:
(1) an inhibitor of UPK1A-AS1 or a pharmaceutically acceptable salt thereof; and
(2) a pharmaceutically acceptable carrier or diluent.
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of a UPK1A-AS1 inhibitor in preparation of an anti-tumor medicine.
Background
Primary Hepatocellular carcinoma (HCC, liver cancer for short) is one of the most common malignant tumors in clinic, the incidence rate is 4 th of the malignant tumor, and the mortality rate is 3 rd. Liver cancer has the characteristics of hidden morbidity and rapid progress, most patients are diagnosed at a late stage, the radical treatment opportunities such as surgical operation and the like are lost, the 5-year survival rate is only 14.1 percent, and the life health of people in China is seriously threatened. Targeted drug therapy such as sorafenib and ranvatinib is a first-line treatment scheme for late-stage liver cancer, but the population benefiting from treatment is few, and patients often have acquired drug resistance after treatment; the total effective rate of the single immune checkpoint inhibitor is only about 20%. Therefore, new anti-liver cancer drugs are urgently needed to be marketed.
Long non-coding RNAs are RNAs with a length of more than 200 nucleotides, and have weak or even lack of protein coding capacity. LncRNA is used as a new pathophysiology regulating factor, can regulate and control the invasion, the metastasis and the proliferation of tumors, and is expected to become a molecular marker and a potential treatment target for judging the prognosis of the tumors.
Disclosure of Invention
The invention aims at providing the application of the UPK1A-AS1 inhibitor in preparing antitumor drugs.
In a second aspect, the present invention is directed to a medicament.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides an application of a UPK1A-AS1 inhibitor or pharmaceutically acceptable salt thereof in preparing an anti-tumor medicament.
Preferably, the UPK1A-AS1 has an accession number (Gene ID) of 100862728.
Preferably, the inhibitor of UPK1A-AS1 is an agent that inhibits expression of UPK1A-AS1 and/or reduces activity of UPK1A-AS 1.
Preferably, the inhibitor of UPK1A-AS1 is at least one of an antisense oligonucleotide targeting UPK1A-AS1, an interfering RNA targeting UPK1A-AS1, a CRISPR targeting UPK1A-AS1, a TALEN targeting UPK1A-AS1, and a zinc finger nuclease targeting UPK1A-AS 1.
Preferably, the antisense oligonucleotide targeting UPK1A-AS1 contains at least one modified nucleotide group that does not result in loss of function of the antisense oligonucleotide targeting UPK1A-AS1, thereby improving the stability and activity of the antisense oligonucleotide.
Preferably, the modified nucleotide group is at least one of (1) to (3):
(1) a phosphate group-modified nucleotide group;
(2) a ribose group-modified nucleotide group;
(3) a base-modified nucleotide group.
Preferably, the phosphate group modification is a modification of the oxygen in the phosphate group, including thio-and boration modifications.
Preferably, the ribose group modification is modification of 2 ' -hydroxyl in the ribose group, and comprises 2 ' -fluorine modification, 2 ' -methoxy modification, 2 ' -methoxyethyl modification, 2 ' -2, 4-dinitrophenol modification, locked nucleic acid modification, 2 ' -amino modification and 2 ' -deoxy modification.
Preferably, the antisense oligonucleotide targeting UPK1A-AS1 is a locked nucleotide targeting UPK1A-AS 1.
Preferably, the locked nucleotide of the targeting UPK1A-AS1 is at least one of (4) to (6); further, at least one of (5) to (6):
(4) a locked nucleotide of sequence AGCAGACCTTCCTAAC (SEQ ID No. 1);
(5) a locked nucleotide of sequence AACAGCACTGTCAAGG (SEQ ID NO. 2);
(6) a locked nucleotide having the sequence of TCTTTGCCCACTTTAC (SEQ ID NO. 3).
Preferably, the interfering RNA targeting UPK1A-AS1 comprises dsRNA, siRNA and shRNA targeting UPK1A-AS 1.
Preferably, the tumor is at least one of liver cancer, ovarian cancer, lung cancer, gastric cancer, colorectal cancer, esophageal cancer, breast cancer, thyroid cancer, cervical cancer, brain tumor and pancreatic cancer; further, liver cancer.
Preferably, the anti-tumor drug further comprises a pharmaceutically acceptable carrier or diluent.
Preferably, the pharmaceutically acceptable carrier is a colloidal dispersion system, a macromolecular complex, a nanocapsule, a nanoparticle, a microsphere, a bead, an oil-in-water emulsion, a micelle, a mixed micelle, or a liposome.
Preferably, the diluent is PBS.
Preferably, the dosage form of the antitumor drug is at least one of a solid preparation, a liquid preparation and a semi-solid preparation.
Preferably, the solid formulation includes tablets, granules, powders and capsules.
Preferably, the liquid formulation comprises an injection.
Preferably, the semi-solid formulation comprises an ointment and a cream.
Preferably, the administration dose is 8-20 mg/kg of mice.
Preferably, the administration mode of the antitumor drug is injection.
In a second aspect of the invention, there is provided a medicament comprising:
(1) an inhibitor of UPK1A-AS1 or a pharmaceutically acceptable salt thereof; and
(2) a pharmaceutically acceptable carrier or diluent.
Preferably, the UPK1A-AS1 has an accession number (Gene ID) of 100862728.
Preferably, the inhibitor of UPK1A-AS1 is an agent that inhibits expression of UPK1A-AS1 and/or reduces activity of UPK1A-AS 1.
Preferably, the inhibitor of UPK1A-AS1 is at least one of an antisense oligonucleotide targeting UPK1A-AS1, an interfering RNA targeting UPK1A-AS1, a CRISPR targeting UPK1A-AS1, a TALEN targeting UPK1A-AS1, and a zinc finger nuclease targeting UPK1A-AS 1.
Preferably, the antisense oligonucleotide targeting UPK1A-AS1 contains at least one modified nucleotide group that does not result in loss of function of the antisense oligonucleotide targeting UPK1A-AS1, thereby improving the stability and activity of the antisense oligonucleotide.
Preferably, the modified nucleotide group is at least one of (1) to (3):
(1) a phosphate group-modified nucleotide group;
(2) a ribose group-modified nucleotide group;
(3) a base-modified nucleotide group.
Preferably, the phosphate group modification is a modification of the oxygen in the phosphate group, including thio-and boration modifications.
Preferably, the ribose group modification is modification of 2 ' -hydroxyl in the ribose group, and comprises 2 ' -fluorine modification, 2 ' -methoxy modification, 2 ' -methoxyethyl modification, 2 ' -2, 4-dinitrophenol modification, locked nucleic acid modification, 2 ' -amino modification and 2 ' -deoxy modification.
Preferably, the antisense oligonucleotide targeting UPK1A-AS1 is a locked nucleotide targeting UPK1A-AS 1.
Preferably, the locked nucleotide of the targeting UPK1A-AS1 is at least one of (4) to (6); further, at least one of (5) to (6):
(4) a locked nucleotide of sequence AGCAGACCTTCCTAAC (SEQ ID No. 1);
(5) a locked nucleotide of sequence AACAGCACTGTCAAGG (SEQ ID NO. 2);
(6) a locked nucleotide having the sequence of TCTTTGCCCACTTTAC (SEQ ID NO. 3).
Preferably, the interfering RNA targeting UPK1A-AS1 comprises dsRNA, siRNA and shRNA targeting UPK1A-AS 1.
Preferably, the pharmaceutically acceptable carrier is a colloidal dispersion system, a macromolecular complex, a nanocapsule, a nanoparticle, a microsphere, a bead, an oil-in-water emulsion, a micelle, a mixed micelle, or a liposome.
Preferably, the diluent is PBS.
Preferably, the dosage form of the antitumor drug is at least one of a solid preparation, a liquid preparation and a semi-solid preparation.
Preferably, the solid formulation includes tablets, granules, powders and capsules.
Preferably, the liquid formulation comprises an injection.
Preferably, the semi-solid formulation comprises an ointment and a cream.
The invention has the beneficial effects that:
the invention discloses the application of a UPK1A-AS1 inhibitor or pharmaceutically acceptable salts thereof in preparing antitumor drugs for the first time, which is based on the discovery that the inhibition of the expression and/or activity of UPK1A-AS1 can inhibit the proliferation of tumor cells and tumor tissues, and has no significant influence on the body weight of an animal to which the drug is administered, and meanwhile, no side effect of the drug treatment is observed during the administration period; shows that: the UPK1A-AS1 inhibitor or its pharmaceutically acceptable salt can inhibit proliferation of tumor tissue, has no side effect, and can be used for preparing antitumor drugs.
The UPK1A-AS1 inhibitor is limited to locked nucleotide targeting UPK1A-AS1, the sequence of the locked nucleotide is limited to TCTTTGCCCACTTTAC, and the locked nucleotide can obviously inhibit the expression (P <0.001) of UPK1A-AS1 in tumor cells and the proliferation (P <0.01) of the tumor cells and tumor tissues, and has more excellent anti-tumor effect.
Drawings
FIG. 1 is a graph of the effect of UPK1A-AS1 inhibitors (locked nucleic acids of UPK1A-AS 1) on the expression level of UPK1A-AS1 in hepatoma cells: wherein A is a graph of the effect of an inhibitor of UPK1A-AS1 (locked nucleic acid of UPK1A-AS 1) on the expression level of UPK1A-AS1 in MHCC-97H of hepatoma cells; b is a graph of the effect of an inhibitor of UPK1A-AS1 (locked nucleic acid of UPK1A-AS 1) on the expression level of UPK1A-AS1 in the hepatoma cell SK-Hep-1.
FIG. 2 is a graph of the effect of UPK1A-AS1 inhibitor (locked nucleic acid of UPK1A-AS 1) on hepatoma cell clonality: wherein A is a visual chart of the influence of a UPK1A-AS1 inhibitor (locked nucleic acid of UPK1A-AS 1) on the clone formation of MHCC-97H of the hepatoma cell; b is a visual chart of the influence of a UPK1A-AS1 inhibitor (locked nucleic acid of UPK1A-AS 1) on the formation of the SK-Hep-1 clone of the hepatoma cell; c is a statistical chart of the effect of UPK1A-AS1 inhibitor (locked nucleic acid of UPK1A-AS 1) on the formation of MHCC-97H clone of hepatoma cells; d is a statistical chart of the effect of UPK1A-AS1 inhibitor (locked nucleic acid of UPK1A-AS 1) on the formation of SK-Hep-1 clone in hepatoma cells.
FIG. 3 is a graph of the effect of UPK1A-AS1 inhibitors (locked nucleic acids of UPK1A-AS 1) on tumor tissue in mice: wherein A is a visual image of the change of tumor tissues in vivo with time after mice are injected with UPK1A-AS1 inhibitor (locked nucleic acid of UPK1A-AS 1); b is a line graph of tumor tissue changes over time in vivo following mouse injection with an inhibitor of UPK1A-AS1 (locked nucleic acid of UPK1A-AS 1); c is a statistical plot of tumor tissue changes over time in vivo following mouse injection with an inhibitor of UPK1A-AS1 (locked nucleic acid of UPK1A-AS 1).
FIG. 4 is a graph of the effect of UPK1A-AS1 inhibitor (locked nucleic acid of UPK1A-AS 1) on mouse body weight: wherein A is a visual depiction of body weight change over time following mouse injection with UPK1A-AS1 inhibitor (locked nucleic acid of UPK1A-AS 1); b is a line graph of body weight over time after mice were injected with an inhibitor of UPK1A-AS1 (locked nucleic acid of UPK1A-AS 1); c is a statistical plot of body weight over time after mice were injected with UPK1A-AS1 inhibitor (locked nucleic acid of UPK1A-AS 1).
Detailed Description
The present invention will be described in further detail with reference to specific examples.
The starting materials used in the examples were prepared by conventional means or purchased from commercial sources, except as otherwise specified.
Experimental materials:
BALB/c nude mice: BALB/c nude mice (4-6 weeks old, male) provided by southern medical university laboratory animal center were bred in SPF animal houses of southern hospital of southern medical university, the squirrel cage and the used utensils were both strictly sterilized, and the mice were fed freely and purified water was drunk; before the experiment, the nude mice were observed to have clinical symptoms for later use.
Example 1 silencing Effect of UPK1A-AS1 inhibitors (locked nucleic acids of UPK1A-AS 1) on UPK1A-AS1 and Effect on hepatoma cell proliferation
1. Experimental Material
Liver cancer cell line: the hepatoma cell strains MHCC-97H and SK-Hep-1 are purchased from Shanghai cell bank of Chinese academy of sciences, and are cultured in DMEM medium containing 10% serum and 5% CO2Culturing in a constant temperature box at 37 ℃, carrying out passage and subsequent experiments when the cell density reaches about 85%, and changing the liquid every 1-2 days.
Medicine preparation: LNA-i-UPK1A-AS1-1 (sequence AGCAGACCTTCCTAAC, SEQ ID NO.1, molecular weight: 5205.2Da, LNA-i-AS1-1), LNA-i-UPK1A-AS1-2 (sequence AACAGCACTGTCAAGG, SEQ ID NO.2, molecular weight: 5280.2Da, LNA-i-AS1-2), LNA-i-UPK1A-AS1-3 (sequence TCTTTGCCCACTTTAC, SEQ ID NO.3, molecular weight: 5139.1Da, LNA-i-AS1-3) and LNA-i-NC (LNA-i-NC), available from Exiqon corporation under the following trade designations: 300600, Design ID: 683533-3; all the bases in LNA-i-UPK1A-AS1-1, LNA-i-UPK1A-AS1-2 and LNA-i-UPK1A-AS1-3 are LNA modified. 2nmol LNA was dissolved in 100. mu.L RNase-free H2And O, preparing 20 mu M of storage solution, storing at-20 ℃, subpackaging and storing, and avoiding repeated freeze thawing.
UPK1A-AS1 expression level assay
(1) Respectively taking 1 × 105MHCC-97H cells and SK-Hep1-1 cells are inoculated in a six-well plate, when the cell fusion degree reaches 70%, LNA-i-UPK1A-AS1-1, LNA-i-UPK1A-AS1-2, LNA-i-UPK1A-AS1-3 and LNA-i-NC are transfected respectively (the final concentration of LNC is 100nM), the culture is continued for 24H, and each treatment is repeated for 3 times;
(2) collecting the above treated cells, discarding the medium, washing the cells with PBS 2 times, then lysing the cells with 1mL Trizol lysate (Takara), precipitating with isopropanol, washing with 75% ethanol, preliminarily extracting total RNA, treating the crude extracted RNA with DNase I, removing genomic DNA mixed in the RNA, to purify the RNA;
(3) RNA reverse transcription was performed according to the instructions of PrimeScirpt reverse transcription kit (Takara), and Real-Time PCR was performed according to the instructions of Real-Time PCR reagent SYBR I Premix Ex TaqTM (Takara) kit, using Roche Light Cycle480 Real-Time PCR system for PCR reaction. The primer sequence of UPK1A-AS1 is AS follows: 5'-AGAGCGGTGGGTTAGGAA-3' (SEQ ID NO.4) and R5'-GGGCAGATGGACCAAGCA-3' (SEQ ID NO. 5).
3. Plate clone formation experiment
(1) Respectively taking 1 × 105The MHCC-97H cells and the SK-Hep1-1 cells are inoculated in a six-well plate until the cell fusion degree reaches 70%, separately transfected with LNA-i-UPK1A-AS1-1, LNA-i-UPK1A-AS1-2, LNA-i-UPK1A-AS1-3 and LNA-i-NC (final LNC concentration 100nM), and cultured for 24h, each treatment being repeated 3 times.
(2) Liver cancer cells (MHCC-97H cells, SK-Hep-1 cells) in logarithmic growth phase were taken, digested with 0.25% pancreatin and blown into single cells, and the cells were suspended in DMEM containing 10% fetal bovine serum for use.
(3) Diluting the cell suspension by gradient multiple, inoculating 100 cells into a six-well plate, gently mixing to uniformly distribute the cells in the six-well plate, and placing at 37 ℃ with 5% CO2And a cell incubator with saturated humidity, culturing for 2 or 3 weeks (wherein MHCC-97H 3 week, SK-Hep-12 week), and changing the solution 1 time per week.
(4) Observing the cells every day, stopping culturing when macroscopic cloning appears in the culture plate, discarding the culture medium, washing the cells with PBS 2 times, fixing with methanol for 15min, removing the fixing solution, adding crystal violet for dyeing for 30min, discarding the crystal violet dye solution, washing the cells with running water, and air-drying.
(5) Clones larger than 10 cells were counted under the microscope.
4. Statistical method
Data analysis was performed using SPSS16.0 statistical software and all results were expressed in mean ± SEM format. A comparison of the differences between the two groups was performed using two independent sample t-tests. P <0.05 indicates that the difference is statistically significant, P < 0.05; denotes P < 0.01; denotes P < 0.001.
5. Results
The results of the expression levels of LncRNA UPK1A-AS1 of liver cancer cells (MHCC-97H and SK-Hep1-1) treated by different LNAs are shown in FIG. 1: the locked nucleic acid of UPK1A-AS1 can inhibit the expression of LncRNA UPK1A-AS1, and particularly LNA-i-UPK1A-AS1-2 and LNA-i-UPK1A-AS1-3 can obviously inhibit the expression of UPK1A-AS 1.
The results of clonogenic experiments on liver cancer cells (MHCC-97H and SK-Hep1-1) treated with different LNAs are shown in FIG. 2: the locked nucleic acid of the UPK1A-AS1 can inhibit the proliferation of liver cancer cells (MHCC-97H and SK-Hep1-1), and particularly the LNA-i-UPK1A-AS1-2 and the LNA-i-UPK1A-AS1-3 can obviously inhibit the proliferation of the liver cancer cells (MHCC-97H and SK-Hep 1-1).
Example 2 UPK1A-AS1 inhibitor (locked nucleic acid of UPK1A-AS 1) for the treatment of BALB/c nude mouse hepatoma subcutaneous tumor model experiment
1. Experimental Material
BALB/c nude mice: BALB/c nude mice (4-6 weeks old, male) provided by southern medical university laboratory animal center were bred in SPF animal houses of southern hospital of southern medical university, the squirrel cage and the used utensils were both strictly sterilized, and the mice were fed freely and purified water was drunk; before the experiment, the nude mice were observed to have clinical symptoms for later use.
Medicine preparation: LNA-i-UPK1A-AS1-3 (sequence TCTTTGCCCACTTTAC, SEQ ID NO.3, molecular weight: 5139.1Da, LNA-i-AS1-3) and LNA-i-NC (LNA-i-NC), available from Exiqon Inc., cat #: 300600, Design ID: 683533-3. Experimental group injection drug configuration: LNA-i-UPK1A-AS 1-350 mg was dissolved in 4mL PBS to prepare 12.5mg/mL stock solution, and the stock solution was stored at-80 ℃. 1mL of LNA-i-UPK1A-AS1-3 stock solution was dissolved completely and then dissolved in 10mL of PBS before injection to prepare 1.25mg/mL of working solution. Injection drug configuration of control group: LNA-i-NC50mg was first dissolved in 4mL PBS to prepare 12.5mg/mL stock solutions, which were stored at-80 ℃. 1mL of LNA-i-NC stock solution was taken, and after complete dissolution, the stock solution was dissolved in 10mL of PBS to prepare 1.25mg/mL of working solution before injection.
Experiment of UPK1A-AS1 inhibitor (locked nucleic acid of UPK1A-AS 1) on BALB/c nude mouse hepatoma subcutaneous tumor model
(1) Constructing a nude mouse subcutaneous tumor model: culturing human hepatocarcinoma cell (MHCC-97H) in vitro, digesting logarithmic growth phase cell with pancreatin, centrifuging at 800rpm × 3min, washing hepatocarcinoma cell with PBS reagent for 3 times, counting, and regulating cell number to 1 × 108and/mL, 100 mu L of liver cancer cell suspension is injected into the back of BALB/c nude mice (4-6 weeks old, male) for subsequent experiments.
(2) Grouping: the tumor length of the tumor-bearing mice reaches about 100-150mm3The body weight and tumor volume of the mice are measured one by one, outliers of the body weight and the tumor volume are eliminated, and the tumor-bearing mice are randomly divided into a control group and an experimental group (6 mice in each group).
(3) And (3) treatment: the experimental groups were administered every other day starting on day 12 after tumor cell inoculation, according to the body weight of the miceInjecting 12.5mg/kg LNA-i-UPK1A-AS1-3 at the position of the neck fold subcutaneously; the control group was administered 12.5mg/kg of LNA-i-NC subcutaneously every other day from day 12 after tumor cell inoculation to the neck fold. The tumor volume was calculated by measuring the major and minor diameters of the tumor with a vernier caliper every day ((major diameter. times. minor diameter))2) And/2), weighing the mice, and observing and recording basic conditions of the mice such as mental condition, skin state, food intake and the like every day.
3. The drug efficacy determination method comprises: mouse tumor volume, and mouse body weight were recorded. After sacrifice and tumor dissection, each group of mice was weighed for subcutaneous tumors. Comparing the weight change, tumor volume and weight difference of the tumor-bearing mice of the control group and the experimental group, and performing statistical analysis on the result by using a Two-way ANOVA method: p <0.05 indicates that the difference is statistically significant,. indicates that P < 0.01; denotes P < 0.001.
The results of mouse tumor weight and volume are shown in fig. 3: and control group (LNA)TM-i-NC) ratio to LNATMThe tumor proliferation (volume) of the i-UPK1A-AS1-3 injection for treating the mice is obviously inhibited (p)<0.001), tumor weight isolated after sacrifice, LNA relative to control groupTMThe tumor weight of i-UPK1A-AS1-3 injection treated mice was significantly reduced (p ═ 0.003); it can be seen that inhibitors of UPK1A-AS1 (locked nucleic acids of UPK1A-AS 1) can inhibit tumor tissue proliferation. The results of the body weights of the mice are shown in FIG. 4 (LNA-i-AS1-3-Before indicates the injection of LNA-i-AS1-3, LNA-i-AS1-3-After indicates the injection of LNA-i-AS1-3, LNA-i-NC-Before indicates the injection of LNA-i-NC, and LNA-i-NC-After indicates the injection of LNA-i-NC): the body weights of the control group and the experimental group of mice did not change significantly, and no side effects of the drug treatment were observed during the drug treatment, and thus, the inhibitor of UPK1A-AS1 (locked nucleic acid of UPK1A-AS 1) had no side effects on the animals and was useful for anti-tumor.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> southern hospital of southern medical university
Application of <120> UPK1A-AS1 inhibitor in preparation of antitumor drugs
<130>
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 16
<212> DNA
<213> Artificial sequence
<400> 1
agcagacctt cctaac 16
<210> 2
<211> 16
<212> DNA
<213> Artificial sequence
<400> 2
aacagcactg tcaagg 16
<210> 3
<211> 16
<212> DNA
<213> Artificial sequence
<400> 3
tctttgccca ctttac 16
<210> 4
<211> 18
<212> DNA
<213> Artificial sequence
<400> 4
agagcggtgg gttaggaa 18
<210> 5
<211> 18
<212> DNA
<213> Artificial sequence
<400> 5
gggcagatgg accaagca 18
- 上一篇：一种医用注射器针头装配设备
- 下一篇：SLC12A5及其抑制剂的应用