阅读说明：本技术 一种与肿瘤细胞对索拉非尼耐药性相关的microRNA及其应用 (MicroRNA related to sorafenib drug resistance of tumor cells and application thereof ) 是由 徐俊杰 蔡秀军 林中杰 季琳 岑栋 梁霄 姜是 夏顺杰 陶力野 于 2020-04-23 设计创作，主要内容包括：本发明公开了一种与肝细胞性肝癌索拉菲尼耐药相关的microRNA及其应用,本发明选取Huh7和HCC-LM3野生及其索拉非尼耐药细胞系作为研究对象,通过二代测序、验证及优选,获得在索拉非尼耐药细胞中低表达的microRNA-378a-3p通过建立荷瘤小鼠的索拉非尼治疗模型结合qPCR方法,进一步确定了miR-378a-3p的表达量与肿瘤细胞对索拉非尼耐药性程度负相关。利用miR-378a-3p的转录因子LXRα激动剂GW3965对细胞耐药性、IGF1R基因的表达程度以凋亡及的影响,确定了GW3965可以促进miR-378a-3p的转录并抑制IGF1R,逆转肿瘤细胞对索拉非尼的耐药。本发明的提出为肿瘤治疗提供新的靶点,为有效地对抗索拉非尼耐药提供了有效技术手段。(The invention discloses a microRNA related to liver cell liver cancer sorafenib drug resistance and application thereof, wherein the invention selects Huh7 and HCC-LM3 wild and sorafenib drug-resistant cell lines thereof as research objects, obtains the microRNA-378a-3p with low expression in sorafenib drug-resistant cells through second-generation sequencing, verification and optimization, and further determines that the expression quantity of miR-378a-3p is negatively related to the sorafenib drug resistance degree of tumor cells by establishing a sorafenib treatment model of tumor-bearing mice and combining a qPCR method. By utilizing the transcriptional factor LXRalpha agonist GW3965 of miR-378a-3p to resist cell drug resistance and the expression degree and apoptosis influence of IGF1R gene, the GW3965 is determined to promote the transcription of miR-378a-3p, inhibit IGF1R and reverse the drug resistance of tumor cells to sorafenib. The invention provides a new target for tumor treatment and provides an effective technical means for effectively resisting sorafenib drug resistance.)

1. A microRNA related to sorafenib drug resistance of tumor cells is characterized in that the microRNA is miR-378a-3p, and the nucleotide sequence of the microRNA is shown in SEQ ID NO. 1.

2. An application of the microRNA detection reagent of claim 1 in preparation of a kit for detecting the drug resistance degree of tumor cells to sorafenib.

3. The use of claim 2, wherein the expression level of miR-378a-3p is inversely proportional to the degree of resistance of the tumor cells to sorafenib.

4. The use of claim 2, wherein the microRNA detection reagent comprises a qPCR detection primer of microRNA, the qPCR detection primer comprises an upstream primer and a universal primer, the sequence of the upstream primer is shown as SEQ ID NO.2, and the sequence of the universal primer is shown as SEQ ID NO. 3.

5. The use of the transcription factor LXR alpha agonist of microRNA as claimed in claim 1 in the preparation of drugs for reversing the resistance of tumor cells to sorafenib.

6. The use of claim 5, wherein the medicament reverses resistance of tumor cells to sorafenib by decreasing IGF1R gene expression, inhibiting the PI3K/AKT pathway.

7. The use of claim 1, wherein the tumor cell is a human hepatoma sorafenib-resistant cell.

8. An application of the transcription factor LXR alpha agonist of the microRNA disclosed by claim 1 in preparation of anti-liver cancer drugs.

9. The use of claim 8, wherein the liver cancer is hepatocellular carcinoma.

10. The use of claim 8, wherein the anti-liver cancer drug comprises a LXR α agonist and sorafenib.

Technical Field

The invention belongs to the field of biotechnology and medicine, and particularly relates to microRNA related to antitumor drug resistance and application thereof.

Background

Liver cancer is one of the most common malignant tumors in China and all over the world. There are about 50 million new cases worldwide per year, of which hepatocellular carcinoma (HCC) accounts for 85%. China is a big hepatitis B country, the incidence rate of HCC is about 30.1/10 ten thousand, and the number of new cases and death cases accounts for more than 50 percent of the whole world. Despite the advances in tumor markers and imaging examinations, surgical level, and various new therapeutic modalities such as intraarterial chemoembolization (TACE), the 5-year survival rate of HCC has improved. Overall, however, the prognosis of HCC is still unsatisfactory. Surgery is the only cure for HCC patients, and in addition, other treatments such as TACE, radio-frequency ablation (RFA), chemotherapy, etc. have certain curative effects. For advanced HCC patients who cannot be treated by surgery, targeted drug therapy is an effective treatment method that can be selected at present. Among them, sorafenib (sorafenib), a multi-target tyrosine kinase inhibitor, is the first clinically approved drug by FDA in the united states for clinically treating late-stage liver cancer at present. The previous research shows that sorafenib can inhibit the growth of tumor cells on one hand and can also inhibit the generation of tumor angiogenesis on the other hand. This provides hope for prolonging the life of patients with advanced liver cancer. However, due to the heterogeneity of tumor patients, sorafenib has an obvious drug resistance phenomenon in the clinical application process. Many studies have shown that sorafenib can only prolong the survival of late-stage liver cancer patients by 2-3 months. Thereafter, most patients are prone to sorafenib resistance.

The drug resistance mechanism of sorafenib is not clear at present, and part of the idea is that tumor patients with mutations in alleles of EGFR, c-JUN and VEGFR are likely to have innate resistance to sorafenib. Other views suggest that many signal pathway key molecules that are involved in cell growth, such as: PI3K/AKT, IGF/FGF, NF-kappa B and the like may be related to the drug resistance formation of sorafenib. Meanwhile, autophagy, tumor microenvironment change, epigenetics and the like are also considered to be possibly related to liver cancer sorafenib drug resistance. However, the leading mechanism or the key gene still is the main problem which puzzles the research of the liver cancer on the sorafenib drug resistance. For sorafenib-resistant HCC patients, an effective treatment method is lacked at present, and how to overcome sorafenib resistance is of great significance for improving HCC patient prognosis.

Recent research shows that the expression difference of microRNA plays a key role in the formation process of the liver cancer to the sorafenib drug resistance. The microRNA is a single-stranded non-coding RNA with the length of only 18-25 nucleotides, is processed from hairpin structure transcripts endogenously generated by cells, can directly degrade or inhibit translation of specific target gene mRNA by combining with a 3 'or 5' UTR region of the target gene mRNA, and plays a role in post-transcriptional regulation. For example, miR-221 can participate in sorafenib drug resistance by combining with the 3' UTR region of Caspase-3 gene mRNA. In addition, a recent document reports that liver cancer cells cause abnormal expression of downstream IGF-1R by reducing miR-122 levels, so that the liver cancer cells are promoted to resist sorafenib drugs. These studies all show that microRNA plays an important role in the formation process of the drug resistance of liver cancer to sorafenib. However, to date, especially in liver cancer research, there is no article to systematically analyze which micrornas are key factors for regulating drug resistance among numerous human micrornas, and it is not clear how to perform personalized treatment by using micrornas.

Therefore, there is an urgent need in the art to find a method capable of enhancing sensitivity of hepatoma cells to chemotherapeutic drugs and to develop corresponding therapeutic drugs.

Disclosure of Invention

The invention aims to find key microRNA for improving the sensitivity of liver cancer cells to anti-sorafenib drugs and develop corresponding therapeutic drugs.

The invention aims to provide a molecular marker miR-378a-3p for judging the drug resistance of an organism to Sorafenib aiming at the phenomenon that the tumor treatment drug Sorafenib often generates drug resistance in the treatment process to cause failure of targeted treatment and even disease relapse, wherein the expression quantity of miR-378a-3p in serum can reflect the relative expression condition of miR-378a-3p in situ tumor, and the drug resistance of the organism to Sorafenib is higher when the expression of miR-378a-3p in serum is lower. Meanwhile, the invention also provides a drug for reversing the drug resistance of the tumor cells to sorafenib by using miR-378a-3p, and particularly relates to application of an agonist of a transcription factor of miR-378a-3p in preparation of a drug for reversing the drug resistance of the tumor cells to sorafenib by down-regulating IGF1R gene expression.

In order to achieve the purpose, the invention adopts the following technical scheme:

according to the invention, a Huh7 and HCC-LM3 wild and sorafenib-resistant cell line thereof are selected for microRNA-seq, and miR-378a-3p with low expression in Huh7 and HCC-LM3 sorafenib-resistant cells is obtained through analysis. Then, after miR-378a-3p mimic is transfected into a drug-resistant cell, the change conditions of growth, drug resistance, expression degree of IGF1R gene and the like before and after cell transfection are detected by applying CCK8 proliferation curve and IC50 analysis, Real-time PCR, flowcytometric, Western Blot and other technical methods, and whether miR-378a-3p can reverse tumor drug resistance by inhibiting IGF1R is definitely interfered; then, GW3965 serving as an agonist of an independent transcription factor LXR alpha of miR-378a-3p is combined with sorafenib in Huh7 and HCC-LM3 sorafenib drug-resistant cells, and the CI value of the combined drug is detected to determine the effect of the combination of the two drugs; and then establishing a sorafenib treatment model of HCC-LM3 wild cells and PDX tumor-bearing mice, and detecting whether the tumor volume, serum ALT, Real-time PCR (polymerase chain reaction) and immunological chemistry of the mice can improve the inhibition effect of sorafenib and reverse tumor drug resistance by combining the two drugs under the combined treatment of GW3965 and sorafenib.

On the basis of the research, the microRNA related to the resistance of tumor cells to sorafenib is found, and is miR-378a-3p, and the nucleotide sequence of the miR-378a-3p is shown in SEQ ID NO. 1.

Furthermore, the invention also provides application of the microRNA detection reagent in preparation of a kit for detecting the drug resistance degree of tumor cells to sorafenib.

Preferably, the reagent for detecting the microRNA is a primer, the primer consists of an upstream primer and a universal primer, and the sequence of the upstream primer is shown as SEQ ID NO.2, and the sequence of the universal primer is shown as SEQ ID NO. 3.

Wherein the expression level of miR-378a-3p is inversely proportional to the drug resistance degree of tumor cells to sorafenib.

Furthermore, the invention also provides application of an agonist GW3965 of a transcription factor LXR alpha of miR-378a-3p in preparation of a drug for reversing the drug resistance of tumor cells to sorafenib.

Preferably, the tumor cells are human liver cancer Huh7 and HCC-LM3 cells. More preferably, the tumor cells are human liver cancer Huh7 and HCC-LM3 cells Sorafenib resistant cells.

In the application, the medicament is combined with the 3' UTR region of IGF1R to inhibit translation, so that the aim of reversing the drug resistance of tumor cells to sorafenib is fulfilled.

Furthermore, the invention also provides application of the transcription factor LXR alpha agonist of the microRNA in preparation of anti-liver cancer drugs.

Wherein the liver cancer is hepatocellular carcinoma.

Wherein, the anti-liver cancer drug comprises LXRalpha agonist and sorafenib.

Compared with the prior art, the invention has the beneficial effects that:

the invention researches the possibility of taking miR-378a-3p as a sorafenib drug-resistant marker by establishing a sorafenib treatment model of a tumor-bearing mouse; meanwhile, the relation between miR-378a-3p and drug-resistant gene expression is explored through transient interference of miR-378a-3 p. Thereby providing scientific basis for searching new diagnostic markers, revealing new therapeutic targets and more effectively resisting sorafenib resistance.

The expression of miR-378a-3p in serum can relatively objectively reflect the expression of miR-378a-3p in situ tumor, and when the expression of miR-378a-3p in serum is relatively low, the drug resistance of the organism to sorafenib is also high. The method provides a new way for judging the tolerance degree of the patient to sorafenib through non-invasive diagnosis of a blood way, and has important significance for clinical personalized medication. Meanwhile, for sorafenib-resistant malignant tumor cells highly expressing IGF1R gene, the GW3965 is used for specifically exciting LXR alpha, so that the expression degree of miR-378a-3p can be increased, the IGF1R in the cells is reduced, and the PI3K/AKT pathway is inhibited, thereby reversing the tumor resistance and improving the tumor treatment efficiency. More importantly, the microRNA agonist mechanism has more clinical transformation value and has very positive significance for deeply understanding the nature of the sorafenib drug resistance and searching drug resistance target points of individualized treatment.

The microRNA discovered by the invention can prompt the effect of a patient on sorafenib treatment, and the agonist of the transcription factor of the microRNA can increase the treatment effect of sorafenib, so that the microRNA has a clinical transformation value.

Drawings

The invention is further explained below with reference to the figures and examples;

figure 1A is a heatmap of mirnas differentially expressed in Huh7 wild-type cells and Huh7 sorafenib-resistant cells, and figure 1B is a volcano plot of mirnas differentially expressed in Huh7 wild-type cells and Huh7 sorafenib-resistant cells;

FIG. 2 is a graph showing the results of validation of 5 different miRNAs in three groups of wild cells and sorafenib-resistant cells, A is a graph showing the results of Huh7 cells, B is a graph showing the results of HCC-LM3 cells, and C is a graph showing the results of SK-Hep-1 cells; in the figure, WT represents a wild-type cell, and SR represents a drug-resistant cell;

FIG. 3 is a graph showing the results of the efficiency verification of miR-378a-3p imic transfection of Huh7 and HCC-LM3 drug-resistant cell strains;

FIG. 4 is a result graph of half inhibitory concentration (IC50) of cells to drugs before and after miR-378a-3p interference detection by a CCK8 method, wherein A is a result graph of Huh7 cells, and B is a result graph of HCC-LM3 cells;

FIG. 5 is a result chart of proliferation of cells under 6 μmol/L sorafenib effect before and after miR-378a-3p interference detection by a CCK8 method, wherein A is a result chart of Huh7 cells, and B is a result chart of HCC-LM3 cells;

FIG. 6 is a graph of the results of the effect on the apoptotic effect of sorafenib after overexpression of miR-378a-3 p;

FIG. 7 is a flow chart of analysis for finding downstream targets of miR-378a-3 p;

FIG. 8A is a Western Blotting graph of miR-378A-3p overexpression in Huh7 and HCCLM3 sorafenib resistant cells resulting in decreased intracellular IGF1R expression; in the figure, WT represents a wild-type cell, SR represents a drug-resistant cell, miR-C represents a drug-resistant cell transfected with miR-378a-3p imic ctrl, 378a-3p represents a drug-resistant cell transfected with miR-378a-3p imic; b is the result chart of IGF 1R-3' UTR fluorescent reporter gene; the C picture is a picture of the detection result of the activity of wild type IGF1R 3'UTR and mutant IGF1R 3' UTR fluoromycin in the sorafenib drug-resistant cells;

FIG. 9 is a graph showing the results of relative expression amounts of miR-378a-3p in each group of cells;

FIG. 10 is a plot of the CI values for GW3965 in combination with sorafenib on Huh7 and HCCLM3 sorafenib resistant cells, A is a plot of the results for Huh7 sorafenib resistant cells, and B is a plot of the results for HCCLM3 sorafenib resistant cells;

FIG. 11 is a schematic view of tumors under VIS after combined use of GW3965 and sorafenib in a HCC-LM3 tumor cell tumor-bearing mouse model, wherein A is a normal saline group, B is a GW3965 group, C is a sorafenib group, and D is a combined drug group, 4 drugs are taken in each group;

FIG. 12 is a graph of tumor volume (FIG. 12A), ALT content in serum (FIG. 12B), miR-378a-3p expression level (FIG. 12C), and mouse body weight (FIG. 12D) of each group after combined use of GW3965 and sorafenib in a HCCLM3 tumor cell-bearing mouse model;

FIG. 13 is a graph of IHC results for various groups of tumor tissues;

FIG. 14 is a schematic representation of the tumors of each group after combined use of GW3965 and sorafenib in a PDX tumor-bearing mouse model, wherein A is tumor tissue and B is mouse liver;

FIG. 15 is a graph showing the results of IHC of tumor tissues of each group after combined use of GW3965 and sorafenib in a PDX tumor-bearing mouse model.

Detailed Description

The present invention is further illustrated by the following experiments in combination with examples, it being understood that these examples are for illustrative purposes only and in no way limit the scope of the present invention.

Experimental materials and reagents related to the present example:

human hepatoma cell lines Huh7, HCC-LM3 and SK-Hep-1 were purchased from the Shanghai cell Bank.

The reagents such as sorafenib, GW3965 and the like are all sold in the market.