| Background:Gastric cancer is one of the most common gastrointestinal malignancies.The incidence of gastric cancer is in the second place and mortality rate in the third in our country.At present,the main treatment of gastric cancer is surgical resection,combined with chemotherapy.Neoadjuvant chemotherapy and postoperative chemotherapy for gastric carcinoma is closely related to the prognosis of gastric cancer and postoperative effective survival.But acquired drug resistance became the main cause of the failure to chemotherapy in clinical.5-Fluorouracil(5-Fu),as the first-line chemotherapy drugs in clinics,is widely used in varieties of solid tumors including gastric cancer.Pharmacological action of 5-Fu is that 5-Fu can be converted into 5F-dUMP to inhibiting DNA thymidylic acid synthetase,and then preventing deoxyuridine acid(dUMP)methylation into DNA thymidylic acid(dTMP),which affect the synthesis of DNA.At the same time,5-Fu can be converted into 5-fluorouracil nucleoside in the body,and interfering protein synthesis in the form of pseudo metabolites as a specific killer for S phase cells.Gastric cancer patients are sensitive to 5-Fu in the initial chemotherapy,but the acquired drug resistance appeared after gradually used 5-Fu,and eventually lead to chemotherapy failure.Therefore,further study the mechanisms of acquired drug resistance to 5-Fu for reversing the chemotherapy drug resistance in clinical and providing a new cancer treatment strategy is very important.microRNA(miRNA)is an endogenous,Single-stranded,non-coding RNA,which inhibit the target gene translation or degrade target mRNA to regulate gene expression.Recent studies have shown that miRNA associated with cancer chemotherapy sensitivity,but the relationship between miRNA and gastric cancer drug resistance is unclear.Aims:The propose of this study is to obtain 5-Fu acquired drug resistance related microRNAs by screening the miRNA expressions between the gastric cancer cell line BGC823 and 5-Fu acquired drug resistance cell line BGC823/5-Fu,on the basis of the application of gene chip.The function and molecular mechanisms of miRNAs in acquired drug resistance gastric cancer cells through bioinformatics analysis and a series of biological function verification is the basis for looking for regulatory molecules associated with drug resistance and finding effective targets and methods to reversing the tumor drug resistance.Methods:1.Establish the 5-Fu resistant gastric cancer cell lines by increasing drug concentration gradient method.2.The miRNA expression profile chip and genome-wide expression profile chip were applied to screening the different expression miRNAs and mRNAs between drug resistant cell lines and the parent cell lines.And then different expression miRNAs and mRNAs were detected by real-time RT-PCR.3.miR-125b was low expressed in drug resistance gastric cancer cells.A series of bioinformatics analysis including target gene prediction,gene ontology annotations and KEGG pathway enrichment analysis was used to miR-125b.Dual luciferase report gene experiment was applied to verify the relationship between miR-125b and the target genes ERBB2 and ERBB3.4.Drug resistant cell line BGC823/5-Fu was transfected miR-125b mimics or miR-125b inhibitors.And then cell proliferation,cell cycle,cell apoptosis and protein expression level of ERBB2,ERBB3,BCL2,BAX,ERK1/2,p-ERK1/2,AKT,p-AKT were detedcted by MTT method,flow cytometry and Western blotting.Results:1.5-Fu acquired drug resistance gastric cancer cell line BGC823/5-Fu was established successfully and resistance index was 18.75.2.29 miRNAs and 1186 mRNA were selected through the gene chip.Application qRT-PCR to test different expressed miRNAs including hsa-miR-125b,hsa-miR-125a,hsa-miR-100,hsa-miR-130a,hsa-miR-221,hsa-miR-194,hsa-miR-192 and different expressed mRNAs including ERBB2,ERBB3,PDGFB,BCL2 and BAX,MDM2,TP53.The results show that there have significant differences in miRNAs and mRNAs between BGC823/5-Fu and BGC823.3.Bioinformatics analysis is applied to forecast the target genes of miR-125b were ERBB2 and ERBB3.Further dual luciferase reporter gene experiments showed that miR-125b has directly targeting combined with ERBB2,ERBB3 gene 3 ’UTR region respectively.4.Overexpression of miR-125b can make sensitivity significantly enhanced to 5-Fu in BGC823/5-Fu(p<0.05),turn the cell cycle from the S phase block to G0/G1 block.Western blotting showed that protein expression of ERBB2,ERBB3,BCL2,ERK1/2,p-ERK1/2,AKT,p-AKT decreased,BAX expression increased(p<0.05).It appeared that BGC823/5-Fu had more resistant to 5-Fu after miR-125b inhibitors transfected.Conclusion:1.A variety of abnormal expression of miRNAs associated with gastric cancer cell 5-Fu acquired drug resistance.2.In the drug resistant cells,miR-125b regulated the expression of target gene ERBB2 and ERBB3 negatively.3.miR-125b can affect the sensitivity of the gastric cancer cells to chemotherapy drugs 5-Fu.Overexpression of miR-125b can enhance the sensitivity of the gastric cancer cells to 5-Fu,and reduced the expression of miR-125b can enhance gastric cancer cells resistance to 5-Fu.This results showed that miR-125b can reverse the drug resistance of gastric cancer cells as a target.4.The mechanism of miR-125b influence the 5-Fu resistance of gastric cancer cells is that the miR-125b can regulate the expression of target genes ERBB2 and ERBB3. |
PDF Full Text Request