Studying On MiRNA-214Promoting Cisplatin Resistance In SGC7901/DDP And Its Mechanism

Background:Gastric cancer is one of the most common malignant tumors of the digestive system. Chemotherapy is the basic way to improve the survival time and life quality of patients with advanced gastric cancer and postoperative patients with gastric cancer. But the drug resistance reduces the effectiveness of chemotherapy. The treatment failure in90%of patients with metastatic tumor is caused by chemotherapy drug resistance. So far, the mechanism related with drug resistance has not yet fully been clear, may be associated with the following factors:increasement of the expression of ABC type membrane carrier protein; P53gene mutation; blockage of cell apoptosis pathway and imbalance of DNA damage repair system.Cisplatin (also called cis-dichlorodiamminoplatinum Ⅱ),an efficient broad-spectrum chemotherapy drug without specific cycle, is the first-line drug used in gastrointestinal tumor. Although cisplatin has strong anti-tumor effect, its effectiveness is restricted by the cisplatin resistance of gastric cancer.MicroRNA(miRNA) is a single-strand non-coding RNA with the length of21-25nt. Researches showed that it is involved in drug resistance of cancer and can influence the effectiveness of the chemotherapy. MiRNA-214is located in the long arm(lq24.3) Dnm3gene of human chromosome1.Studies showed miRNA-214is related to the growth and proliferation of cells. It can promote the invasion and migration ability of gastric cancer cells. But so far, the studying on the relationship of miRNA-214and cisplatin resistance of tumor has only down in ovarian cancer. In our experiment, the mechanism of miRNA-214promoting cisplatin resistance in SGC7901/DDP was studied for the first time. Objective:To explore the effect of microRNA-214in promoting cisplatin (DDP) resistance of gastric cancer SGC7901/DDP cell line and its mechanism, and search for a new molecular therapeutic target to guide the therapy.Methods:1、Using quantitative real-time PCR to detect miRNA-214expression difference in parental SGC7901cells and DDP-resistant SGC7901/DDP cells;2、Using Liposome transfection method to instantaneous transfect gastric cancer cells SGC7901/DDP and using quantitative real-time PCR to detect the expression variation of miRNA-214in DDP-resistant SGC7901/DDP cells after transient transfecting miRNA-214inhibitors;3、Using MTT to detect the sensitivity variation of SGC7901/DDP cells to DDP drug after transfecting miRNA-214inhibitor;4、Using Western blot to detect the influence of transfecting miRNA-214inhibitor on the target gene-PTEN protein expression level.Results:1、Real-time RT-PCR showed that the expression of miRNA-214in DDP-resistance cell line SGC790/DDP was significantly higher than that of the parent cell line SGC7901. It is2.320±0.106times to the parent cell line SGC7901(P<0.01).2、The transient transfection efficiency of SGC790/DDP cells is about80%, when observed under the fluorescence microscope after transfecting with miRNA-214inhibitors for24h;3、Real-time RT-PCR showed that the ΔCt of miRNA-214in miRNA-214inhibitors transfected SGC7901/DDP cells was0.187±0.031times of that in blank control cells, and ΔCt of miRNA-214in negative control transfected SGC7901/DDP cells was0.869±0.025times of that in blank control cells (P<0.05); 4、MTT showed the inhibitory rate of SGC7901/DDP cells transfected with miRNA-214inhibitors was significantly down-regulated, compared to the negative control. The inhibitory rate of SGC7901/DDP cells transfected with miRNA-214inhibitors (for the DDP concentration2.5,5,10,20,40and80mg/L) were (20.67±0.50)%,(36.87±1.29)%,(56.50±0.53)%,(77.43±1.55)%,(88.93±0.65)%,(93.87±2.40)%, respectively. The inhibitory rate of negative control cells were (16.97±0.95)%,(31.07±0.59)%,(47.87±1.16)%,(58.37±1.40)%,(79.50±1.87)%,(90.37±0.55)%, respectively. The IC50of DDP was (7.766±0.109) mg/L in miRNA-214inhibitors transfected SGC7901/DDP cells, and was (15.310±1.640) mg/L in negative control cells. The IC50of DDP was significantly down-regulated after miRNA-214inhibitors transfecting (P<0.05);5、Western blot showed the expression lever of PTEN in SGC7901/DDP cells at72h after transfecting was significantly up-regulated in transfected cells than in control cells, up-regulating to1.80±0.02times (P<0.05).Conclusion:1、MiRNA-214plays a certain role in promoting the resistance of SGC7901/DDP cells to DDP, down-regulating miRNA-214may reverse the resistance of SGC7901/DDP cells to DDP;2、Regulating the expression of PTEN may be one of the mechanisms that miRNA-214takes part in DDP resistance of SGC7901cells.