| Background:Gastric cancer is one of the major causes of mortality in the world, and is the most frequent cancer-related cause of death second only to lung cancer. Chemotherapy is one of the important means of treatment of gastric cancer. Gastric cancer cells developing resistance to chemotherapy is one of the main reasons for therapy failure. Patients that are less responsive to chemotherapy have a poor prognosis. Thus, it is important to identify the molecular mechanisms underlying the drug resistance of gastric cancer cells.MicroRNAs (miRNA) are endogenous small noncoding RNAs that post-transcriptionally regulate genes expression by adjusting mRNA translation and degradation, and plays an important role in cancer occurrence and development. Recent studies have shown that dysregulation of miRNA might modulate the drug sensitivity and resistance of cancer cells. Comparing the miRNA expression spectrum, the specific miRNA that is related to drug resistance of cancer cells can be recognized and used as therapeutic targets. It may become the new way of targeted therapy and improve therapeutic effect. However the mechanism of miRNA modulating drug resistance of gastric cancer cells is still not clear. So it is very important to investigate the specific miRNA that is related to drug resistance of gastric cancer and clarify the mechanism. This study would examine the different miRNA exression profiles of two kinds of drug-resistant gastric cancer cells SGC7901/DDP and BGC823/5-FU and the parental cells, and explore the effect and action mechanism of miRNA on the drug resistance.Purpose:In the two types of drug-resistant gastric cancer cells SGC7901/DDP and BGC823/5-FU, we aimed to analyze the miRNA expression and screen drug resistance related specific miRNA, to study the effect of miRNA on the drug-resistant gastric cancer cell lines, to find out the possible targets of miRNA, and to explore the role of molecular mechanism, so as to create a new way of targeted therapy.Methods:Different miRNA expression levels between gastric cancer cells SGC7901 and SGC7901/DDP, BGC823 and BGC823/5-FU were analyzed by miRNA microarray and validated by real-time quantitative polymerase chain reaction. SGC7901/DDP and BGC823/5-FU cells with their parental cells were transfected with miR-17-5p antibodies and tested the drug sensitivity by CCK-8 assay to vincristine, adriamycin, 5-fluorouracil and cisplatin. Bioinformatics tools PicTar and Miranda algorithms were used to predict miR-17-5p target genes. The Luciferase report plasmid pGL3-p21-3’-UTR was usesd to verify that p21 is one of the target genes of miR-17-5p. The mechanism of miR-17-5p on modulating drug resistance in cancer cells was elucidated by western blots analysis, flow cytometry assay and the cell count methods.Results:Compared with the parental SGC7901 cells, the expression level of miR-17-5p was obviously up-regulated in SGC7901/DDP cells (P<0.01), while in BGC823/5-FU cells the expression of miR-17-5p was obviously up-regulated which is compared with BGC823 cells (P<0.01). These results were validated by real-time quantitative polymerase chain reaction (P<0.01). Down-regulation of miR-17-5p could sensitize SGC7901/DDP and BGC823/5-FU cells to vincristine, adriamycin,5-fluorouracil and cisplatin (P<0.05). Target gene predictive software combined with dual fluorescent reporter gene system results verified that p21 is one of the target genes of miR-17-5p. MiR-17-5p could reduce the relative luciferase activity of pGL3-p21-3’-UTR (P<0.05). Down-regulation of miR-17-5p increased p21 protein level, inhibited cell growth and promoted cell apoptosis in SGC7901/DDP and BGC823/5-FU cells (P<0.05). Down-regulation of miR-17-5p in SGC7901/DDP and BGC823/5-FU cells could significantly decrease the Gl-phase cells and increase the S-phase cells, causing G1/S cell cycle arrest (P<0.05).Conclusions:Compared with the parental cells, the expression level of miR-17-5p was obviously up-regulated in SGC7901/DDP and BGC823/5-FU cells. Down-regulation of miR-17-5p could sensitize SGC7901/DDP and BGC823/5-FU cells to anticancer drugs. It also could inhibite the cell growth, promoted cell apoptosis and cause G1/S cell cycle arrest in SGC7901/DDP and BGC823/5-FU cells. MiR-17-5p could play a role in cell growth and apoptosis via targeting p21. This study found that down-regulation of miR-17-5p could obviously reduce the resistance of gastric cancer cells, which might be cells to to provide theoretical support and new molecular targets to overcome drug resistance of gatric cancer cells. |
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