The Role And Possible Mechanisms Of MicroRNA In Multidrug Resistance Of Gastric Cancer

【Background】The development of multidrug resistance (MDR) in cancer cells is the primary cause for failed chemotherapy in gastric cancer patients. It is now well acknowledged that mechanisms responsible for MDR in gastric cancer are likely to be multifaceted and extremely intricate. New MDR-related molecules keep emerging owing to the great advances in genomics and proteomics approaches, but the understanding to gastric cancer MDR still remains incomplete. It is worth noticing that while great efforts have been made in identifying differentially expressed genes between drug-resistant and parental/ drug-sensitive cells, and then exploring its downstream signal pathway, much less insight has been gained into the regulatory networks that establish such altered gene expression states. If the'crime boss'in the MDR molecule network could be identified by inversely exploring upstream regulators, strategies targeting them may pave a new road for chemotherapy of gastric cancer.microRNAs (miRNAs) are a newly discovered class of small noncoding RNAs encoded by the genomes of a wide range of multicellular organisms and negatively regulate gene expression at posttranscriptional level. It is predicted that each miRNA could regulate about 200 genes and the ~1000 miRNAs in the human body may regulate the expression of up to 1/3 of human protein-coding genes, which implies the potential influence of miRNAs on almost every genetic pathway. Indeed, miRNAs have been shown to play crucial roles in many physiological and pathological processes, including cancer development and progression. Thus, we postulated that miRNA could be such class of modulators located in the upstream of MDR molecule network, which regulate the expression of numerous MDR-related genes, hence the MDR phenotype of gastric cancer cells. Until now, there has been no report on the potential role of miRNAs in MDR of gastric cancer.【Aims】To investigate the regulatory role of miRNAs in MDR of gastric cancer cells and the underlying mechanisms, with the aim of better elucidating the mechanisms of gastric cancer MDR and laying a foundation for formulating novel MDR reversing strategies.【Methods】1. The differentially expressed miRNAs between multidrug-resistant gastric cancer cell line SGC7901/VCR and its parental cell line SGC7901 were identified by miRNA profiling of these two cell lines using miRNA microarray; 2. The results obtained by microarray profiling were validated using real-time RT-PCR analysis; 3. The specific miRNA precursor for miR-15b/miR-16/let-7a was respectively transfected into SGC7901/VCR cells to up-regulate its expression and the specific antisense inhibitor for miR-15b/miR-16/let-7a was transfected into SGC7901 cells to down-regulate its expression; 4. the effect of miR-15b/miR-16/let-7a on in vitro drug sensitivity of gastric cancer cells (SGC7901,SGC7901/VCR) was determined by MTT assay; 5. The effect of miR-15b/miR-16/let-7a on in vivo drug sensitivity of gastric cancer cells (SGC7901/VCR) was determined by subrenal capsule xenograft assay (SRCA); 6. The putative target genes of miR-15b/miR-16/let-7a were predicted using bioinformatics website or software; 7. The putative target genes of miR-15b/miR-16/let-7a were validated by Western blot, RT-PCR and luciferase reporter assay; 8. The effect of enforced miR-15b or miR-16 expression on drug-induced apoptosis in SGC7901/VCR cells was evaluated using AnnexinV/PI staining flow cytometry and caspase-3/7 activity assay; 9. The effect of let-7a on DNA repair capacity in SGC7901/VCR cells was assessed by host cell reactivation assay.【Results】1. There are 12 miRNAs differentially expressed (>2 fold) in multidrug-resistant gastric cancer cell line SGC7901/VCR and its parental cell line SGC7901. The up-regulated miRNAs in SGC7901/VCR cell line were miR-302b and miR-492 and the down-regulated miRNAs were let-7a,miR-15b,miR-16,miR-17-5p,miR-20a,miR-23b,miR-106a,miR-106b,miR-196a and miR-320. Among them, some have been implicated in cancer, or known as'oncomir', but their involvement in MDR of gastric cancer has yet been reported; 2. In accordance with the microarray data, real-time RT-PCR showed decreased miR-15b, miR-16 and let-7a levels in SGC7901/VCR cells compared with its counterpart; 3. In vitro drug sensitivity assay demonstrated that overexpression of miR-15b/miR-16/let-7a sensitized SGC7901/VCR cells to different anticancer drugs whereas inhibition of them conferred SGC7901 cells multidrug resistance; 4. SRCA in vivo drug sensitivity assay demonstrated that overexpression of miR-15b/miR-16/let-7a sensitized SGC7901/VCR cells to CDDP in vivo; 5. miR-15b and miR-16 belong to miR-15/16 family and exhibit structural homology, thus they potentially target same genes by in silico analysis. Among the numerous putative target genes for miR-15b, miR-16 and let-7a, some are well-defined MDR-related molecules, e.g. Bcl-2 for miR-15b and miR-16, and Ras for let-7a; 6. Compared with precursor control-transfected cells, Bcl-2 protein level in miR-15b or miR-16 precursor-transfected cells were decreased significantly, whereas no visible difference was observed in mRNA level. miR-15b and miR-16 caused a decrease in relative luciferase activity by binding to the target site in Bcl-2 3'UTR which was cloned immediately downstream of luciferase gene coding region. As expected, this suppression was abolished by disrupting the target site; 7. Compared with inhibitor control-transfected cells, H-Ras protein level in let-7a antisense inhibitor-transfected cells were increased significantly, whereas no visible difference was observed in mRNA level. let-7a caused a decrease in relative luciferase activity by binding to the target site in H-Ras 3'UTR which was cloned immediately downstream of luciferase gene coding region; 8. AnnexinV/PI staining flow cytometry and caspase-3/7 activity assay showed that miR-15b or miR-16 precursor-transfected SGC7901/VCR cells exhibited much higher sensitivity to VCR-induced apoptosis compared to precursor control-transfected ones; 9. Host cell reactivation assay revealed that DNA repair capacity in let-7a precursor-transfected SGC7901/VCR cells were greatly depressed compared with precursor control-transfected ones.【Conclusions】1. miRNAs are differentially expressed in multidrug-resistant gastric cancer cell line and its parental cell line, which suggests that miRNAs may play a role in the development of MDR in gastric cancer cells; 2. miR-15 and miR-16 were demonstrated to modulate MDR phenotype in gastric cancer cells and the underlying mechanism is that miR-15 and miR-16 regulate target gene Bcl-2, thereby modulating the sensitivity of gastric cancer cells to drug-induced apoptosis; 3. let-7a was demonstrated to modulate MDR phenotype in gastric cancer cells and the underlying mechanism is that let-7a regulates target gene H-Ras, thereby modulating DNA repair capacity of gastric cancer cells.