Research On The Abnomal Expression And The Role Of MiR-100in Gastric Cancer Carcinogenesis

Backgroud:MicroRNAs (miRNAs) are endogenous, small non-coding RNAs consisting of19-22nucleotides. Recently more attention has been paid to their roles in the development and progression of diaseases, especially in human cancers. Growing evidence clearly demonstrated that miRNAs could function as oncogenes and tumor suppressor genes in human carcinogenesis. Gastric cancer (GC) is the fourth most common cancer worldwide. Dispite considerable improvement in the diagnosis and treatment of GC, much more research need to be done to clarify the underling mechanism and improve the patiens’ prognosis. In the present study, we detected the expression of miR-100in GC tissues and revealed the biological function of miR-100in GC. Moreover, we demonstrated the molecular mechanism of miR-100to inhibite tumor metastasis as a tumor suppressor in GC.Methods:GC tissue samples and clinical data of patients were obtained from Qi Lu Hospital. Total RNAs were extracted from83cases of GC tissuers,17cases of nontumorous gastric tissues and two gastric cancer cell lines SGC7901and BGC823, and reversely transcribed into cDNA. The relative expression of miR-100was determined by real-time PCR. After transient transfection of miR-100, migration ability and invasiveness of GC cells were tested by migration and invasion assays, respectively. MTS and EdU proliferation assays were used to detect the growth ability of the experimental cells. In addition, the effect of miR-100on cell apoptosis was detected by flow cytometry. ZBTB7A was identified as a target gene of miR-100using luciferase assay and further confirmed by western blotting and immunohistochemistry. To determine the transcription region of pri-miR-100, primers for5’-RACE were designed according to the sequence4.0kb upstream of the miR-100gene and5"-RACE assay was performed.Results:Compared with nontumorous tissues, miR-100expression was significantly reduced in GC tissues and four GC cell lines. Interestingly, we found that the expression of miR-100was dramatically decreased in primary GC tissues with lymph node metastasis than GC tissues without lymph node metastasis, but our data showed no significant difference in miR-100expression between lymph node metastatic tissues and the matched primary GC tissues. And we demonstrated that the migration and invasion ability of SGC7901and BGC823cell lines was significantly attenuated after transfection of miR-100. MTS and EdU proliferation assays and flow cytometry analysis demonstrated that miR-100has no effect on cell growth and cell apoptosis in vitro. However, animal experiment revealed that in vivo tumorigenesis abilities of SGC7901cells could be remarkably inhibited by overpression of miR-100using lentiviral transfection. And the expression of miR-100was inversely correlated with tumor size and tumor tages in GC. We used three algorithms (Targetscan,Miranda and Pictar) to help identify miR-100targets in human gastric cancers and four putative targets (FUR, ZBTB7A, CTNNB1and CLDN4) were selected for further analysis. We cloned3’-UTRs of the targets into a luciferase construct pmirGLO. ZBTB7A was identified as a direct target of miR-100and the luciferase assay showed that miR-100transfected group significantly reduced the luciferase activity of pmirGL0-ZBTB7A compared to the negative control group in BGC823and SGC7901cells. Western-blot and immunohistochemistry also confirmed that ZBTB7A is the target gene of miR-100. Interestingly, our5’-RACE results showed that3.8kb upstream of miR-100gene was the transcription starting site of pri-miR-100.Conclusion:MiR-100was downregulated in GC tissues and four GC cell lines, especially in GC tissues with lymph node metastasis. Our results showed that miR-100might serve as a tumor suppressor, and thus involves in the tumorigenesis and development of GC by inducing the accumulation of oncogene ZBTB7A. Moreover, we found that the fragment3.8kb upstream of miR-100gene was the transcription starting site of pri-miR-100, which provide important foundation for research on the regualtion mechanism of miR-100in GC.