MicroRNA-27b,microRNA-101,and MicroRNA-128 Inhibit Angiogenesis By Down-regulating Vascular Endothelial Growth Factor C Expression In Gastric Cancers

Backgroud:Gastric cancer is the fourth most common cancer worldwide. Despite considerable improvements in curative surgery, the patients with metastasis still have a poor prognosis. Metastasis is one of the predominant routes of tumor cell spread in patients with gastric carcinoma. VEGF-C, a member of the platelet-derived growth factor family, correlates significantly with proliferation and migration of blood vascular endothelial cells, as well as lymphangiogenesis. It has been reported that VEGF-C secreted from cancer cells may directly promote cancer cells’ migration and invasion capabilities by the autocrine action. VEGF-C plays an important role in stimulating the development or progression of tumors. However, the mechanisms underlying the regulation of VEGF-C expression remain largely unknown. MiRNAs is one of the hotspots in recent cancer research. Recent evidences revealed that miRNAs function as repressors of its target genes, participating in regulating the translation of protein or/and the stability of mRNAs. The aim of our sudy was to discuss the mechanism regulating VEGF-C expression and detect the effect of the three miRNAs targeted to VEGF-C, including miRNA-27b, miRNA-101 and miRNA-128 on migration, invasion capabilities of gastric cancer cells and migration, proliferation and tube formation in HUVECs probably in vitro.Methods:GC tissue samples and clinical data of patients were obtained from Qi Lu Hospital. The expression of VEGF-C, MVD and LVD in gastric cancer was detected by immunohistochemistry and the correlation between VEGF-C and clinicopathological parameters, MVD or LVD was analyzed. VEGF-C was identified as a target gene of miR-27b, miR-101 and miR-128 using luciferase assay and further confirmed by western blotting. After transient transfection of miR-27b, miR-101 or miR-128, migration and invasion ability of GC cells and migration capabilities of HUVECs were tested by migration and invasion assays, respectively. EdU proliferation assays were used to detect the growth ability of HUVECs. Tube formation assay were performed to test the abilities of HUVECs angiongenesis. Total RNAs were extracted from 102 cases of GC tissuers,12 cases of nontumorous gastric tissues and three gastric cancer cell lines MKN-45, BGC-823 and SGC-7901 and reversely transcribed into cDNA. The relative expression of miR-27b, miR-101 and miR-128 was determined by real-time PCR.Results:Our data also showed that high VEGF-C immunostaining significantly associated with bigger tumor size, advanced TNM classification, clinical stage, and higher microvessel density (MVD) or lymphatic density (LVD).The three miRNAs expression levels were also proved to be inversely correlated with MVD. Using miRanda, TargetScan, miRWalk forecasting software, we chose 5 potential miRNAs for further investigation. The luciferase assay showed that miR-27b, miR-101 and miR-128 transfection significantly reduced the luciferase activity compared to the negative control group. Western blot was used to further validate that miR-27b, miR-101 and miR-128 could significantly inhibit the expression of VEGF-C protein. These data suggest that miR-27b, miR-101 and miR-128 could directly target to VEGF-C. Overexpression of miR-27b, miR-101 or miR-128 suppressed the migration, invasion activity in gastric cancer cells and the migration, proliferation activity and tube formation in HUVEC in vitro. Compared with nontumorous tissues, miR-27b, miR-101 and miR-128 expression was significantly reduced in GC tissues and three GC cell lines. However, our data showed no significant difference in miR-27b, miR-101 and miR-128 expression between clinicopathological parameters.Conclusions:Our study determinated miR-27b, miR-101 and miR-128 inhibited migration, invasion capabilities of gastric cancer cells and migration, proliferation and tube formation in HUVECs by decreasing the expression of VEGF-C in vitro. We also conclude that miR-27b, miR-101 or miR-128 could inhibit angiogenesis by down-regulating VEGF-C expression in gastric cancers and suppress development of gastric cancers.