Effects of miR-944 on the Proliferation and Migration of Glioma Cellsby Targeting RAB1A
ZHU Yingting, WANG Yuan, LUO Ming, CHEN Jie, LUO Zhihua*
This study aims to discuss the effects of miR-944 on proliferation and migration of glioma cells by targeting RAB1A (Rab family protein 1A). Cancer tissues and adjacent tissues of glioma patients were collected, normal control cells NHA and glioma cell lines (A172, LN229, SHG-44, U251, U-87 MG) in vitro were cultured, and qRT-PCR experiments were used to test miR-944 and RAB1A mRNA expression in tissues and cells. The relationship between miR-944, RAB1A and the clinical prognosis of glioma was analyzed through the TCGA database. U251 cells were grouped into Control group, miR-mimic-NC group, miR-944-mimic group, miR-944- mimic+pcDNA3.1-NC group, and miR-944-mimic+pcDNA3.1-RAB1A group. qRT-PCR experiments were used to test miR-944 and RAB1A mRNA in U251 cells. Cell viability was assessed using the CCK-8 assay; the number of colonies formed by each group of cells was measured via the colony formation assay; and the migratory capacity of each group of cells was evaluated using the Transwell assay. Western blot was performed to detect the expression of RAB1A and EMT (epithelial mesenchymal transition) related proteins in U251 cells. Dual luciferase assay was used to determine the targeting relationship between miR-944 and RAB1A. The effect of miR-944 on the in vivo growth of glioma cells was analyzed through xenograft tumor experiments. For adjacent cancer tissues, the miR-944 in glioma tissues was clearly lower (P<0.05), while RAB1A mRNA was clearly higher (P<0.05). Compared with the normal control cells NHA, the levels of miR-944 in the glioma cell lines (A172, LN229, SHG-44, U251, U-87 MG) were significantly decreased (P<0.05), and the expression level of RAB1A mRNA was significantly increased (P<0.05); in the TCGA database, both miR-944 and RAB1A were related to the prognosis of glioma patients (P<0.05). For the miR-mimic-NC group, the miR-944-mimic group showed clearly decreased RAB1A mRNA and protein, cell survival rate, number of colonies, migration rate, N-cadherin, MMP-9, Vimentin, Fibronectin, Snail, and Slug in U251 cells (P<0.05), and clearly increased miR-944, E-cadherin and ZO-1 (P<0.05). For the miR-944-mimic+pcDNA3.1-NC group, the miR-944-mimic+pcDNA3.1-RAB1A group showed clearly increased RAB1A mRNA and protein, cell survival rate, number of colonies, migration rate, N-cadherin, MMP-9, Vimentin, Fibronectin, Snail, and Slug in U251 cells (P<0.05), and clearly reduced E-cadherin and ZO-1 (P<0.05). For the co-transfection group of RAB1A-WT and miR-mimic-NC, the relative luciferase activity in the co-transfection group of RAB1A-WT and miR-944-mimic was significantly reduce (P<0.05). The results of the in vivo experiments showed that compared with the miR-mimic-NC group, the tumor volume and mass, as well as the expression levels of RAB1A mRNA and protein in the tumor tissue in the miR-944-mimic group were significantly reduced (P<0.05), and the level of miR-944 was significantly increased (P<0.05); compared with the miR-944-mimic+pcDNA3.1-NC group, the tumor volume and mass, as well as the expression levels of RAB1A mRNA and protein in the tumor tissue in the miR-944-mimic+pcDNA3.1-RAB1A group were significantly increased (P<0.05). miR-944 may inhibit the proliferation and migration of glioma cells by targeting RAB1A and hindering the EMT process.
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