Targeted-regulation of MRAS expression by microRNA-148a-3p and its effects on invasion and migration of gastric cancer HGC-27 cells

WANG Jianguo; SUN Peida; LIU Haiyan; CHEN Lin

doi:10.3969/j.issn.1009-0460.2017.10.003

Abstract：Objective To investigate the effect of microRNA-148a-3p (miR-148a-3p) on invasion,migration and expression of muscle RAS oncogene homolog(MRAS) in gastric cancer HGC-27 cells.Methods The cryopreserved gastric cancer cell line HGC-27 was recovered and cultured to the logarithmic growth phase.The miR-148a-3p analog and its negative control (miR-NC) were transfected into HGC-27 cells (miR-148a-3p transfection group and miR-NC group),and HGC-27 cells without transfection were chosen as blank control (untransfection group).The level of miR-148a-3p was detected by real-time quantitative PCR (QPCR) at 48 h after transfection.Transwell and scratch test were used to compare the number of penetrating-membrane cells and relative migration distance at 48 h after transfection in each group to evaluate the migration and invasion ability.The mRNA and protein levels of MRAS in each group at 48 h after transfection were detected by QPCR and Western blotting,respectively.Double luciferase reporter gene test was employed to verify the targeting regulation between miR-148a-3p and MRAS.Results QPCR detection showed that miR-148a-3p level of miR-148a-3p transfection group was 2.612±0.213,higher than 0.954±0.098 of untransfected group and 0.983±0.196 of miR-NC group,and the difference was statistically significant (P＜0.05).Scratch results show that the relative migration distance of miR-148a-3p group was 0.615±0.019,lower than 1.021±0.019 of untransfection group and 0.948±0.022 of miR-NC group (P＜0.05).Transwell assay showed that the transmembrane cell number in miR-148a-3p transfected group was 83± 17,less than 124± 17 of untransfection group and 136±26 of miR-NC group (P＜0.05).QPCR and Western blotting results showed that mRNA and protein levels of MRAS in miR-148a-3p transfection group were 0.614±0.057 and 0.553±0.049,lower than 1.106±0.024 and 0.824±0.091 of untransfected group and 1.095 ± 0.031 and 0.784± 0.121 of miR-NC group (P ＜ 0.05).MiR-148a-3p significantly decreased the luciferase activity of wild-type MRAS-3'untranslated region plasmid transfected cells,but had no significant inhibitory effect on the luciferase activity of mutant MRAS plasmid transfected cells.Conclusion MiR-148a-3p may inhibit the invasion and migration of gastric cancer HGC-27 cells by targeting MRAS.