目的 探讨微小RNA-148a-3p(miR-148a-3p)对胃癌HGC-27细胞侵袭迁移及肌RAS癌基因同源基因(MRAS)表达的影响.方法 将冻存的胃癌细胞株HGC-27复苏并常规培养至对数生长期,将miR-148a-3p模拟物及其阴性对照(miR-NC)分别转染HGC-27细胞(miR-148a-3p转染组和miR-NC组),以未行转染的HGC-27细胞为空白对照(未转染组),采用实时定量PCR(QPCR)检测各组转染48 h后的miR-148a-3p水平,划痕实验和Transwell侵袭实验分别比较各组HGC-27细胞转染48 h后的迁移相对距离和穿膜细胞数目以评价迁移和侵袭情况,QPCR和Western blotting分别检测各组转染48 h后MRAS的mRNA和蛋白水平,双荧光素酶报告基因实验验证miR-148a-3p与MRAS之间的靶向调节作用.结果 QPCR检测显示转染48 h后miR-148a-3p转染组的miR-148a-3p水平为2.612±0.213,高于未转染组的0.954±0.098和miR-NC组的0.983±0.196,差异有统计学意义(P＜0.05);划痕实验结果显示,miR-148a-3p转染组的相对迁移距离为0.615±0.019,低于未转染组的1.021±0.019和miR-NC组的0.948±0.022,Transwell侵袭实验显示miR-148a-3p转染组的穿膜细胞数目为(83±17)个,少于未转染组的(124±17)个和miR-NC组的(136±26)个,差异有统计学意义(P＜0.05).QPCR和Westernblotting结果显示,miR-148a-3p转染组MRAS mRNA和蛋白水平分别为0.614±0.057和0.553±0.049,均低于未转染组的1.106±0.024和0.824±0.091及miR-NC组的1.095±0.031和0.784±0.121,差异有统计学意义(P＜0.05).miR-148a-3p显著降低野生型MRAS-3'非翻译区质粒转染细胞的荧光素酶活性,但对突变型质粒转染细胞的荧光素酶活性无明显影响.结论 MiR-148a-3p可能通过靶向调控MRAS来抑制胃癌HGC-27细胞的侵袭和迁移.%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.
展开▼
