摘要
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miRNC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Western blotting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 oneogene homolog（MRAS） in gastric cancer HGC-27 cells. Methods The eryopreserved 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 trans- fected 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 luciferasc reporter gene test was em- ployed 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 untransfec- tion 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±.049, lower than 1. 106±.024 and 0. 824±. 091 of un- transfected group and l. 095 ±0. 031 and 0. 784± 0. 121 of miR-NC group �
出处
《临床肿瘤学杂志》
CAS
2017年第10期874-879,共6页
Chinese Clinical Oncology
