微小RNA 99a 5p靶向调控IGF1R表达及对肝癌HepG2细胞侵袭迁移的影响#br##br#

目的探讨微小RNA 99a 5p（miR 99a 5p）对肝癌细胞HepG2侵袭和迁移的影响及对胰岛素样生长因子1受体（IGF1R）的靶向调控作用。方法收集本院2014年1月至2017年3月经手术切除的90对癌组织及癌旁组织，采用实时定量PCR（QPCR）检测以上组织中miR 99a 5p及IGF1R mRNA水平；采用脂质体法向HepG2细胞分别转染miR 99a 5p模拟物（过表达组）和miR 99a 5p阴性对照（NC组），采用QPCR法检测转染48 h后各组的miR 99a 5p水平以评价转染效率；分别采用Transwell和划痕实验比较各组的侵袭和迁移情况；QPCR和Western blotting检测各组的IGF1R mRNA和蛋白水平；双荧光素酶报告基因实验验证miR 99a 5p对IGF1R的靶向作用。结果QPCR检测结果显示，癌组织的miR 99a 5p水平为0823±0039，低于癌旁组织的1016±0013，而癌组织的IGF1R mRNA水平为1329±0024，高于癌旁组织的1035±0026，差异均有统计学意义（P＜005）。过表达组转染48 h后的miR 99a 5p水平为1487±0027，均高于对照组的1021±0013和NC组的1019±0025，差异有统计学意义（P＜005）。对照组、NC组和过表达组的愈合率分别为（605±24）％、（584±33）％和（421±29）％，穿膜细胞数分别为（921±37）个、（952±49）个和（367±45）个，过表达组的愈合率和穿膜细胞数均少于其余两组（P＜005）；过表达组的IGF1R mRNA和蛋白水平均低于其余两组（P＜005）。双荧光素酶报告基因试验结果显示，miR 99a 5p显著抑制野生型IGF1R 3’ UTR质粒转染细胞的荧光素酶活性（P＜001），但是对突变型IGF1R 3’ UTR荧光素酶活性无显著影响。结论miR 99a 5p在肝癌中表达降低，且可抑制肝癌细胞的侵袭迁移过程，可能是通过靶向调控IGF1R来实现，可作为肝癌的潜在治疗靶点。

Targeted regulation of microRNA 99a 5p on IGF1R expression and its effect on invasion and migration of HepG2 cells#br#

ObjectiveTo investigate the effect of microRNA 99a 5p （miR 99a 5p） on the invasion and migration of hepatoma cells HepG2 and its regulatory role on insulin like growth factor 1 receptor （IGF1R）.MethodsNinety pairs of cancerous and paracancerous tissues surgically removed in our hospital were collected from January 2014 to March 2017. Real time quantitative PCR （QPCR） was used to detect the level of miR 99a 5p and IGF1R mRNA in the above tissues. HepG2 cells were transfected with miR 99a 5p mimics （overexpression group） and miR 99a 5p negative control （NC group） by liposome method. QPCR method was used to detect the miR 99a 5p level of each group after transfection for 48 h to evaluate the transfection efficiency. The invasion and migration of each group were evaluated by Transwell and scratch test， respectively. The mRNA and protein levels of IGF1R were detected by QPCR and Western blotting， respectively. The dual luciferase reporter gene test was applied to verify the targeting effect of miR 99a 5p on IGF1R.ResultsThe results of QPCR showed that the level of miR 99a 5p in the cancer tissues was 0823±0039， lower than 1016±0013 in normal paracancerous tissues， while the level of IGF1R mRNA in the cancer tissues was 1329±0024， higher than 1035±0026 in normal paracancerous tissues （P＜005）. The miR 99a 5p level in the overexpression group was 1487±0027， higher than 1021±0013 of control group and 1019±0025 of NC group （P＜005）. The healing rates of control group， NC group and overexpression group were （605±24）％， （584±33）％ and （421±29）％， and the numbers of membrane cells were 921±37， 952±49 and 367±45， respectively. Compared with other two groups， the healing rates and numbers of membrane cells of overexpression group decreased （P＜005）. The mRNA and protein levels of IGF1R in overexpression group were lower than those in other two groups （P＜005）. The results of double fluorescence report showed that miR 99a 5p significantly inhibited the luciferase activity of cells transfected with the wild type IGF1R 3’ UTR plasmid （P＜001）， but had no significant effect on the activity of the mutant IGF1R 3’ UTR luciferase activity.ConclusionThe expression of miR 99a 5p is reduced in hepatoma cancer. MiR 99a 5p can inhibit the invasion and migration of hepatoma cells. It may be realized by targeting IGF1R， which can be used as a potential target for the treatment of hepatoma cancer.