摘要
目的探索微小RNA-30a（rmR-30a）在肝细胞癌（HCC）中的表达及调控HCC细胞增殖的相关分子机制。方法收集配对的HCC及癌旁组织30对，分别采用实时荧光定量PCR和Western印迹法检测叉头框蛋白A1（FOXAl）RNA和蛋白表达，四甲基偶氮唑蓝比色法（MTT）检测HepG2细胞增殖，萤光素酶报告基因检测验证miR-30a与FOXAl的靶点关系，MTT和Westernb10t法检测转染miR-30a后Hep02细胞增殖及FOXAI蛋白的表达。两组数据分析比较采用f检验，多组数据分析比较采用单因素方差分析，P〈0．05为差异有统计学意义。结果miR-30a在HCC组织中的相对表达量为1．049±0．380显著低于癌旁组织的1．982±1．013，f=3．985，P〈0．001，差异有统计学意义。过表达miR-30a72h后，空白对照组HeDG2细胞增殖能力为0．821±0．006，miR-30a—NC组为0．816±0．013，miR-30a组为0．546±0．020，过表达miR-30a能显著降低HepG2细胞的增殖，F=3．396，P〈0．05，差异有统计学意义。FOXAl是miR-30a的靶基因，其蛋白表达被miR-30a负调控，荧光素酶的活性在野生型FOXA1—3。UTR载体中的表达量为1．221±0．024，在突变型FOXA1-3。UTR载体中的表达量为2．658±0．031，F=6．737，P〈0．05，差异有统计学意义。过表达miR-30a能显著抑制FOXAl在HepG2细胞中的蛋白水平，FOXAI在空白对照组相对表达量为1．019±0．016，miR-30a—NC组为1．022±0．017，miR-30a组为0．227±0．021，F=45．43，P〈0．05，差异有统计学意义。上调FOXAI的蛋白水平能够逆转miR-30a对HepG2细胞增殖的抑制作用，HeDG2细胞的增殖能力在miR-30a组为0．524±0．023，在miR-30a＋FOXAl组为0．843±0．019，f=2．507，P〈0．05，差异有统计学意义。结论miR-30a对HCC细胞增殖的抑制作用是通过负调控FOXAl的表达而实现。
Objective To investigate the expression of microRNA-30a （miR-30a） in hepatocellular carcinoma （HCC） and related molecular mechanisms in regulating HCC cell proliferation. Methods A total of 30 pairs of HCC and adjacent tissue samples were collected, and quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression of forkhead-box protein A1 （FOXAI）. Methyl thiazolyl tetrazolium （MTT） assay was used to evaluate the proliferation of HCC cells, luciferase reporter gene assay was performed to verify the target relationship between miR-30a and FOXA1, and MTT assay and Western blot were used to measure the proliferation of HepG2 cells and the protein expression of FOXA1 after miR-30a transfection. The t-test was used for comparison of data between two groups, and a one-way analysis of variance was used for comparison of data between multiple groups. P 〈 0.05 was considered statistically significant. Results HCC tissue had significantly lower relative expression of miR-30a than adjacent tissue （1.049 ± 0.380 vs 1.982 ± 1.013, t = 3.985, P 〈 0.001）. At 72 hours after miR-30a overexpression, there was a significant difference in the proliferative capacity of HepG2 cells between the blank control group, the miR- 30a-NC group, and the miR-30a group （0.821 ± 0.006 vs 0.816 ± 0.013 vs 0.546± 0.020,F= 3.396,P 〈 0.05）, suggesting that miR-30a overexpression significantly inhibited the proliferation of HepG2 cells. F OXA 1 was a target gene of miR-30a and its protein expression was negatively regulated by miR-30a, and there was a significant difference in luciferase activity between wild-type and mutant FOXA1-3＇UTR vectors （1.221 ± 0.024 vs 2.658± 0.031, F = 6.737, P 〈 0.05）. In HepG2 cells, miR-30a overexpression significantly inhibited the protein expression of FOXA1, and there was a significant difference in the relative expression of FOXA1 between the blank control group, the miR-30a-NC group, and the miR-30a group （1.019 ± 0.016 vs 1.022±0.0
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2017年第9期706-711,共6页
Chinese Journal of Hepatology
基金
湖南省自然科学基金（12JJ6082）
湖南省科技厅科技计划（2012FJ435,2015JC3009）
