Yang Yue-Feng, Wang Fei, Xiao Jun-Jie, Song Yang, Zhao Ying-Ying, Cao Yan, Bei Yi-Hua, Yang Chang-Qing
Division of Gastroenterology and Hepatology, Digestive Disease Institute, Tongji Hospital, Tongji University School of Medicine Shanghai 200065, China.
Regeneration Laboratory, School of Life Science, Shanghai University Shanghai 200444, China ; Experimental Center of Life Sciences, Shanghai University Shanghai 200444, China.
Int J Clin Exp Med. 2014 Apr 15;7(4):893-902. eCollection 2014.
Hepatocellular carcinoma (HCC) represents the third leading cause of cancer-related death worldwide. Increasing evidence suggests that microRNAs, a novel class of non-coding RNAs that function as endogenous suppressors of gene expression, are deregulated in HCC. Although microRNA-222 (miR-222) overexpression has been described in HCC, the role of miR-222 and its target genes in the proliferation of hepatocellular carcinoma cells remain poorly elucidated.
HepG2 cells were transfected with miR-222 mimic, inhibitor or their negative controls. Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) and EdU incorporation assay. Flow cytometry was performed to assess the effects of miR-222 on HepG2 cell cycle progression. MiR-222 and putative targets genes (p27 and p57) expression levels were determined using qRT-PCR and/or Western blot.
MiR-222 overexpression induced an enhancement of HepG2 cell proliferation in vitro, paralleling with an altered cell cycle progression via increased cell population in S phase. P27 expression, other than p57, was negatively regulated by miR-222 overexpression at post-transcriptional level in HepG2 cells. Transfection of either small interfering RNA (siRNA) for p27 or miR-222 mimic increased HepG2 cell proliferation rate, whereas co-transfection of p27 siRNA and miR-222 mimic did not further enhance HepG2 cell proliferation in comparison with the cells transfected with p27 siRNA or miR-222 mimic alone, validating that p27 is a target gene of miR-222 during HepG2 cell proliferation.
This study suggests that miR-222 overexpression promotes HepG2 cell proliferation by downregulating p27.
肝细胞癌（HCC）是全球癌症相关死亡的第三大主要原因。越来越多的证据表明，微小RNA（一类新型的非编码RNA，作为基因表达的内源性抑制因子发挥作用）在HCC中表达失调。尽管已有研究报道HCC中存在微小RNA-222（miR-222）过表达的情况，但miR-222及其靶基因在肝癌细胞增殖中的作用仍未得到充分阐明。
用miR-222模拟物、抑制剂或其阴性对照转染HepG2细胞。通过细胞计数试剂盒-8（CCK-8）和EdU掺入试验评估细胞增殖。进行流式细胞术以评估miR-222对HepG2细胞周期进程的影响。使用qRT-PCR和/或蛋白质免疫印迹法测定miR-222和假定靶基因（p27和p57）的表达水平。
miR-222过表达在体外诱导HepG2细胞增殖增强，同时通过增加S期细胞数量改变细胞周期进程。在HepG2细胞中，miR-222过表达在转录后水平负调控p27的表达，而非p57。转染针对p27的小干扰RNA（siRNA）或miR-222模拟物均可提高HepG2细胞增殖率，然而，与单独转染p27 siRNA或miR-222模拟物的细胞相比，共转染p27 siRNA和miR-222模拟物并未进一步增强HepG2细胞增殖，这证实了在HepG2细胞增殖过程中p27是miR-222的靶基因。
本研究表明，miR-222过表达通过下调p27促进HepG2细胞增殖。
