Wu Wei-Lu, Wang Wei-Ya, Yao Wen-Qing, Li Gan-Di
Department of Pathology, West China Hospital of Sichuan University, Chengdu 610041, P.R. China.
Int J Mol Med. 2015 Dec;36(6):1713-9. doi: 10.3892/ijmm.2015.2379. Epub 2015 Oct 16.
miR-16 is known to be abnormally expressed in hepatocellular carcinoma (HCC) cells, and the overexpression of miR-16 inhibits the proliferation, invasion and metastasis of various cancer cells. MicroRNAs (miRNAs or miRs) are closely related to the proliferation, invasion and metastasis of HCC. The present study aimed to explore the effects of miR-16 on the proliferation, invasion and metastasis of HCC cells, and to elucidate the mechanisms involved. A cell line with moderate levels of miR‑16 expression was selected from the SMMC-7721, HepG2, SK-Hep-1 and Huh‑7 HCC cells and validated by reverse transcription-PCR (RT-PCR). The effects of miR‑16 on HCC cell viability were determined by MTT assay; cell migration and invasion were determined by Transwell cell invasion assay, and apoptosis was determined by flow cytometery (FCM). Western blot analysis was used to measure the expression levels of the apoptosis-related proteins, Bax, Bcl-2, matrix metalloproteinase (MMP)-2, MMP-9, as well as to examine epithelial-mesenchymal transition (EMT), and E-cadherin, vimentin, and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway-related protein expression. The mRNA expression levels of miR‑16 were highest in the SMMC-7721 cells and lowest in the SK-Hep‑1 and Huh‑7 cells; moderate levels were observed in the HepG2 cells. The HepG2 cell line was selected as the cell line for use in the follow-up experiments, where we measured cell viability, and the expression of PI3k/Akt, Bax, Bcl-2, MMP-2 and MMP-9, and E-cadherin and vimentin. miR‑16 overexpression significantly inhibited the proliferation, invasion and metastasis of the HepG2 cells, as shown by western blot analysis. This was achieved through the upregulation of Bax expression, the downregulation of Bcl-2 expression and the decrease in the expression of MMP-2 and MMP-9. In addition the expression of E-cadherin increased and vimentin expression decreased. miR‑16 overexpression inhibited PI3K expression and Akt phosphorylation. The results of this study suggest that the overexpression of miR‑16 inhibits the proliferation, invasion and metastasis of HepG2 HCC cells, and that these effects are associated with the PI3K/Akt signaling pathway.
已知miR-16在肝癌（HCC）细胞中异常表达，miR-16的过表达可抑制多种癌细胞的增殖、侵袭和转移。微小RNA（miRNA或miR）与HCC的增殖、侵袭和转移密切相关。本研究旨在探讨miR-16对HCC细胞增殖、侵袭和转移的影响，并阐明其相关机制。从SMMC-7721、HepG2、SK-Hep-1和Huh-7 HCC细胞中筛选出miR-16表达水平适中的细胞系，并通过逆转录聚合酶链反应（RT-PCR）进行验证。采用MTT法检测miR-16对HCC细胞活力的影响；采用Transwell细胞侵袭试验检测细胞迁移和侵袭能力，采用流式细胞术（FCM）检测细胞凋亡情况。采用蛋白质免疫印迹分析检测凋亡相关蛋白Bax、Bcl-2、基质金属蛋白酶（MMP）-2、MMP-9的表达水平，以及检测上皮-间质转化（EMT）、E-钙黏蛋白、波形蛋白和磷脂酰肌醇3-激酶（PI3K）/Akt信号通路相关蛋白的表达。miR-16的mRNA表达水平在SMMC-7721细胞中最高，在SK-Hep-1和Huh-7细胞中最低；在HepG2细胞中观察到中等水平。选择HepG2细胞系用于后续实验，检测细胞活力、PI3k/Akt、Bax、Bcl-2、MMP-2和MMP-9以及E-钙黏蛋白和波形蛋白的表达。蛋白质免疫印迹分析显示，miR-16过表达显著抑制HepG2细胞的增殖、侵袭和转移。这是通过上调Bax表达、下调Bcl-2表达以及降低MMP-2和MMP-9的表达来实现的。此外，E-钙黏蛋白表达增加，波形蛋白表达降低。miR-16过表达抑制PI3K表达和Akt磷酸化。本研究结果表明，miR-16过表达抑制HepG2 HCC细胞的增殖、侵袭和转移，且这些作用与PI3K/Akt信号通路有关。
