miR-214通过靶向调控E2F3抑制肝癌细胞的增殖

目的探讨miR-214通过靶向调控E2F转录因子3(E2F3)抑制肝癌细胞的增殖。方法 RT-PCR法检测细胞株SMMC-7721、Hep G2、SK-Hep-1和Huh 7中miR-214的表达量,并利用脂质体转染miR-214 NC及miR-214 mimics。采用MTT法检测miR-214对肝癌细胞活力的影响;Hoechst染色试剂盒检测miR-214对细胞凋亡的影响,流式细胞术检测miR-214对细胞周期的影响;Western blot及RT-PCR法检测miR-214对肝癌细胞中E2F3蛋白及mRNA表达量的影响,并通过荧光素酶报告基因进行验证。结果 SMMC-7721、SK-Hep-1、Huh 7及Hep G2中miR-214的表达量分别为(0.83±0.08)、(0.32±0.03)、(0.33±0.03)、(0.08±0.01),其中Hep G2中miR-214表达量最低,因此选用Hep G2作为后续实验细胞株。Hep G2细胞转染miR-214 NC及miR-214 mimics、miR-214mimics组中miR-214表达量(0.65±0.06)明显高于miR-214 NC组(0.14±0.01),miR-214 mimics组细胞活力(0.35±0.03)明显低于miR-214 NC组(0.69±0.06),miR-214 mimics组细胞凋亡率(36.37±3.43)%明显高于miR-214 NC组(3.74±0.34)%,miR-214 mimics组G1期明(57.79±5.78)显长于miR-214 NC组(45.319±4.53),miR-214 mimics组中E2F3蛋白(0.23±0.02)、(0.24±0.02)]及mRNA表达量明显低于miR-214 NC组(0.98±0.09)、(0.99±0.10)],差异均具有统计学意义(P0.01)。结论 miR-214过表达能通过下调E2F3表达抑制肝癌细胞的增殖。

Inhibition of the proliferation of hepatocellular carcinoma cells by miR-214 via regulation of E2F3 expression

Objective To explore the effect of inhibition of miR-214 expression on the proliferation of hepatocellular carcinoma cells via regulation of E2F3 expression. Methods The expression of miR-214 in SMMC-7721, HepG2, SK-Hep-1 and Huh 7 cells was examined by RT-PCR. Hepatocellular carcinoma cells were transfected with miR-214 NC and miR-214 mimics with liposomes. The expression of miR-214 was detected by RT-PCR. The cell viability was detected by MTT assay. Cell apoptosis was detected by Hoechst staining. Cell cycle was detected by flow cytometry. Western blot, RT-PCR and dual luciferase reporter gene assay were used to detect whether E2F3 was a downstream target gene of miR-214. Results The expression of miR-214 in SMMC-7721, HepG2, SK-Hep-1 and Huh 7 cells was 0.83±0.08, 0.32±0.03, 0.33±0.03, and 0.08±0.01, respectively. The expression of miR-214 in the HepG2 cells was the lowest, so HepG2 cells were selected as the subsequent experimental cell line. Compared with the miR-214 NC group, the expression of miR-214 (0.65±0.06 vs. 0.14±0.01) was up-regulated, the cell viability (0.35±0.03 vs. 0.69±0.06) was decreased, cell apoptosis rate (36.37±3.43)% vs. (3.74±0.34)%] was increased, the G1 phase of the cell cycle (57.79±5.78 vs. 45.319±4.53) was prolonged, the expression of E2F3 protein (0.23±0.02 vs. 0.98±0.09) and mRNA (0.24±0.02 vs. 0.99±0.10) was significantly down-regulated in the miR-214 mimics group (P<0.01). Conclusion miR-214 mimics suppress the HepG2 cell proliferation via targeted down-regulation of E2F3 expression.