紫草素抑制肝癌细胞增殖的机制研究

Mechanism of shikonin inhibiting the proliferation of hepatocellular carcinoma cell

目的探讨紫草素抑制肝细胞肝癌(HCC)细胞增殖的分子机制。方法将人HCC细胞株HepG2分为对照组、紫草素低剂量组、紫草素高剂量组和miR-181c空白组、miR-181c抑制组和miR-181c转染组,培养基中分别加入工作浓度为2.5和5.0μmol/L紫草素溶液以及miR-181c抑制物和miR-181c模拟物转染处理。采用细胞计数(CCK-8)法检测细胞增殖率,流式细胞术检测细胞凋亡率;qRT-PCR法检测细胞中miR-181c和非染色体结构维护凝缩蛋白I复合体G亚基(NCAPG)mRNA相对表达量,Western blot法检测细胞NCAPG和B淋巴细胞瘤-2(Bcl-2)蛋白相对表达量。结果与对照组相比,高剂量组、低剂量组细胞增殖率显著降低(均P<0.05);与低剂量组相比,高剂量组显著降低(P<0.05)。与miR-181c空白组相比,miR-181c抑制组细胞增殖率显著升高,miR-181c转染组显著降低(均P<0.05)。高剂量组细胞凋亡率明显高于低剂量组和对照组(均P<0.05);与miR-181c空白组相比,miR-181c抑制组细胞凋亡率显著降低(P<0.05),miR-181c转染组显著升高(P<0.05)。高剂量组miR-181c mRNA相对表达量明显高于低剂量组和对照组(均P<0.05);与miR-181c空白组相比,miR-181c抑制组miR-181c mRNA相对表达量显著降低(P<0.05),miR-181c转染组显著升高(P<0.05)。高剂量组NCAPG mRNA相对表达量、NCAPG和Bcl-2蛋白相对表达量均明显低于低剂量组和对照组(均P<0.05);与miR-181c空白组相比,miR-181c抑制组NCAPG mRNA相对表达量、NCAPG和Bcl-2蛋白相对表达量均显著升高(P<0.05),miR-181c转染组均显著降低(均P<0.05)。结论紫草素可上调HCC细胞中miR-181c表达从而抑制其增殖。

Objective To investigate the molecular mechanism of shikonin in inhibiting the proliferation of hepatocellular carcinoma(HCC)cells.Methods HepG2 human HCC cells were divided into six groups:control,low-dose shikonin,high-dose shikonin,miR-181c negative control(NC),miR-181c-inhibitor,and miR-181c-mimic groups.Cells were treated respectively with 2.5 or 5.0μmol/L concentrations of shikonin,and transfected with miR-181c inhibitor or miR-181c mimics,respectively.Cell proliferation was detected using the cell counting kit-8(CCK-8)assay,while apoptosis was assessed by flow cytometry.The relative mRNA expression levels of miR-181c and non-SMC condensinⅠcomplex subunit G(NCAPG)were measured by qRT-PCR,and the relative protein expression levels of NCAPG and B-cell lymphoma 2(Bcl-2)were determined by Western blot.Results Compared with the control group,the cell proliferation rate was significantly reduced in both the high-dose and low-dose shikonin groups(both P<0.05),and that of the high-dose group reduced more remarkably than the low-dose group(P<0.05).In comparison to the miR-181c NC group,the cell proliferation rate significantly increased in the miR-181c inhibitor group,while significantly decreased in the miR-181c mimic group(both P<0.05).The high-dose group exhibited a significantly higher apoptosis rate than both the low-dose group and control group(both P<0.05).Compared with the miR-181c NC group,the apoptosis rate significantly decreased in the miR-181c inhibitor group,while significantly increased in the miR-181c mimic group(both P<0.05).The relative expression level of miR-181c mRNA in the high-dose group was significantly higher than that of the low-dose and control groups(both P<0.05).Compared to the miR-181c NC group,the miR-181c mRNA relative expression level significantly decreased in the miR-181c inhibitor group,while significantly increased in the miR-181c mimic group(both P<0.05).The relative expression levels of NCAPG mRNA,NCAPG and Bcl-2 proteins in the high-dose group were significantly lower than those of the low-dose group and control group(all P<0.05).Compared to the miR-181c NC group,the relative expression levels of NCAPG mRNA,NCAPG and Bcl-2 proteins significantly increased in the miR-181c inhibitor group,while significantly decreased in the miR-181c mimic group(all P<0.05).Conclusion Shikonin can upregulate miR-181c expression in HCC cells,thereby inhibiting the proliferation of HCC cells.