山药多糖通过miR-98-5p/TGFβR1分子轴调控肝癌细胞凋亡的机制研究

Chinese Yam polysaccharide regulating apoptosis of hepatocellular carcinoma cells through miR-98-5p/TGFβR1 axis

目的探究山药多糖(CYPS)通过miR-98-5p/TGFβR1分子轴调控肝癌(HCC)细胞凋亡的分子机制。方法qPCR检测miR-98-5p在不同HCC细胞系中的表达情况,使用不同浓度的CYPS处理Huh-7细胞,CCK-8检测细胞增殖活力,Annexin V-FITC/PI流式细胞术检测细胞凋亡,Western blotting检测TGFβR1和细胞凋亡相关蛋白Caspase-3、Caspase-8、Bcl-2、Bax的表达,双荧光素酶报告基因系统验证miR-98-5p与TGFβR1的靶向关系。结果与人正常肝细胞HL-7702相比,miR-98-5p在HCC细胞系中表达升高(均P<0.05),且在Huh-7细胞中的表达高于其他HCC细胞(P<0.05);CYPS处理可明显抑制Huh-7细胞的增殖活力并诱导细胞凋亡(均P<0.05),且10-3 kg/L CYPS对细胞增殖活力的抑制作用比其他浓度明显。双荧光素酶报告基因实验证实,miR-98-5p靶向调控TGFβ1。与10-3 kg/L CYPS组相比,miR-98-5p mimics可下调10-3 kg/L CYPS对细胞增殖活力(P<0.05)的抑制作用和对细胞凋亡(P<0.01)的促进作用,CYPS+miR-98-5p mimics+pcDNA-TGFβR1组中细胞增殖活力与CYPS+miR-98-5p mimics组相比明显降低(P<0.05),细胞凋亡水平明显升高(P<0.01)。结论CYPS可下调miR-98-5p,促进TGFβR1的表达,抑制Huh-7细胞增殖并诱导细胞凋亡。

Objective To explore the mechanism of Chinese Yam polysaccharide(CYPS)regulating hepatocellular carcinoma(HCC)cells apoptosis through the molecular axis of miR-98-5p/TGFβR1.Methods The expression of miR-98-5p in different HCC cell lines was detected by qPCR.Huh-7 cell was treated with CYPS at different concentrations,cell proliferation activity was detected by CCK-8,apoptosis rate was detected by Annexin V-FITC/PI flow cytometry,and the expressions of TGFβR1 and apoptosis-related proteins Caspase-3,Caspase-8,Bcl-2 and Bax were detected by Western blotting.The dual luciferase reporter gene system was used to verify the targeted relationship between miR-98-5p and TGFβR1.Results Compared with human normal hepatocytes HL-7702,the expression of miR-98-5p was increased in HCC cell lines(all P<0.05),and the expression in Huh-7 cell was higher than other cells(P<0.05).CYPS treatment could significantly inhibit the proliferation of Huh-7 cell(all P<0.05)and induce cell apoptosis(all P<0.05),and the inhibitory effect of 10-3 kg/L CYPS on cell proliferation was more obvious than other concentrations.The dual luciferase reporter gene experiment confirmed that miR-98-5p targeted and regulated TGFβR1.Compared with the 10-3 kg/L CYPS group,miR-98-5p mimics could down-regulate the inhibitory effect of 10-3 kg/L CYPS on cell proliferation(P<0.05)and the promotion of cell apoptosis(P<0.01).The cell proliferation activity in the 10-3 kg/L CYPS+miR-98-5p mimics+pcDNA-TGFβR1 group was significantly lower than that in the 10-3 kg/L CYPS+miR-98-5p mimics group(P<0.05),while the level of cell apoptosis was significantly increased(P<0.01).Conclusion CYPS can inhibit the proliferation of Huh-7 cell,induce apoptosis,down-regulate miR-98-5p and promote the expression of TGFβR1.