LncRNA MIR100HG靶向miR-142-5p诱导肝癌细胞对索拉菲尼的耐药机制研究

Experimental study on the role and molecular mechanism of LncRNA MIR100HG in the drug resistance of liver cancer cells to sorafenib

目的探讨LncRNA MIR100HG在肝癌对索拉菲尼耐药性中的作用及分子机制。方法建立耐索拉菲尼的肝癌细胞株Huh7/SFB,以肝癌细胞Huh7为对照,实时荧光定量PCR(qRT-PCR)检测细胞中MIR100HG和miR-142-5p表达水平,双荧光素酶基因报告实验验证MIR100HG和miR-142-5p调控关系。转染MIR100HG的小干扰RNA至Huh7/SFB细胞抑制MIR100HG表达,四甲基噻唑蓝染色法(MTT)检测抑制MIR100HG表达对索拉菲尼诱导的Huh7/SFB细胞增殖的影响,流式细胞仪检测抑制MIR100HG表达对索拉菲尼诱导的Huh7/SFB细胞凋亡的影响,蛋白印迹(Western Blot)检测抑制MIR100HG表达对索拉菲尼诱导的Huh7/SFB细胞细胞周期蛋白D1(CyclinD1)、p21、B淋巴细胞瘤-2(Bcl-2)和B淋巴细胞瘤-2相关蛋白(Bax)表达的影响。结果与Huh7细胞比较,索拉菲尼对Huh7/SFB细胞的抑制率降低(P<0.05)。索拉菲尼对Huh7/SFB细胞的半数抑制率(IC50值)约是Huh7细胞的15倍。与Huh7细胞比较,Huh7/SFB细胞中MIR100HG水平升高(P<0.05),miR-142-5p水平降低(P<0.05)。MIR100HG靶向负调控miR-142-5p表达。抑制MIR100HG表达可提高索拉菲尼对Huh7/SFB细胞的抑制率,促进索拉菲尼诱导的Huh7/SFB细胞凋亡及p21和Bax蛋白表达,抑制CyclinD1和Bcl-2蛋白表达。抑制miR-142-5p表达可逆转抑制MIR100HG表达对索拉菲尼诱导的Huh7/SFB细胞存活率、凋亡及相关蛋白p21、Bax、CyclinD1和Bcl-2表达的影响。结论抑制MIR100HG表达可能通过负调控miR-142-5p表达降低肝癌细胞对索拉菲尼的耐药性。

Objective To investigate the role and molecular mechanism of LncRNA MIR100HG in the drug resistance of liver cancer cells to sorafenib.Methods The hepatoma cell line Huh7/SFB that was resistant to sorafenib was established,and the expression levels of MIR100HG and miR-142-5p in cells were detected by quantitative real-time PCR(qRT-PCR)with Huh7 as a control.The dual luciferase gene reporter assay was used to identify the regulatory correlation between MIR100HG and miR-142-5p.The small interfering RNA of MIR100HG was transfected into Huh7/SFB cells to inhibit MIR100HG expression,MTT assay was used to detect the effects of expression of MIR100HG on the proliferation of sorafenib-induced Huh7/SFB cell,and flow cytometry was used to detect the effects of inhibiting MIR100HG expression on the apoptosis of sorafenib-induced Huh7/SFB cell,moreover,Western Blot was used to detect the effects of inhibiting MIR100HG expression on the expressions of CyclinD1,p21,Bcl-2 and Bax.Results As compared with that on Huh7 cells,the inhibition rate of sorafenib on Huh7/SFB cells was decreased(P<0.05).The half-inhibition rate(IC50 value)of sorafenib on Huh7/SFB cells was about 15 times as high as that on Huh7 cells.As compared with those in Huh7 cells,MIR100HG levels were significantly increased in Huh7/SFB cells(P<0.05),however,miR-142-5p levels were significantly decreased(P<0.05).MIR100HG could targetedly negatively regulate the expression of miR-142-5p.To inhibit MIR100HG expression could increase the inhibition rate of sorafenib on Huh7/SFB cells,promote apoptosis of sorafenib-induced Huh7/SFB cell as well as the expression levels of p21 and Bax protein,and inhibit the expressions of CyclinD1 and Bcl-2 protein.Moreover to inhibi the expression of miR-142-5p reversed the effects of inhibition of MIR100HG expression on the survival rate,apoptosis rate,and expression of related proteins p21,Bax,CyclinD1 and Bcl-2.Conclusion To inhibit the expression of MIR100HG may decrease the drug resistance of liver cancer cells to sorafenib by negatively regulating the expression of miR-142-5p.