蛋白酶激活受体2对肝癌细胞增殖及凋亡的影响

Effect of protease activated receptor 2 on proliferation and apoptosis of hepatocellular carcinoma cells

目的探讨蛋白酶激活受体2(protease activated receptor 2,PAR2)对肝癌细胞增殖及凋亡的影响。方法 RT-qPCR检测100例肝癌组织及相应的癌旁组织中PAR2 mRNA的表达,并分析肝癌患者临床病理参数与PAR2表达水平之间的相关性。RT-qPCR及Western blot检测原代肝细胞、永生化肝细胞及肝癌细胞中PAR2 mRNA及蛋白的表达。采用siRNA靶向沉默PAR2的表达,MTS法及软琼脂集落形成实验检测沉默PAR2对肝癌细胞增殖的影响,流式细胞术及PARP的剪切检测沉默PAR2对肝癌细胞凋亡的影响。RT-qPCR筛选PAR2调控的凋亡靶基因,并用Western blot进行验证。结果肝癌组织中PAR2 mRNA的相对表达量为(1.86±1.26),明显高于癌旁组织(0.96±0.66,P<0.001),且PAR2在肝癌组织中表达上调与肿瘤的长径以及分期均相关(P=0.002)。PAR2在肝癌细胞中的表达高于永生化肝细胞和原代肝细胞(P<0.05)。沉默PAR2抑制肝癌细胞SK-Hep-1和PLC/PRF/5的活性(P<0.01)及集落形成能力(P<0.01),沉默PAR2导致SK-Hep-1和PLC/PRF/5细胞凋亡比例分别增加109%(P<0.01)和112%(P<0.01);且沉默PAR2增加了Bax mRNA和蛋白表达水平。结论 PAR2可通过影响Bax的表达而影响肝癌细胞的增殖与凋亡。

Objective To demonstrate the role of protease activated receptor 2(PAR2)in the proliferation and apoptosis of hepatocellular carcinoma(HCC)cells.Methods Firstly,the mRNA expression level of PAR2 in the cancer and para-cancerous tissues of 100 HCC patients were detected by RT-qPCR.Then the association between multiple clinicopathologic parameters and PAR2 mRNA level in HCC tissues were illustrated by statistical analysis.Secondly,the mRNA and protein levels of PAR2 in primary hepatocyte(PHH cells),immortalized hepatocyte(MIHA cells)and hepatoma cell lines(PLC/PRF/5,SK-Hep-1,HepG2 and Hep3 B)were determined by RT-qPCR and Western blotting.Thirdly,the proliferation of HCC cells was analyzed by MTS and soft agar assay after the silence of PAR2.Moreover,the apoptosis of HCC cells was analyzed by flow cytometry and the cleavage of PARP after the silence of PAR2.Finally,the apoptosis target genes were screened by RT-qPCR,and then validated by Western blotting.Results The relative mRNA level of PAR2 was 1.86±1.26 in HCC tissues,which was significantly higher than that in the adjacent tissues(0.96±0.66,P<0.001),and the up-regulation of PAR2 in HCC tissues was positively correlated with tumor size and tumor stage(P=0.002).Also,the expression level of PAR2 in hepatoma cell lines was significantly higher than those in immortalized hepatocytes and primary hepatocytes(P<0.05).Importantly,silence of PAR2 inhibited the cell viability and colony forming ability in SK-Hep-1 and PLC/PRF/5 cells(both P<0.01),and also induced the apoptotic rate increased to 109%and 112%in SK-Hep-1 and PLC/PRF/5 cells(P<0.01),separately.What’s more,the silence of PAR2 up-regulate the expression of Bax at mRNA and protein levels.Conclusion PAR2 affects the proliferation and apoptosis of HCC cells through regulating the expression of Bax.