SUMO调控XBP1介导的内质网应激对肝癌进展的影响机制

Mechanism of SUMO regulating XBP1 mediated endoplasmic reticulum stress on the progression of liver cancer

目的探讨SUMO特异性蛋白酶1(SENP1)调控内质网应激转录调节因子X-盒结合蛋白1(XBP1)参与肝癌细胞增殖的分子机制。方法收集2012年1月至2020年1月内蒙古自治区人民医院肝胆外科收治的180例原发性肝癌患者的病理样本,检测肝癌、癌旁组织及不同肝癌细胞株中SENP1及XBP1的表达情况;分析肝癌组织中SENP1阳性表达与患者临床病理特征的相关性。免疫荧光与流式细胞技术检测SENP1 siRNA对XBP1及凋亡的影响。免疫沉淀检测肝癌细胞株中XBP1表面SUMO1表达情况及SENP1 siRNA对XBP1的SUMO化的影响。结果SENP1在肝癌及癌旁组织中的表达水平分别为16.332±4.371、6.840±2.238,差异具有统计学意义(t=-5.073,P=0.017)。XBP1在肝癌及癌旁组织中的表达水平分别为6.641±2.482、16.051±4.452,差异具有统计学意义(t=3.592,P=0.032)。肝癌组织中SENP1的表达与分期(χ^2=6.724,P=0.010)和是否转移(χ^2=6.265,P=0.012)相关。免疫荧光染色结果发现,XBP1表达水平在L02(0.509±0.219)、MHCC97-L(0.092±0.022)和HCCLM3(0.086±0.014)细胞间的表达差异有统计学意义(F=6.378,P=0.004),其中MHCC97-L和HCCLM3细胞显著低于在L02细胞的表达(P=0.023;P=0.021);SENP1在L02、MHCC97-L、HCCLM3细胞中的表达水平分别为0.109±0.079、0.802±0.392、0.921±0.352,差异具有统计学意义(F=7.783,P=0.004),其中MHCC97-L及HCCLM3细胞的表达均显著高于L02细胞(P=0.039;P=0.016);SENP1 siRNA转染MHCC97-L、HCCLM3细胞后,XBP1的表达水平均升高(0.462±0.192,t=3.664,P=0.022;0.524±0.203,t=3.383,P=0.028);SENP1的表达水平均下降(0.153±0.093,t=2.790,P=0.049;0.165±0.104,t=3.568,P=0.023)。流式细胞术结果显示,L02、MHCC97-L、HCCLM3、MHCC97-L+SENP1 siRNA及HCCLM3+SENP1 siRNA细胞的凋亡率分别为(20.80±3.11)%、(2.02±1.20)%、(0.12±0.01)%、(7.01±1.80)%、(6.20±2.01)%,差异具有统计学意义(F=1.025,P=0.030);MHCC97-L、HCCLM3细胞的凋亡率明显低于L02细胞(P=0.040;P=0.010);MHCC97-L+SENP1 siRNA、HCCLM3+SENP1 siRNA细胞凋亡率分别较MHCC97-L、HCCLM3细胞明显升高(均P=0.009)。免疫沉淀结果发现,XBP1在L02、MHCC97-L、HCCLM3、MHCC97-L+SENP1 siRNA及HCCLM3+SENP1 siRNA的表达水平分别为11.943±5.043、7.467±1.903、2.051±0.913、9.532±3.012、8.731±3.102,SUMO1表达水平分别为10.158±4.005、5.871±3.075、1.941±0.907、8.658±4.878、7.169±4.677,差异均有统计学意义(F=11.730,P=0.010;F=8.548,P=0.001);XBP1及SUMO1在MHCC97-L(P=0.028;P=0.038)、HCCLM3(P<0.001;P<0.001)细胞中的表达均较L02细胞下调,XBP1在HCCLM3+SENP1 siRNA细胞中的表达较HCCLM3上调(P=0.001),SUMO1在MHCC97-L+SENP1 siRNA、HCCLM3+SENP1 siRNA细胞中的表达分别较MHCC97-L(P=0.045)、HCCLM3(P=0.039)上调。结论SENP1 siRNA可通过上调XBP1的SUMO化修饰促进肝癌细胞的凋亡。

Objective To investigate the molecular mechanism of SUMO specific protease 1(SENP1)regulating endoplasmic reticulum stress transcription regulator X-box binding protein 1(XBP1)in the proliferation of liver cancer cells.Methods The pathological samples of 180 patients with primary liver cancer in the Department of Hepatobiliary Surgery of Inner Mongolia People's Hospital from January 2012 to January 2020 were collected.The expressions of SENP1 and XBP1 in liver cancer,adjacent tissues and different liver cancer cell lines were detected.The correlation between SENP1 positive expression and clinicopathological features of liver cancer patients was analyzed.Immunofluorescence and flow cytometry were used to detect the effect of SENP1 siRNA on XBP1 and apoptosis.SUMO1 expression on XBP1 surface was detected and the effect of SENP1 siRNA on SUMO formation of XBP1 was detected by immunoprecipitation.Results The expression levels of SENP1 in liver cancer and adjacent tissues were 16.332±4.371 and 6.840±2.238,with a statistically significant difference(t=-5.073,P=0.017).The expression levels of XBP1 in liver cancer and adjacent tissues were 6.641±2.482 and 16.051±4.452,with a statistically significant difference(t=3.592,P=0.032).The expression of SENP1 was correlated with stage(χ^2=6.724,P=0.010)and metastasis(χ^2=6.265,P=0.012).Immunofluorescence staining showed that the expressions of XBP1 in L02(0.509±0.219),MHCC97-L(0.092±0.022)and HCCLM3(0.086±0.014)cells were significantly different(F=6.378,P=0.004),while the expression of XBP1 in MHCC97-L and HCCLM3 cells was significantly lower than that in L02 cells(P=0.023;P=0.021).The expression levels of SENP1 in L02,MHCC97-L and HCCLM3 cells were 0.109±0.079,0.802±0.392 and 0.921±0.352,with a statistically significant difference(F=7.783,P=0.004),while the expression level of SENP1 in MHCC97-L and HCCLM3 cells was significantly higher than that in L02 cells(P=0.039;P=0.016).After transfection of SENP1 siRNA into MHCC97-L and HCCLM3 cells,the expressions of XBP1 increased(0.462±0.192,t=3.664,P=0.022;0.524±0.203,t=3.383,P=0.028);the expressions of SENP1 decreased(0.153±0.093,t=2.790,P=0.049;0.165±0.104,t=3.568,P=0.023).The results of flow cytometry showed that the apoptosis rates of L02,MHCC97-L,HCCLM3,MHCC97-L+SENP1 siRNA and HCCLM3+SENP1 siRNA cells were(20.80±3.11)%,(2.02±1.20)%,(0.12±0.01)%,(7.01±1.80)%,(6.20±2.01)%,with a statistically significant difference(F=1.025,P=0.030).The apoptosis rate of MHCC97-L and HCCLM3 cells was significantly lower than that of L02 cells(P=0.040;P=0.010),the apoptosis rate of MHCC97-L+SENP1 siRNA and HCCLM3+SENP1 siRNA cells was significantly higher than that of MHCC97-L and HCCLM3 cells(both P=0.009).Immunoprecipitation results showed that the expression levels of XBP1 in L02,MHCC97-L,HCCLM3,MHCC97-L+SENP1 siRNA,HCCLM3+SENP1 siRNA cells were 11.943±5.043,7.467±1.903,2.051±0.913,9.532±3.012,8.731±3.102,and SUMO1 expression levels were 10.158±4.005,5.871±3.075,1.941±0.907,8.658±4.878,7.169±4.677,and the differences were statistically significant(F=11.730,P=0.010;F=8.548,P=0.001).The expressions of XBP1 and SUMO1 in MHCC97-L(P=0.028;P=0.038)and HCCLM3(P<0.001;P<0.001)cells were lower than those in L02 cells,XBP1 expression in HCCLM3+SENP1 siRNA cells was higher than that in HCCLM3 cells(P=0.001),and SUMO1 expression in MHCC97-L+SENP1 siRNA cells and HCCLM3+SENP1 siRNA cells respectively was higher than that in MHCC97-L(P=0.045)and HCCLM3(P=0.039)cells.Conclusion SENP1 siRNA can promote the apoptosis of liver cancer cells by up regulating SUMO modification of XBP1.