微小RNA-570-3p靶向TPX2爪蟾运动蛋白样蛋白2靶向蛋白调控肝胆管癌细胞凋亡的研究

MicroRNA-570-3p targeted targeting protein for Xenopus kinesin-like protein 2 to regulate apoptosis in hepatobiliary duct cancer cells

目的探讨微小RNA(miR)-570-3p调控肝胆管癌细胞凋亡的潜在机制。方法纳入2019年1月至2020年1月于山西省人民医院收治确诊为肝胆管癌患者30例,收集胆管癌患者癌组织样本(胆管癌组)和癌旁组织样本(癌旁组)。通过实时荧光定量聚合酶链反应(Real-time PCR)检测30例肝胆管癌患者癌组和癌旁组中miR-570-3p的表达量,同时过表达或敲低miR-570-3p后,Annexin染色测肝胆管癌细胞的凋亡。过表达或敲低miR-570-3p后,检测这些潜在底物的表达量过表达或敲低潜在底物,检测其对肝胆管癌细胞凋亡的影响。通过t检验分析组间差异。胆管癌组与癌旁组使用Mann-Whitney检验分基因表达量差异。结果胆管癌组中miR-570-3p的相对表达量低于癌旁组(0.76±0.14比1.91±0.34,U=42.123,P<0.05)。过表达miR-570-3p后,过表达组的肝胆管癌细胞RB3的凋亡水平高于对照组(0.79±0.20比0.21±0.05,t=4.873,P<0.05);敲低miR-570-3p后,敲低组中爪蟾运动蛋白样蛋白2靶向蛋白(TPX2)和磷脂酰肌醇特异性磷酯酶CB1(PLCB1)的表达水平均高于对照组(1.34±0.28比0.32±0.28,t=4.462,P<0.05;0.39±0.13比0.14±0.04,t=3.184,P<0.05),过表达miR-570-3p后,过表达组中TPX2和PLCB1的表达水平均低于对照组(1.34±0.28比2.06±0.33,t=2.882,P<0.05;0.39±0.13比1.43±0.39,t=4.382,P<0.05);敲低TPX2和PLCB1后,敲低TPX2组中肝胆管癌细胞RBE的凋亡水平高于对照组(0.72±0.13比0.21±0.05,t=6.342,P<0.05),而敲低PLCB1组中肝胆管癌细胞RBE的凋亡水平与对照组比较差异无统计学意义(0.23±0.04比0.21±0.05,t=0.541,P>0.05)。过表达TPX2后,过表达TPX2组中肝胆管癌细胞RBE的凋亡水平低于对照组(0.04±0.01比0.21±0.06,t=4.841,P<0.05);同时,胆管癌组中TPX2的相对表达量高于癌旁组(1.25±0.15比0.59±0.04,U=85.152,P<0.05)。结论 miR-570-3p靶向TPX2的3端非编码区,减少了TPX2的表达,最终增加了肝胆管癌细胞的凋亡水平。

Objective To investigate the potential mechanism of microRNA(miR-570-3p)in the regulation of apoptosis in hepatobiliary duct cancer cells.Methods Thirty patients diagnosed with hepatobiliary carcinoma admitted to Shanxi Provincial People′s Hospital from January 2019 to January 2020 were included,and cancer tissue samples(bile duct carcinoma group)and paracancerous tissue samples(paracancerous group)of patients with bile duct carcinoma were collected.The expression level of miR-570-3p in cancer group and paracancerous group of 30 patients with hepatobiliary carcinoma was detected by real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).Annexin staining was used to detect apoptosis of hepatobiliary carcinoma cells.After overexpression or knockdown of miR-570-3p,the expression levels of these potential substrates were detected to detect the effect of overexpression or knockdown of miR-570-3p on apoptosis of hepatobiliary carcinoma cells.SPSS 22.0 software was used for analysis.The differences between groups were analyzed by t test.Mann-Whitney was used to test the difference of subgene expression between the cholangiocarcinoma group and the paracancerous group.Results The relative expression level of miR-570-3p in the cholangiocarcinoma group was lower than that in the paracancerous group(0.76±0.14 vs.1.91±0.34,U=42.123,P<0.05).After the overexpression of miR-570-3p,the apoptosis level of RB3 of hepatobiliary carcinoma cells in the overexpression group was higher than that in the control group(0.79±0.20 vs.0.21±0.05,t=4.873,P<0.05).After knocking down miR-570-3p,targeting protein for Xenopus kinesin-like protein 2(TPX2)and Phospholipase C Beta 1(B1)in the knockdown group The expression level of PLCB1 was higher than that of control group(1.34±0.28 vs.0.32±0.28,t=4.462,P<0.05;0.39±0.13 vs.0.14±0.04,t=3.184,P<0.05).After overexpression of miR-570-3p,the expression levels of TPX2 and PLCB1 in the overexpression group were lower than those in the control group(1.34±0.28 vs.2.06±0.33,t=2.882,P<0.05.0.39±0.13 vs.1.43±0.39,t=4.382,P<0.05);After knockdown of TPX2 and PLCB1,the apoptosis level of RBE of hepatobiliary carcinoma cells in the knockdown of TPX2 group was higher than that in the control group(0.72±0.13 vs.0.21±0.05,t=6.342,P<0.05).There was no significant difference in RBE apoptosis level between PLCB1 knockdown group and control group(0.23±0.04 vs.0.21±0.05,t=0.541,P>0.05).After the overexpression of TPX2,it was found that the apoptosis level of RBE in the overexpression of TPX2 group was lower than that in the control group(0.04±0.01 vs.0.21±0.06,t=4.841,P<0.05).Meanwhile,the relative expression level of TPX2 in cholangiocarcinoma group was higher than that in paracancerous group(1.25±0.15 vs.0.59±0.04,U=85.152,P<0.05).Conclusion MiR-570-3p targets the 3-terminal non-coding region of TPX2,reduces the expression of TPX2,and ultimately increases the apoptosis level of hepatobiliary carcinoma cells.Therefore,miR-570-3p and TPX2 can be used as potential targets for the treatment of hepatobiliary carcinoma.