羧基张力蛋白素对MHCC97肝癌细胞上皮-间充质转化和侵袭的影响

Effect of carboxytensin on epithelial mesenchymal transition and invasion of MHCC97 hepatoma cells

目的观察羧基张力蛋白素(CTEN)对肝癌细胞上皮-间充质转化和侵袭的影响。方法选取2017年6月到2020年1月郑州大学附属南阳市中心医院收集的120例肝癌组织和癌旁组织作为研究对象,采用蛋白质印迹法(Western blot)分析肝癌组织和癌旁组织中CTEN蛋白表达水平。采用慢病毒介导对照短发卡RNA(shRNA)和CTEN shRNA在肝癌细胞株MHCC97中构建CTEN敲低细胞株(CTEN KD组)和对照细胞株(对照组)。采用Transwell分析两组细胞的侵袭能力。采用Western blot分析两组细胞EMT标记分子表达水平。采用酶联免疫吸附试验(ELISA)分析两组基质金属蛋白酶(MMP)-9和MMP-2表达水平。计量数据比较采用t检验。结果与癌旁组织CTEN mRNA和蛋白表达水平(1.08±0.21、1.25±0.21)比较,肿瘤组织中CTEN mRNA和蛋白表达水平(3.14±0.37、2.74±0.19)显著增加,差异有统计学意义(t=3.447、2.149,P<0.05)。与对照组细胞CTEN mRNA和蛋白表达水平(1.00±0.17、1.12±0.16)比较,CTEN KD组细胞CTEN mRNA和蛋白表达水平(0.25±0.09、0.21±0.08)显著下降,差异有统计学意义(t=2.228、2.548,P<0.05)。与对照组细胞迁移数量[(125.29±21.05)个]比较,CTEN KD组细胞迁移数量[(51.28±5.39)个]显著下降,差异有统计学意义(t=3.849,P<0.05)。与对照组细胞上皮标志物E-钙黏蛋白(E-cadherin)(0.19±0.07)和间质标志物[波形蛋白(Vimentin)和Snail]蛋白表达水平(1.22±0.19、0.87±0.15)比较,CTEN KD组细胞E-cadherin表达水平(1.19±0.18)显著增加(t=4.012,P<0.05),而Vimentin和Snail蛋白表达水平(0.71±0.16、0.20±0.08)显著下调,差异有统计学意义(t=2.147、3.017,P<0.05)。与对照组细胞MMP-9和MMP-2蛋白表达水平[(78.69±5.99)、(60.82±4.09)μg/L]比较,CTEN KD组细胞MMP-9和MMP-2蛋白表达水平[(28.18±4.80)、(31.49±4.96)μg/L]显著下降,差异有统计学意义(t=2.912、2.612,P<0.05)。结论CTEN在肝癌组织中表达上调,参与肝癌细胞的迁移和上皮-间充质转化。

Objective To investigate the effect of carboxytensin(CTEN)on epithelial mesenchymal transition(EMT)and migration of hepatocellular carcinoma cells.Methods Totally,120 cases of liver cancer tissues and adjacent tissues in our hospital from June 2017 to January 2020 were selected as the research objects,and the expression level of CTEN protein in liver cancer tissues and adjacent tissues was analyzed by real time and Western blotting.CTEN knockdown cell lines(CTEN KD group)and control cell lines(control group)were established by lentivirus mediated control short hairpin RNA(shRNA)and CTEN shRNA.The invasion ability of the two groups were analyzed by Transwell.The expression levels of EMT markers were analyzed by Western blotting.Matrix metalloproteinase(MMP)-2 and MMP-9 expression level were analyzed by enzyme linked immunosorbent assay(ELISA).Results Compared with the expression levels of CTEN mRNA and protein in tumor tissues(1.08±0.21,1.25±0.21),the expression levels of CTEN mRNA and protein in tumor tissues(3.14±0.37,2.74±0.19)significantly increased(t=3.447,2.149,P<0.05).The expression of CTEN mRNA and protein(1.00±0.17,1.12±0.16)in control group significantly increased than that of CTEN KD group(0.25±0.09,0.21±0.08,t=2.228,2.548,P<0.05).Compared with the control group[(125.29±21.05)cells],the number of cell migration in CTEN KD group[(51.28±5.39)cells]significantly decreased(t=3.849,P<0.05).Compared with control group(0.19±0.07),the expression levels of E-cadherin in CTEN KD group(1.19±0.18)significantly increased(t=4.012,P<0.05).Compared with Vimentin and snail of control group(1.22±0.19,0.87±0.15),the expression levels of Vimentin and snail in CTEN KD group(0.71±0.16,0.20±0.08)significantly decreased(t=2.147,3.017,P<0.05).Compared with the control group[(78.69±5.99),(60.82±4.09)μg/L],the protein expression levels of MMP-9 and MMP-2 in CTEN KD Group[(28.18±4.80),(31.49±4.96)μg/L]significantly decreased(t=2.912,2.612,P<0.05).Conclusion The expression of CTEN up-regulated in HCC tissues,which is involved in the migration of HCC cells and EMT.