华支睾吸虫CsLegumain促胆管癌细胞迁移和侵袭的机制研究

Research on the mechanism of Clonorchis sinensis CsLegumain promoting the migration and invasion of cholangiocarcinoma cells

目的探讨华支睾吸虫CsLegumain在胆管癌细胞的增殖、侵袭和转移中的作用及其分子机制。方法构建真核表达质粒pcDNA3.1(+)-CsLegumain并转染胆管癌细胞,建立CsLegumain过表达细胞株。通过CCK8试验、细胞划痕试验、Matrigel Transwell试验分析CsLegumain在胆管癌细胞的增殖、侵袭和转移中的作用。通过实时荧光定量PCR及Western blot分别检测CsLegumain影响的靶分子转录水平和蛋白表达水平。结果成功构建CsLegumain稳定表达细胞株。CCK8试验显示,CsLeguamin对RBE细胞的增殖无显著影响(均P>0.05)。划痕实验中,过表达CsLegumain的RBE细胞其运动能力与对照组细胞相比显著增强,细胞间的距离明显变窄(均P<0.05)。Transwell试验显示,过表达CsLegumain的RBE细胞穿过基质小室的数量较对照组细胞数量多(P<0.05)。过表达CsLegumain组RBE细胞中E-cadherin和N-cadherin的转录水平及其蛋白表达水平均较对照组细胞显著降低(均P<0.05)。结论CsLeguamin对胆管癌细胞的增殖无显著影响,但具有促进胆管癌细胞迁移和侵袭能力,可能的机制是通过调控E-cadherin和N-cadherin的表达诱导EMT促进胆管癌细胞的迁移和侵袭。

Objective To investigate the role of Clonorchis sinensis CsLegumain in the proliferation,invasion and metastasis of cholangiocarcinoma and its molecular mechanism.Methods The plasmid pcDNA3.1(+)-CsLegumain was constructed and transfected into cholangiocarcinoma cells to establish a cell line with CsLegumain overexpression.The role of CsLegumain in the proliferation,invasion and metastasis of cholangiocarcinoma was investigated by CCK8 assay,scratch wound healing assay and matrigel transwell assay respectively.The transcription level and protein expression level of target molecules affected by CsLegumain were detected by real-time quantitative PCR and Western blotting,respectively.Results CsLegumain expression cell line was successfully constructed.The CCK8 assay showed that CsLeguamin had no significant influence on the proliferation of RBE cellsfrom 24 h to 72 h after culture(P>0.05)however enhanced the cells motor ability and narrowed the distance between cells significantly which detected by scratch wound healing assay 48 h later(P<0.05).Transwell experiments also showed that the number of RBE cells overexpressing CsLegumain passing through the stromal compartment was five times as many as the control group(P<0.05).The transcriptional and protein level of E-cadherin and N-cadherin in CsLegumain expressing cells were significantly lower than those in control cells detected by qRT-PCRand western blotting(P<0.05).Conclusion CsLeguamin had no significant influence on the proliferation of RBE cells but promoted the migration and invasion ability of them through downregulating the expression of E-cadherin and N-cadherin which might induce EMT.