PCK1影响肝癌细胞迁移能力的研究

Effect of PCK1 on migration of hepatocarcinoma cells

摘要:

目的：构建编码磷酸烯醇丙酮酸羧化激酶（phosphoenolpyruvate carboxykinase，PEPCK）的基因PCK1重组腺病毒和基因敲除2种细胞模型，并初步观察PCK1过表达和敲除后对肝癌细胞迁移能力的影响。方法：PCK1 cDNA克隆到pAdTrack-TO4载体，构建重组腺病毒质粒，在HEK293细胞中包装、扩增成高滴度重组腺病毒AdPCK1；利用CRISPR/Cas9系统在PLC/PRF/5肝癌细胞系中筛选出PCK1基因敲除的稳定细胞株。首先，在过表达模型将Huh7细胞设为2组：AdGFP组和AdPCK1组，AdGFP组为感染腺病毒AdGFP的对照组，AdPCK1组为感染腺病毒AdPCK1的实验组。在敲除模型设Parental组和KO组，Parental组是PLC/PRF/5细胞作为对照组，KO组是PCK1基因敲除稳定细胞株作为实验组。Western blot技术检测2种模型蛋白表达量，采用划痕实验观察细胞迁移能力，进一步经qRT-PCR检测影响肝癌细胞的迁移关键分子。结果：重组腺病毒AdPCK1构建成功，经Western blot鉴定PCK1在Huh7细胞中的表达。向导RNA（single guide RNA，sgRNA）寡核苷酸双链成功插入酶切后的LentiCRISPR-V2质粒载体中且测序正确；经Western blot鉴定PCK1敲除的细胞株筛选成功。划痕结果显示，PCK1过表达模型在48 h的细胞迁移率为（66.300±0.383）%，与AdGFP组[（42.900±3.833）%]相比明显增加（t=10.540，P=0.000）；PCK1敲除KO组在48 h的细胞迁移率为（59.40±5.68）%，与亲本细胞组[（79.00±5.20）%]相比明显减少（t=4.420，P=0.012）。qRT-PCR结果显示，PCK1过表达后，相较于对照组AdGFP，Cdh1相对表达量为2.733±0.501（t=5.989，P=0.004），相对表达增高；PCK1敲除后，相较于亲本细胞组，Cdh1相对表达量为0.664±0.017（t=34.290，P=0.000），相对表达减少。结论：PCK1过表达后可明显抑制肝癌细胞的迁移能力，而敲除后促进其迁移，证实PCK1可能作为抑癌基因参与肝癌的发生和发展。

Abstract:

Objective：To construct overexperssion and knockout model of phosphoenolpyruvate carboxykinase1（PCK1） gene and to in－vestigate the effects of PCK1 on migration of hepatocarinoma cell. Methods：The cDNA of PCK1was cloned into plasmid pAdTrack-TO4. Recombinant adenovirus plasmid pAdTrack-PCK1 was transfected into HEK293 cells to package and generate high titer recom－binant adenoviruses encoding PCK1（AdPCK1）. The PCK1 knockout cell lines using CRISPR/Case9 system were selected in PLC/PRF/5 cells. First，the Huh7 cells were set into two groups in the overexpression model，one of which was AdGFP group as control group infected with adenovirus AdGFP，and the other of which was AdPCK1 group as experimental group infected with adenovirus AdPCK1. The knockdown model included two groups，one of which was Parental group as control group using PLC/PRF/5 cells，and the other of which was KO group as experimental group using PCK1 knockout cells. Two models were confirmed by Western blot. The effect on the migration of hepatocarcinoma cells was observed by wound assay. Further，Cdh1 were analyzed by qRT-PCR. Results：AdPCK1 was successfully constructed and Western blot showed PCK1 overexpression in AdPCK1 infected cells. Western blot showed that PLC/PRF/5 cell lines with PCK1 knockout were successfully estab－lished by the usage of CRISPR/Cas9 system. Wound assay showed PCK1 significantly inhibited the migration of hepato－carcinoma cells compared with control group. The wound clo－sure rate of AdPCK1 group was （66.300±0.383）% compared with that of （42.900±3.833）% in AdGFP control（t=10.540，P=0.000）. The wound closure rate of KO group was （59.40±5.68）% compared with that of （79.00±5.20）% in parental cell control（t=4.420，P=0.012）. In addition，qRT-PCR showed that the expressions of Cdh1 genes in EMT signal pathway were signifi－cantly inhibited by the gene of PCK1 than control. qRT-PCR showed that AdPCK1 compared with those of AdGFP control group，the relative expression of Cdh1 was 2.733±0.501（t=5.996，P=0.004），and KO group compared with those of parental cell，the relative expression of Cdh1 was 0.664±0.017（t=34.290，P=0.000）. Conclusion：PCK1 inhibited the migration of hepatocarcinoma cells through regulating the expression of Cdh1 genes in EMT pathway，suggesting that PCK1 may be involved in the initiation and progress of hepatocarcinoma.