赣南医科大学学报2025，Vol.45Issue(9)：830-836,7.DOI:10.3969/j.issn.1001-5779.2025.09.002
敲除PTEN基因对人胆管上皮细胞H69生物学功能的影响及机制初探
Biological effect of PTEN knockout on human cholangiocyte cell line H69 and its underlying mechanisms
摘要
Abstract
Objective:To establish phosphatase and tensin homolog(PTEN)gene stable knockout human cholangiocyte cell line H69 using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system,and to investigate the effect of PTEN knockout on altered cell biological behaviors and its underlying mechanisms.Methods:Firstly,sgRNA oligos targeting PTEN gene were designed.The constructed recombinant plasmids and empty vectors were respectively packaged with lentivirus,and then infected with H69 cells.Subsequently,PTEN stable knockout H69 cell line were constructed by screening using puromycin,and then the expression of PTEN protein was detected via western blot(WB)assay.The morphological changes and growth of H69 cells with PTEN knockout were observed,and cell proliferation ability was detected using cell proliferation assay and cell colony formation assay.Finally,the canonical downstream signal molecules of PTEN genes were explored and detected using WB.Results:WB analysis of PTEN protein knockout efficiency showed that PTEN protein expression was undetectable in single-cell clones infected with sgRNA 2 lentivirus,while partial PTEN protein expression was still present in single-cell clones infected with sgRNA 1 lentivirus,suggesting the successful construction of a stable PTEN genes knockout H69 cell line.The results of the colony formation assay indicated that the number of cell colonies in the PTEN-KO group was significantly increased compared with the WT group,with a statistically significant difference(P＜0.05).The MTS assay results showed that the proliferation ability of cells in the PTEN-KO group was significantly enhanced compared with the WT group,with a statistically significant difference(P＜0.05).Morphological observation of cells revealed that cells in the PTEN-KO group were more elongated and spindle-shaped,with fuller cytoplasm and partial pseudopodia formation compared with the WT group.Compared with the WT group,the expression levels of phosphorylated AKT proteins(including p-AKT 308 and p-AKT 473)in the PTEN-KO group were significantly increased,while the total AKT protein levels did not change significantly.The expression levels of phosphorylated ERK protein and total ERK protein did not show significant changes,suggesting that the knockout of the PTEN gene significantly activated the AKT signaling pathway but had no significant effect on the ERK signaling pathway.Compared with the WT group,the expression levels of N-cadherin,Vimentin,Snail,and Slug in the PTEN-KO group were significantly upregulated.Conclusion:PTEN loss can promote cell proliferation and trigger morphological changes,which may be related to the activation of AKT pathway and induction of EMT.关键词
PTEN磷酸水解酶/人胆管上皮细胞/细胞增殖/AKT信号通路/CRISPR/Cas9系统Key words
PTEN phosphohydrolase/Human biliary epithelial cells/Cell proliferation/AKT signaling pathway/CRISPR/Cas9 system分类
医药卫生引用本文复制引用
邵玉,余晖豪,周新瑞,刘静,杨燕..敲除PTEN基因对人胆管上皮细胞H69生物学功能的影响及机制初探[J].赣南医科大学学报,2025,45(9):830-836,7.基金项目
安徽省高校自然科学研究重大项目(2023AH040291) （2023AH040291）
安徽省高校自然科学研究重点项目(2024AH051288) （2024AH051288）
蚌埠医科大学自然科学重点项目(2023byzd077) （2023byzd077）
