摘要
目的通过小鼠肠癌类器官模型,探讨角蛋白23在结直肠癌中的作用及机制。方法从2023年6月至2023年12月,实验所用小鼠结肠癌类器官源自郑州大学医学科学院再生医学中心。采用慢病毒转染的方法构建KRT23基因的过表达和敲降细胞模型。将转染KRT23过表达载体的细胞命名为oeKRT23组,对应转染空载体PCDH的细胞为PCDH组;将转染KRT23特异性shRNA载体的细胞命名为shKRT23组,对应转染空载体PLKO的细胞为PLKO组。感染后分别采用实时定量反转录聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)验证基因表达效率,并进行类器官成形和增殖实验,观察KRT23对类器官形态和生长的影响。检测上皮-间充质转化(EMT)相关蛋白[E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、锌指蛋白(Snail)]及磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路相关蛋白[PI3K、Akt、磷酸化PI3K(p-PI3K)、磷酸化Akt(p-Akt)]的表达变化。组间比较使用t检验。结果RT-qPCR结果显示,shKRT23组KRT23 mRNA表达低于PLKO组(0.34±0.07比1.02±0.23,t=4.293,P<0.01),oeKRT23组高于PCDH组(19.40±0.50比1.00±0.04,t=63.291,P<0.01)。Western blot结果显示shKRT23组KRT23蛋白表达低于PLKO组(0.50±0.15比1.17±0.04,t=7.586,P<0.01),oeKRT23组高于PCDH组(0.83±0.08比0.52±0.04,t=5.944,P<0.01)。类器官成形实验结果显示,shKRT23组类器官成形数量低于PLKO组(21.56±3.25比28.78±1.18,t=3.617,P<0.05),而oeKRT23组高于PCDH组(41.67±7.04比28.44±4.25,t=2.786,P<0.05)。类器官增殖实验结果显示,shKRT23组类器官增殖期细胞百分比低于PLKO组(26.37±0.95比36.17±2.12,t=7.300,P<0.01),而oeKRT23组高于PCDH组(47.70±2.46比36.00±0.76,t=7.888,P<0.01)。Western blot结果显示,shKRT23组类器官的E-cadherin表达量(1.31±0.17比0.67±0.15,t=2.869,P<0.05)高于PLKO组,N-cadherin、Vimentin、Snail、p-Akt和p-PI3K蛋白表达量低于PLKO组(1.41±0.24比3.13±0.38、0.67±0.02比1.23±0.22、1.01±0.11比3.11±0.47、0.33±0.04比0.68±0.06、0.80±0.05比0.79±0.10,t=7.768、2.865、9.467、2.812、10.850,P<0.05),差异有统计学意义;oeKRT23组类器官的E-cadherin表达量(0.23±0.06比1.38±0.16,t=17.710,P<0.01)低于PCDH组,N-cadherin、Vimentin、Snail、p-Akt和p-PI3K蛋白表达量高于PCDH组(0.49±0.07比0.27±0.05、0.51±0.05比0.16±0.04、0.51±0.08比0.21±0.07、0.84±0.05比0.43±0.04、1.04±0.07比0.38±0.07,t=3.306、5.462、4.679、8.112、12.920,P<0.05),差异有统计学意义。结论KRT23能够磷酸化PI3K/Akt通路影响EMT过程,促进结直肠癌的进展。
Objective To investigate the role and mechanism of keratin 23 in colorectal cancer using a mouse intestinal cancer organoid model.Methods From June 2023 to December 2023,the murine colorectal cancer organoids used in the experiments were derived from the Center for Regenerative Medicine,Zhengzhou University Institute of Medical Sciences.Lentiviral transduction was employed to establish KRT23 gene overexpression and knockdown cell models.Cells transduced with the KRT23 overexpression vector were designated as the oeKRT23 group,with the corresponding empty vector control designated as thePCDH group.Cells transduced with KRT23-specific shRNA were designated as the shKRT23 group,with the corresponding empty vector control designated as the PLKO group.After infection,real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)and Western blotting were performed to verify gene expression levels.Organoid formation and proliferation were evaluated to assess the effects of KRT23.The expression of epithelial-mesenchymal transition(EMT)-related proteins(E-cadherin,N-cadherin,Vimentin,Snail)and phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt)pathway proteins(PI3K,Akt,phosphorylated-PI3K,and phosphorylated-Akt)was detected.Comparisons between groups were performed using the t-test.Results RT-qPCR results showed that KRT23 mRNA expression in the shKRT23 group was significantly lower than in the PLKO group(0.34±0.07 vs.1.02±0.23,t=4.293,P<0.01),while the oeKRT23 group exhibited significantly higher expression than the PCDH group(19.40±0.50 vs.1.00±0.04,t=63.291,P<0.01).Western blotting confirmed reduced KRT23 protein expression in the shKRT23 group compared to the PLKO group(0.50±0.15 vs.1.17±0.04,t=7.586,P<0.01)and elevated expression in the oeKRT23 group compared to the PCDH group(0.83±0.08 vs.0.52±0.04,t=5.944,P<0.01).Organoid formation assays revealed fewer formed organoids in the shKRT23 group than in the PLKO group(21.56±3.25 vs.28.78±1.18,t=3.617,P<0.05),whereas the oeKRT23 group showed increased formation(41.67±7.04 vs.28.44±4.25,t=2.786,P<0.05).The results of the organoid proliferation assay showed that the percentage of proliferating cells in the shKRT23 group was lower than that in the PLKO group(26.37±0.95 vs.36.17±2.12,t=7.300,P<0.01),while the percentage in the oeKRT23 group was higher than that in the PCDH group(47.70±2.46 vs.36.00±0.76,t=7.888,P<0.01).Western blotting analysis revealed that the expression level of E-cadherin in the shKRT23 group organoids was higher than that in the PLKO group(1.31±0.17 vs.0.67±0.15,t=2.869,P<0.05),while the expression levels of N-cadherin,Vimentin,Snail,phosphorylated Akt(p-Akt),and phosphorylated PI3K(p-PI3K)were significantly lower than those in the PLKO group(1.41±0.24 vs.3.13±0.38;0.67±0.02 vs.1.23±0.22;1.01±0.11 vs.3.11±0.47;0.33±0.04 vs.0.68±0.06;0.80±0.05 vs.0.79±0.10;t=7.768,2.865,9.467,2.812,10.850 respectively;P<0.05).In contrast,the E-cadherin expression level in the oeKRT23 group organoids was lower than that in the PCDH group(0.23±0.06 vs.1.38±0.16,t=17.710,P<0.01),while the expression levels of N-cadherin,Vimentin,Snail,p-Akt,and p-PI3K were significantly higher(0.49±0.07 vs.0.27±0.05;0.51±0.05 vs.0.16±0.04;0.51±0.08 vs.0.21±0.07;0.84±0.05 vs.0.43±0.04;1.04±0.07 vs.0.38±0.07;t=3.306,5.462,4.679,8.112,12.920 respectively;P<0.05).Conclusion KRT23 promotes colorectal cancer progression by phosphorylating the PI3K/Akt pathway and regulating the epithelial-mesenchymal transition process.
作者
夏坤锟
黑志军
尹少军
白耀文
李震
刘珍珍
Xia Kunkun;Hei Zhijun;Yin Shaojun;Bai Yaowen;Li Zhen;Liu Zhenzhen(Department of Colorectal and Anorectal Surgery,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;Department of Chinese Medicine,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)
出处
《中华实验外科杂志》
2025年第7期1306-1309,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(81903694)
河南省高层次人才国际化培养资助项目
河南省医学科技攻关计划省部共建重点项目(SBGJ 202103060)
河南省中青年卫生健康科技创新人才优青项目(YQRC2024009)
河南省教育厅高校重点项目(25A310021)。
