靶向调控PTEN和PI3K对肾母细胞瘤细胞增殖和凋亡的影响  

Effect of targeted regulation of phosphatase and tensin homolog and phosphatidylinositol 3-kinase on the proliferation and apoptosis of nephroblastoma cells

作　　者：耿耿[1] 鹿洪亭[2] 李庆浩 明明 GENG Geng;LU Hongting;LI Qinghao;MING Ming(Department of Pediatric Surgery,The Affiliated Taian City Central Hospital of Qingdao University,Taian 271000,China)

机构地区：[1]青岛大学附属泰安市中心医院儿童外科,山东泰安271000 [2]青岛大学附属妇女儿童医院

出　　处：《精准医学杂志》2024年第2期114-119,共6页Journal of Precision Medicine

基　　金：中国高校产学研创新基金-北创助教项目(2021BCF01003)。

摘　　要：目的探讨靶向调控张力蛋白同源物(PTEN)和磷脂酰肌醇激酶(PI3K)对肾母细胞瘤细胞增殖和凋亡的影响及其机制。方法选取15例肾母细胞瘤患儿的术后肿瘤组织及癌旁正常组织,采用免疫印迹实验及实时荧光定量PCR方法检测组织中PTEN和PI3K蛋白及mRNA的表达水平;将肾母细胞瘤SK-NEP-1细胞分为对照组(A组,转染NC siRNA及Flag空载体)、PTEN过表达组(B组,转染NC siRNA及Flag-PTEN表达载体)、PI3K敲低组(C组,转染siPI3K及Flag空载体)、联合靶向组(D组,转染siPI3K及Flag-PTEN)。采用免疫印迹实验检测各组转染24 h后SK-NEP-1细胞中PI3K、蛋白激酶A(AKT)及磷酸化蛋白激酶A(p-AKT)的表达水平;采用CCK-8实验检测干预第24、48、72小时时SK-NEP-1细胞增殖情况;采用流式细胞术分析各组SK-NEP-1细胞转染24 h后细胞凋亡率及细胞周期。结果与癌旁正常组织相比,肾母细胞瘤肿瘤组织中PI3K蛋白及mRNA表达水平均显著升高(t=22.862、7.098,P<0.05),PTEN蛋白及mRNA表达水平均显著降低(t=25.634、8.379,P<0.05);体外细胞实验结果显示,B、C、D组SK-NEP-1细胞中p-AKT蛋白水平明显低于A组(t=8.386~11.900,P<0.05);CCK-8实验显示,干预第48、72小时时,B、C、D组SK-NEP-1细胞吸光度值明显低于A组(t=5.163~8.647,P<0.05),干预第72小时时,D组SK-NEP-1细胞吸光度值明显低于B、C组(t=3.982、4.021,P<0.05);B、C、D组SK-NEP-1细胞早期凋亡率和晚期凋亡率均明显高于A组(t=4.673~9.563,P<0.05),D组SK-NEP-1细胞早期凋亡率和晚期凋亡率明显高于B、C组(t=5.829~8.075,P<0.05);B、C、D组SK-NEP-1细胞G 1期细胞比例明显高于A组(t=7.518~14.747,P<0.05),S期细胞比例明显低于A组(t=8.029~13.451,P<0.05),D组SK-NEP-1细胞G 1期细胞比例明显高于B、C组(t=9.930、9.732,P<0.05),S期细胞比例明显低于B、C组(t=10.281、9.927,P<0.05)。结论相比于单一靶向PTEN或PI3K,联合靶向调控PTEN和PI3K可更显著抑制肾母细胞瘤细胞生长,促进肾母细胞�Objective To investigate the effect and mechanism of targeted regulation of phosphatase and tensin homolog(PTEN)and phosphatidylinositol 3-kinase(PI3K)on the proliferation and apoptosis of nephroblastoma cells.Methods Posto-perative tumor tissue and normal paracancerous tissue were collected from 15 children with nephroblastoma,and Western blotting and quantitative real-time PCR were used to measure the protein and mRNA expression levels of PTEN and PI3K in tissue.Nephroblastoma SK-NEP-1 cells were divided into control group(group A,transfected with NC siRNA and Flag empty vector),PTEN overexpression group(group B,transfected with NC siRNA and Flag-PTEN expression vector),PI3K knockdown group(group C,transfected siPI3K and Flag empty vector),a nd combined targeting group(group D,transfected siPI3K and FLAG-PTEN).Western blotting was used to measure the expression levels of PI3K,protein kinase A(AKT),and phosphorylated AKT(p-AKT)in SK-NEP-1 cells at 24 h after transfection;CCK-8 assay was used to observe the proliferation of SK-NEP-1 cells at 24,48,and 72 h of intervention;flow cytometry was used to observe apoptosis rate and cell cycle at 24 h after transfection.Results Compared with normal paracancerous tissue,nephroblastoma tumor tissue showed significant increases in the protein and mRNA expression levels of PI3K(t=22.862,7.098,P<0.05)and significant reductions in the protein and mRNA expression levels of PTEN(t=25.634,8.379,P<0.05).The results of in vitro cell experiments showed that compared with group A,groups B,C,and D had a significantly lower protein expression level of p-AKT(t=8.386-11.900,P<0.05).CCK-8 assay showed that groups B,C,and D had a significantly lower absorbance value of SK-NEP-1 cells than group A at 48 and 72 h of intervention(t=5.163-8.647,P<0.05),and at 72 h of intervention,group D had a significantly lower absorbance value of SK-NEP-1 cells than groups B and C(t=3.982,4.021,P<0.05).Groups B,C,and D had significantly higher early and late apoptosis rates of SK-NEP-1 cells than group A(t=

关 键 词：WILMS瘤 细胞增殖 细胞凋亡 细胞周期 PTEN磷酸水解酶 磷酸肌醇3-激酶类 

分 类 号：R737.11[医药卫生—肿瘤]