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

Abstract

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=4.673-9.563,P<0.05), and group D had significantly higher early and late apoptosis rates of SK-NEP-1 cells than groups B and C (t=5.829-8.075,P<0.05). Compared with group A, groups B, C, and D had a significantly higher proportion of SK-NEP-1 cells in G1 phase (t=7.518-14.747,P<0.05) and a significantly lower proportion of cells in S phase (t=8.029-13.451,P<0.05), and compared with groups B and C, group D had a significantly higher proportion of SK-NEP-1 cells in G1 phase (t=9.930,9.732,P<0.05) and a significantly lower proportion of cells in S phase (t=10.281,9.927,P<0.05). Conclusion Compared with the targeted regulation of PTEN or PI3K alone, the targeted regulation of PTEN and PI3K can significantly inhibit the growth of nephroblastoma cells and promote cell apoptosis, possibly by inhibiting the PI3K/AKT signaling pathway and blocking cell cycle.

Keywords

• wilms tumor|cell proliferation|apoptosis|cell cycle|pten phosphohydrolase|phosphatidylinositol 3-kinases