临床小儿外科杂志2024，Vol.23Issue(11)：1054-1061,8.DOI:10.3760/cma.j.cn101785-202305039-010
miR-20a-5p对人肾母细胞瘤增殖、迁移、侵袭及凋亡的影响
Study on the effect of miR-20a-5p on proliferation,migration,invasion,and apoptosis in human nephro-blastoma
摘要
Abstract
Objective miR-20a-5p on proliferation,migration,invasion and apoptosis of human Wilms tumor and its mechanism.Methods Human embryonic kidney cell line 293T and human Wilms tumor lung metastasis cell line WIT49 were cultured in vitro.miR-20a-5p mimics and Mir-20a-5p inhibitor genes were con-structed and transfected into WIT49 cells,respectively,and a control group was set up,namely miR-20a-5p mimics,NC mimics,miR-20a-5p inhibitors and NC inhibitors.The bioinformatics software TargetScan was used to predict that the target gene of miR-20a-5p might be Nuclear factor kappa-B inhibitor beta(NFKBIB),The human embryonic kidney cell line 293T was transfected with miR-20a-5p,negative control,NFKBIB wild type target plasmid with 3'UTR fragment and NFKBIB mutant target plasmid with 3'UTR fragment,respectively.Dual luciferase assay was used to compare the luciferase activity of miR-20a-5p overexpression wild type,overexpres-sion control wild type,miR-20a-5p overexpression mutant,and overexpression control mutant cells,and to verify that miR-20a-5p had a binding site with NFKBIB.real time fluorescent quantitative polymerase chain reaction(real time fluorescent quantitative polymerase chain reaction,qRT-PCR was used to detect the expression levels of miR-20a-5p and NFKBIB in 293T,WIT49 and WIT49 cells after transfection.Cell Counting Kit-8(CCK-8),wound healing assay,Transwell migration and invasion assay and flow cytometry apoptosis assay were used to detect the proliferation,migration,invasion and apoptosis inhibition ability of cells in each group.western blot(WB)was used to detect the expression of Nuclear factor kappa-B(Nf-κb)and nuclear factor Kappa-B(IκBβ)in 293T cells,WIT49 cells and WIT49 cells after transfection.NF-κB)p65 expression level.Results The results of dual luciferase assay showed that compared with the overexpression control wild type,the lucifer-ase activity of miR-20a-5p overexpression wild type was down-regulated[(0.44±0.39)vs.(1.01±0.06)],and the difference was statistically significant(P＜0.01);qRT-PCR results showed that the expression of miR-20a-5p in WIT49 was significantly higher than that in 293T[(1.97±0.79)vs.(1.01±0.20)],and the ex-pression of NFKBIB was lower than that in 293T[(0.26±0.08)vs.(1.02±0.26)],the expression of miR-20a-5p in the miR-20a-5p mimics was higher than that in the NC mimics[(2.30±0.97)vs.(1.02±0.23)],and the expression of NFKBIB was lower than that in the NC mimics[(0.53±0.20)vs.(1.01±0.17)],the expression of miR-20a-5p in the miR-20a-5p inhibitors was lower than that in the NC inhibitors[(0.45±0.14)vs.(1.01±0.15)],and the expression of NFKBIB in the miR-20a-5p inhibitors was higher than that in the NC inhibitors[(2.47±1.06)vs.(1.00±0.09)],and the differences were statistically significant(P＜0.01).The results of CCK-8 experiment showed that the optical density value of miR-20a-5p mimics at 24h(0.36±0.03)and 48h(1.76±0.03)was higher than that of NC mimics at 24h(0.18±0.01)and 48 h(0.87±0.04).The optical density value of miR-20a-5p inhibitors at 24h(0.04±0.05),48h(0.61±0.01),and 72h(1.80±0.03)was lower than that of NC inhibitors at 24h(0.11±0.01),48h(0.85±0.01),and 72h(2.72±0.06).The differences were statistically significant(P＜0.01).Scratch test results showed that the scratch healing rate of miR-20a-5p mimics(53.99±0.28)％was higher than that of NC mimics(34.87±0.08)％,and the scratch healing rate of miR-20a-5p inhibitors(16.56±0.09)％was low-er than that of NC inhibitors(34.74±0.15)％.The differences were statistically significant(P＜0.01).Tr-answell migration assay showed that the number of cells penetrating the microporous membrane in the miR-20a-5p mimics was(88.67±11.20),which was higher than that in the NC mimics(51.00±7.45).The number of cells penetrating the microporous membrane in the miR-20a-5p inhibitors was(23.67±7.59).Compared with the NC inhibitors(53.67±10.35),the differences were statistically significant(P＜0.01).Transwell in-vasion assay showed that the number of cells penetrating the microporous membrane in the miR-20a-5p mimics(64.00±18.75)was higher than that in the NC mimics(24.33±7.59),and the number of cells penetrating the microporous membrane in the miR-20a-5p inhibitors(3.33±1.44).Compared with NC inhibitors(25.00±4.30),the differences were statistically significant(P＜0.01).The results of apoptosis experiment showed that the percentage of apoptotic cells in the miR-20a-5p mimics(1.87±0.89)％was lower than that in the NC mimics(6.42±0.48)％,and the percentage of apoptotic cells in the miR-20a-5p inhibitors(11.33±0.91)％was higher than that in the NC inhibitors(6.07±0.58)％.The differences were statistically signifi-cant(P＜0.01).WB results The expression of IκBβ in 293T cells was higher than WIT49[(17.81±0.08)vs.(9.42±0.05)],and p65 expression was lower than WIT49[(1.54±0.01)vs.(2.02±0.02)].The ex-pression of IκB[3 in miR-20a-5p mimics was lower than that in over-expression control group[(6.45±0.01)vs.(9.35±0.02)],and the expression of p65 was higher than that in over-expression control group[(3.33±0.01)vs.(2.46±0.03)],The expression of IκBβ in miR-20a-5p inhibitors was higher than that in NC inhib-itors[(15.87±0.04 vs.9.32±0.05)],and the expression of p65 in miR-20a-5p inhibitors was lower than that in NC inhibitors[(1.82±0.02)vs.(2.46±0.02)],and the differences were statistically significant(P＜0.01).Conclusions miR-20a-5p may promote the proliferation,migration and invasion of human Wilms tumor and inhibit its apoptosis by regulating NFKBIB to activate the NF-κB pathway.关键词
微小RNA-20a-5p/肾母细胞瘤/核因子κB/外科手术/儿童Key words
miR-20a-5p/Wilms Tumor/NF-κB/Surgical Procedures,Operative/Child引用本文复制引用
王雅琦,李万富,阿依古再丽·麦麦江,艾尼娃·克然木,樊珈榕,刘辉,帕洛克·迪力木拉提..miR-20a-5p对人肾母细胞瘤增殖、迁移、侵袭及凋亡的影响[J].临床小儿外科杂志,2024,23(11):1054-1061,8.基金项目
自治区区域协同创新专项(科技援疆计划)(2020E0267) Regional Collaborative Innovation Special Fund(Science and Technology Aid Program for Xinjiang)(2020E0267) （科技援疆计划）
