Wang Yaqi,Li Wanfu,Ayiguzaili Maimaijiang,et al.Study on the effect of miR-20a-5p on proliferation,migration,invasion,and apoptosis in human nephroblastoma[J].Journal of Clinical Pediatric Surgery,2024,(11):1054-1061.[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
- Title:
- Study on the effect of miR-20a-5p on proliferation,migration,invasion,and apoptosis in human nephroblastoma
- 关键词:
- 微小RNA-20a-5p; 肾母细胞瘤; 核因子κB; 外科手术; 儿童
- Keywords:
- miR-20a-5p; Wilms Tumor; NF-κB; Surgical Procedures; Operative; Child
- 摘要:
- 目的 探讨miR-20a-5p对人肾母细胞瘤增殖、迁移、侵袭及凋亡的影响及作用机制。方法 体外培养人胚肾细胞系293T及人肾母细胞瘤肺转移灶细胞系WIT49;采用miR-20a-5p模拟物及其抑制基因构建慢病毒载体转染至WIT49细胞,并设立对照组(即miR-20a-5p过表达组、过表达对照组、miR-20a-5p低表达组、低表达对照组);采用生物信息学软件TargetScan预测miR-20a-5p的靶基因可能为核因子κB抑制因子β(nuclear factor kappa-B inhibitor beta,NFKBIB),分别将miR-20a-5p、阴性对照与携带3'UTR片段的NFKBIB野生型目的质粒、携带3'UTR片段的NFKBIB突变型目的质粒混合后转染至人胚肾细胞系293T,通过双荧光素酶实验比较miR-20a-5p过表达野生型、过表达对照野生型、miR-20a-5p过表达突变型、过表达对照突变型细胞荧光素酶活性,验证miR-20a-5p与NFKBIB的结合位点;采用实时荧光定量聚合酶链反应(real time fluorescent quantitative polymerase chain reaction,qRT-PCR)检测293T、WIT49及转染后各组WIT49细胞内miR-20a-5p及NFKBIB的表达水平;采用细胞计数试剂盒(Cell Counting Kit-8,CCK-8)、划痕愈合实验、Transwell迁移、侵袭实验及流式细胞凋亡实验检测各组细胞增殖、迁移、侵袭及凋亡抑制能力;采用蛋白质印迹法(western blot,WB)检测293T、WIT49及转染后各组WIT49细胞内核因子κB抑制蛋白β(I kappaB beta,IκBβ)及核因子κB(nuclear factor kappa-B,NF-κB) p65的表达水平。结果 与过表达对照野生型相比,miR-20a-5p过表达野生型荧光素酶活性下调;WIT49中miR-20a-5p表达显著高于293T,NFKBIB表达低于293T;miR-20a-5p过表达组中miR-20a-5p表达高于过表达对照组,NFKBIB表达低于过表达对照组;miR-20a-5p低表达组中miR-20a-5p表达低于低表达对照组,NFKBIB表达高于低表达对照组;miR-20a-5p过表达组光密度值24 h(0.36±0.03)、48 h(1.76±0.03),高于过表达对照组24 h(0.18±0.01)、48 h(0.87±0.04);miR-20a-5p低表达组光密度值24 h(0.04±0.05)、48 h(0.61±0.01)、72 h(1.80±0.03),低于低表达对照组24 h(0.11±0.01)、48 h(0.85±0.01)、72 h(2.72±0.06);miR-20a-5p过表达组划痕愈合率(53.99±0.28)％,高于过表达对照组(34.87±0.08)%;miR-20a-5p低表达组划痕愈合率(16.56±0.09)％,低于低表达对照组(34.74±0.15)%;Transwell迁移实验结果miR-20a-5p过表达组穿透微孔膜的细胞个数(88.67±11.20)高于过表达对照组(51.00±7.45),miR-20a-5p低表达组穿透微孔膜的细胞个数(23.67±7.59)低于低表达对照组(53.67±10.35);Transwell侵袭实验结果miR-20a-5p过表达组穿透微孔膜的细胞个数(64.00±18.75)高于过表达对照组(24.33±7.59);miR-20a-5p低表达组穿透微孔膜的细胞个数(3.33±1.44)低于低表达对照组(25.00±4.30);凋亡实验结果miR-20a-5p过表达组凋亡细胞(1.87±0.89)%,低于过表达对照组(6.42±0.48)％;miR-20a-5p低表达组凋亡细胞％(11.33±0.91)％,高于低表达对照组(6.07±0.58)％;293T中IκBβ表达高于WIT49,p65表达低于WIT49;miR-20a-5p过表达组IκBβ表达低于过表达对照组,p65表达高于过表达对照组;miR-20a-5p低表达组IκBβ表达高于低表达对照组,p65表达低于低表达对照组;上述指标差异均具有统计学意义(P＜0.01)。结论 miR-20a-5p可能是通过调控NFKBIB激活NF-κB通路促进人肾母细胞瘤增殖、迁移、侵袭,并抑制其凋亡。
- 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 constructed 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,overexpression 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 luciferase activity of miR-20a-5p overexpression wild type was down-regulated, 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,and the expression of NFKBIB was lower than that in 293T,the expression of miR-20a-5p in the miR-20a-5p mimics was higher than that in the NC mimics,and the expression of NFKBIB was lower than that in the NC mimics,the expression of miR-20a-5p in the miR-20a-5p inhibitors was lower than that in the NC inhibitors, and the expression of NFKBIB in the miR-20a-5p inhibitors was higher than that in the NC inhibitors,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 48h (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 lower than that of NC inhibitors (34.74±0.15) %.The differences were statistically significant (P<0.01).Transwell 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 invasion 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 significant (P<0.01).WB results The expression of IκBβ in 293T cells was higher than WIT49,and p65 expression was lower than WIT49.The expression of IκBβ in miR-20a-5p mimics was lower than that in over-expression control group,and the expression of p65 was higher than that in over-expression control group, The expression of IκBβ in miR-20a-5p inhibitors was higher than that in NC inhibitors,and the expression of p65 in miR-20a-5p inhibitors was lower than that in NC inhibitors, 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.
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备注/Memo
收稿日期:2023-5-23。
基金项目:自治区区域协同创新专项(科技援疆计划)(2020E0267)
通讯作者:李万富,Email:13699982169@163.com