CAJ | 학술논문

目的研究E2F3基因、miR-17-5p和miR-20a在膀胱尿路上皮癌细胞株中的表达及相互影响,探讨E2 F3基因与miR-17-5p和miR-20a在膀胱癌发生中的作用. 方法用pcDNA3.1-HA-E2F3和pAAV-siRNA-E2F3质粒在膀胱癌细胞株5637中分别过表达和敲低E2F3基因,用miR-17-5p和miR-20a的模拟物和反义寡核苷分别过表达和敲低miR-17-5p和miR-20a后,实时荧光定量PCR法检测miR-17-5 p、miR-20a和E2F3基因的变化,蛋白质印迹法检测E2F3蛋白水平的变化.结果 E2F3基因过表达后,miR-17-5p和miR-20a的2-△ △Ct值分别为2.26±0.30和4.04 ±0.51,与对照组(1.00±0.00)比较差异有统计学意义(P＜0.05)；敲低E2F3后,miR-17-5p和miR-20a的2-△△Ct值分别为0.49±0.02和0.65 ±0.04,与对照组(1.00±0.00)比较差异有统计学意义(P＜0.05)；同时过表达miR-17-5p和miR-20a,E2F3的mRNA水平明显下调,蛋白质平均灰度值为55.31 ±7.89,对照组为103.67±13.61,与对照组比较差异有统计学意义(P＜0.05)；同时敲低miR-17-5p和miR-20a后,E2 F3的mRNA比对照显著上调,蛋白质平均灰度值为295.68±19.25,与对照组平均灰度值为103.67±13.6l比较差异有统计学意义(P＜0.05). 结论 E2F3基因可上调miR-17-5p和miR-20a的表达,miR-17-5p和miR-20a可以下调E2F3基因的表达.E2F3基因、miR-17-5p和miR-20a在膀胱尿路上皮癌中形成调节环路,协调膀胱肿瘤细胞的生长.
목적 연구E2F3기인、miR-17-5p화miR-20a재방광뇨로상피암세포주중적표체급상호영향,탐토E2 F3기인여miR-17-5p화miR-20a재방광암발생중적작용. 방법 용pcDNA3.1-HA-E2F3화pAAV-siRNA-E2F3질립재방광암세포주5637중분별과표체화고저E2F3기인,용miR-17-5p화miR-20a적모의물화반의과핵감분별과표체화고저miR-17-5p화miR-20a후,실시형광정량PCR법검측miR-17-5 p、miR-20a화E2F3기인적변화,단백질인적법검측E2F3단백수평적변화.결과 E2F3기인과표체후,miR-17-5p화miR-20a적2-△ △Ct치분별위2.26±0.30화4.04 ±0.51,여대조조(1.00±0.00)비교차이유통계학의의(P＜0.05)；고저E2F3후,miR-17-5p화miR-20a적2-△△Ct치분별위0.49±0.02화0.65 ±0.04,여대조조(1.00±0.00)비교차이유통계학의의(P＜0.05)；동시과표체miR-17-5p화miR-20a,E2F3적mRNA수평명현하조,단백질평균회도치위55.31 ±7.89,대조조위103.67±13.61,여대조조비교차이유통계학의의(P＜0.05)；동시고저miR-17-5p화miR-20a후,E2 F3적mRNA비대조현저상조,단백질평균회도치위295.68±19.25,여대조조평균회도치위103.67±13.6l비교차이유통계학의의(P＜0.05). 결론 E2F3기인가상조miR-17-5p화miR-20a적표체,miR-17-5p화miR-20a가이하조E2F3기인적표체.E2F3기인、miR-17-5p화miR-20a재방광뇨로상피암중형성조절배로,협조방광종류세포적생장.
Objective To explore the correlation and role of E2F3 gene,miR-17-5p and miR-20a in the cell lines of transitional cell carcinoma of bladder. Methods The plasmids of pcDNA3.1-HA-E2F3 and pAAV-siRNA-E2F3 were used to overexpress and knockdown E2F3.The mimics of miR-17-5p,miR-20a and their anti-miRNA oligonucleotides were used to overexpress and screen miR-17-5p and miR-20a.The expression levels of E2F3 gene,miR-17-5p and miR-20a were detected by quantitative real-time PCR,and E2F3 protein were detected by Western blot. Results When E2F3 was overexpressed,the 2- △△Ct of miR-17-5p and miR-20a were 2.26 ± 0.30 and 4.04 ± 0.51,it was statistically significant to compared with control (P ＜ 0.05) ; when E2F3 was knockdown,the 2 △△Ct of miR-17-5p and miR-20a were 0.49 ± 0.02and 0.65 ± 0.04 (P ＜ 0.05) ; when miR-17-5p and miR-20a were overexpressed simultaneously,the level of E2F3 mRNA was significantly decreased,the average E2F3 protein gray scale was 55.31 ± 7.89,the control was 103.67 ± 13.61 (P ＜ 0.05 ) ; when miR-17-5p and miR-20a were knockdown simultaneously,the E2F3 mRNA was significantly increased,the E2F3 protein gray scale was 295.68 ± 19.25,the control was 103.67 ± 13.61 ( P ＜ 0.05 ). Conclusions miR-17-5p and miR-20a could be up-regulated by E2F3 gene,and the E2F3 gene could be down-regulated by miR-17-5p and miR-20a.The regulatory feedback loop of E2F3 gene,miR-17-5p and miR-20a exists in transitional cell carcinoma of bladder. The loop maybe plays a key role in the development of bladder cancer.