膀胱尿路上皮癌细胞株E2F3基因与miR-17—5p和miR-20a的相互作用

The correlation of E2F3, miR-17-5p and miR-20a in transitional cell carcinoma of bladder

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E2F3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-17-5p、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值分别为2．26±0．30和4．044-0．51，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miR17—5P和miR-20a后，E2F3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±13．61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。

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-AACt 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 of miR-17-5p and miR-20a were 0.49 ±0.02 and 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 E2F3gene, 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.