The Role Of Regulated Loop E2F3/miR-17-5p/miR-20a In Transitional Cell Carcinoma Of Bladder

Objective:The mir-17-92cluster has been implicated in tumorigenesis, the microRNAs (miRNAs) from the cluster can function as tumour suppressors or oncogenes depends on the tissular and cellular contexts. Also it is known that E2F3gene acts as an oncogene of bladder cancer. The promoter of the mir-17-92cluster possibly contains E2F transcription factor binding sites. Therefore we postulate that E2F3as the primary E2F family member, may occupies the promoter in the biogenesis of bladder cancer. Although the role of mir-17-92cluster in bladder cancer is not clear, the miRNAs often regulate the expression of their downstream factors via a tempo-spatial pattern. In this study we propose to explore the correlation of the expression levels of E2F3, miR-17-5p and miR-20a in bladder cancer tissue samples. And we attempt to decipher whether there is an autoregulatory feedback loop of E2F3/miR-17-5p/miR-20a in human bladder cancer. Vrification of such a regulatory loop may present us a treatment target or some potential molecular drugs of the bladder cancer.Methods and materials:A total of3parts were included in the study. Part1:To collect fresh clinical tissues specimens from bladder cancer patients (transitional cell carcinoma, TCC) who were performed radical or partial cystectomy and the bladder cancer cell lines5637, T24cell lines were included in the study. Using TRIzol reagent, we isolate total RNA from tissue samples and cell lines. Real-time fluorescent quantitative reverse transcript polymerase chain reaction (qRT-PCR) was used to assay the miRs and E2F3changes in tissues and cell lines, and E2F3protein were detected by Western blot. Part2:The plasmid of pcDNA3.1-HA-E2F3and pAAV-siRNA-E2F3were used to enforce overexpression or knockdown of E2F3. qRT-PCR was used to detect the miRs and E2F3changes in cell lines and E2F3protein were detected by Western blot, after transfection of the vector above. The growth curve of5637cell line was drawed according to the MTT data after transfection; Part3:The mimics of miR-17-5p, miR-20a and their anti-miRNA oligonucleotides (AMO) were selected to overexpression or knockdown of miR-17-5p and miR-20a. The expression levels of E2F3cell lines were measured by qRTPCR, and E2F3protein were detected by Western blot after transfection. The proliferation of5637cell line after transfection was analyzed by MTT method.Results:miR-17-5p, miR-20a, E2F3gene and E2F3protein were significantly amplification in the specimens from bladder cancer (P<0.05), and with advanced tumor grade, E2F3gene and E2F3protein showed increasing. The5637cells showed significantly high expression of E2F3compared with T24cells (P<0.05). When transfected pcDNA3.1-HA-E2F3, the E2F3protein were significantly increased and the miR-17-5p and miR-20a were significantly increased; transfected pAAV-siRNA-E2F3, the E2F3protein were significantly decreased, and the miR-17-5p and miR-20a were significantly decreased.The E2F3protein were significantly decreased when transfected miR-17-5p, miR-20a, whereas The E2F3protein was significantly increased when transfected the AMO of which miRNA member.Conclusions:E2F3and E2F3protein level could be down-regulated by miR-17-5p and miR-20a, whereas enforced overexpression of E2F3has been shown to activate transcription of miR-17-5p and miR-20a. The autoregulatory feedback loop of E2F3/miR-17-5p/miR-20a exists in human bladder cancer. E2F3/miR-17-5p/miR-20a constraint to each other and coordinate the growth of bladder tumor.