Research Of C-Myb Upregulating Syncytin-1Expression To Be Involved In The Tumorigenesis Of Bladder Cancer By Binding To Mutated3’LTR

ObjectiveHuman endogenous retroviruses (HERV) as a potent "oncogene" element could be involved in tumorigenesis. In the present study, molecular biology and molecular modeling methods were used to investigate that c-Myb bound to mutated-LTR to upregulate the expression of the HERV-W env (syncytin-1) gene in bladder tumorigenesis. The machanism of HERV in bladder cancer was discussed from the perspective of tumor virology.Methods1.82samples of bladder cancer and matched adjacent bladder tissues were collected and human bladder cell lines (SV-HUC-1, T24and5637) were cultured. Using semi-quantitative RT-PCR, real-time quantitative RT-PCR, western blot and immunohistochemistry, we detected the expression of syncytin-1gene in bladder tumor tissues.2. Flow cytometry, MTT assay, colony formation assay and xenograft tumor assay were used for detecting the role of syncytin-1gene.3. The sequence of HERV-W3’LTR and5’LTR were indentified by PCR amplification and sequence alignment.4. Molecular cloning and PCR site-directed mutation techniques were used to construct several combination vectors. Luciferase asssy was used to detect the effect of3’LTR mutations on the syncytin-1promoter activity in human bladder cell lines (SV-HUC-1、 T24、5637).5. Transcription factors search program was used to predicte that potential transcription factors binding sites in the3’LTR. EMS A, ChIP and computer simulation were carried out to identifiy the interaction between the candidate proteins and3’LTR (normal and mutated). Meanwhile, computer simulation were used to prodict the key amio acids and nucleotides for the interaction between proteins and3’LTR. Next EMSA was used to identify the importance of these key amio acids and nucleotides.6. Co-transfection and luciferase assay were carried out to observe the role of candidate proteins (c-Myb and HOXA5) for the syncytin-1gene promoter activity when the3’LTR mutated.7. Semi-quantitative RT-PCR, real-time quantitative RT-PCR and Western-blot were used to detect the expression of syncytin-1gene with overexpression or lowexpression of the potential transcription factors.Results1. In all the82samples, high expression of syncytin-1was detected in75.6%of the bladder tumor samples, compared to6.1%of tumor adjacent tissues. Syncytin-1overexpression was associated with high stage and high grade of bladder cancer.2. Flow cytometry, MTT assay showed that syncytin-1gene promoted cell proliferation. Colony formation assay and xenograft tumor assay suggested that syncytin-1gene overexpression had oncogenic potential.3. No mutation in HERV-W5’LTR was found in all the bladder tumor tissues. However, two point mutations in3’LTR were found from87.8%of the bladder tumor tissues(P<0.05).3’LTR mutations were associated with high stage and high grade of bladder cancer. The overexpression of syncytin-1gene was significantly associated with3’LTR mutations in all bladder tumors (P<0.05)4. Mutations in3’LTR could increase syncytin-1expression and syncytin-1promoter activity in human bladder cell lines (SV-HUC-1、 T24.5637).5. Program transcription element search system results showed that the HOXA5-binding site was missing, while an additional c-Myb-binding site emerged following mutation of the142site. Electrophoretic mobility shift assay (EMSA), ChIP and molecular dynamics simulation results identified that c-Myb could bind to3’LTR instead of HOXA5when the mutations occurred. Luciferase assay results demonstrated that c-Myb and HOXA5could increase syncytin-1promoter activity after dual-point mutations happened.6. The computer simulation and EMSA results also suggested that c-Myb Glu132, c-Myb Lys182and HOXA5Arg197were key amino acids for interactions between c-Myb or HOXA5and the3’LTR. At the same time, EMSA confirmed that the first point mutation site (142T>C) in3’LTR was the important base for its interaction with transcription factors.7. Overexpression of c-Myb could upregulate the expression of syncytin-1gene. However, overexpression of HOXA5gene did not upregulate the expression of the syncytin-1gene. RNA interference experiments confirmed that knockdown of c-Myb could decrease the expression of the syncytin-1gene and knockdown of HOXA5could not effect syncytin-1expression significantly.Conclusion1. Our study revealed that the higher expression of syncytin-1and the mutations of HERV-W3’LTR were closely correlative with bladder cancer.3’LTR mutations could upregulate the expression of syncytin-1gene.2. Syncytin-1gene can promote cell proliferation and induce tumor formation. Transcription factor c-Myb increased the syncytin-1expression in bladder cancer by binding to mutated3’LTR, but not binding to normal3’LTR.3. Above all, these results demonstrated that c-Myb could upregulate the expression of syncytin-1to participate in the bladder tumorigenesis and development by binding to mutated3’LTR. Syncytin-1overexpression might be a potential indicator of bladder cancer risk. Our study would provide new ideas for the mechnism of HERV-W family in bladder cancer pathogenesis.