gms | German Medical Science

Introduction: The truncated version of the full length Vimentin, Vim3, has, due to the extra c-terminal ending a different function. Vim3 is a possible biomarker for the detection of prostate cancer. Furthermore, increased Vim3 levels could also be analyzed in urine samples from bladder patients. Here, the involvement of Vim3 in target gene expression was analyzed.

Materials and Methods: It was possible to detect Vim3 via Western blot in nuclear extracts of testicular cancer cells (TCam2), prostate cancer cells (BPH, LNCap, PC3, and DU145) as well as in urothelial cancer cells (HTB-9). Due to the extra Vim3 c-terminal ending a hypothetical DNA interaction site was analyzed.

Pull-down assays with Vim3 agarose conjugated antibodies were performed, RNA and DNA was isolated and specific PCRs were performed. A miR-371a-3p specific non-radioactive electromobility shift assay was established. Vim3 siRNA experiments were established in all used cell lines and possible targets were analyzed via Western Blot and PCR.

Results: Performing pull-downs with Vim3 agarose conjugated antibodies, 13 wnt-pathway involved genes could be identified interacting with Vim3 on DNA level. Furthermore, the hypothetical interaction site was identified within the promoter region of the miR-371a-3p and MAPK p38. In a non-radioactive electromobility shift assay a clear shift was identified for the miR-371a-3p promoter region, indicating that Vim3 is important for the activation of the transcription. For the identification of the hypothetical DNA interacting sites on the MAPK p38 promoter, specific primers were designed and a specific Vim3 pull-down assay was performed, followed by PCR. In cell culture experiments the direct involvement of Vim3 was analyzed via siRNA against Vim3 and the determination of the possible target genes.

Conclusion: Concluding this, Vim3 could be a possible new therapeutic target for drug development.