Jun Zhang1, Jing Xu1, Guoqing Wang1, Ping Sun1, Tao Yan1, Xixia Zhao2. 1. Department of Gynecology Oncology, Shaanxi Provincial Tumor Hospital, No. 309 Yanta West Road, Xi'an, 710061, Shaanxi, China. 2. Department of Gynecology Oncology, Shaanxi Provincial Tumor Hospital, No. 309 Yanta West Road, Xi'an, 710061, Shaanxi, China. zhaoxixia17986@126.com.
Abstract
AIM: Breast cancer 2, early onset (BRCA2) has been reported to be associated with familial breast and ovarian cancer. Several proteins interact with conserved regions of BRCA2, which play significant roles in DNA damage repair and centrosomal localization. This study was aimed to identify a novel protein, Wilms tumor 1 interacting protein (WTIP), which might interact with the conserved regions of BRCA2, as well as the functional role of silencing of WTIP in response to centrosomal localization. MATERIALS AND METHODS: Hela S3 cells were used in our study. A yeast two-hybrid screening was used to identify a novel BRCA2-interacting protein. Coimmunoprecipitation and glutathione S-transferase (GST) pull-down assays were performed to detect protein-protein interaction between BRCA2 and hemaglutinin (HA)-WTIP. The expression of WTIP was silenced by short hairpin RNA (shRNA) and the levels of WTIP were confirmed by Western blot. Immunofluorescence microscopy was performed to study the centrosome localization. The functional role of knocking down WTIP expression in response to centrosomal localization was then investigated. RESULTS: The results showed that there was an interaction between WTIP and BRCA2 (amino acids 2750-2864) in Hela S3 cells. We found that WTIP interacted with BRCA2 in both exogenous and endogenous level. The expression levels of WTIP were significantly decreased by siRNA compared to the control group. Downregulation of WTIP abolished BRCA2 centrosome localization and abnormal cell division. CONCLUSION: This study indicates that WTIP interacts with BRCA2 and might be responsible for BRCA2 centrosome localization in cervical cancer cell.
AIM: Breast cancer 2, early onset (BRCA2) has been reported to be associated with familial breast and ovarian cancer. Several proteins interact with conserved regions of BRCA2, which play significant roles in DNA damage repair and centrosomal localization. This study was aimed to identify a novel protein, Wilms tumor 1 interacting protein (WTIP), which might interact with the conserved regions of BRCA2, as well as the functional role of silencing of WTIP in response to centrosomal localization. MATERIALS AND METHODS: Hela S3 cells were used in our study. A yeast two-hybrid screening was used to identify a novel BRCA2-interacting protein. Coimmunoprecipitation and glutathione S-transferase (GST) pull-down assays were performed to detect protein-protein interaction between BRCA2 and hemaglutinin (HA)-WTIP. The expression of WTIP was silenced by short hairpin RNA (shRNA) and the levels of WTIP were confirmed by Western blot. Immunofluorescence microscopy was performed to study the centrosome localization. The functional role of knocking down WTIP expression in response to centrosomal localization was then investigated. RESULTS: The results showed that there was an interaction between WTIP and BRCA2 (amino acids 2750-2864) in Hela S3 cells. We found that WTIP interacted with BRCA2 in both exogenous and endogenous level. The expression levels of WTIP were significantly decreased by siRNA compared to the control group. Downregulation of WTIP abolished BRCA2 centrosome localization and abnormal cell division. CONCLUSION: This study indicates that WTIP interacts with BRCA2 and might be responsible for BRCA2 centrosome localization in cervical cancer cell.
Entities:
Keywords:
Breast cancer 2, early onset; Centrosome localization; Cervical cancer; Wilms tumor 1 interacting protein
Authors: Sethu M Madhavan; Martha Konieczkowski; Leslie A Bruggeman; Megan DeWalt; Jane K Nguyen; John F O'Toole; John R Sedor Journal: Am J Physiol Renal Physiol Date: 2022-07-21