BLCAP arrests G1/S checkpoint and induces apoptosis through downregulation of pRb1 in HeLa cells

Zhao,Min; Zhang,Li; Qiu,Xiaoping; Zeng,Fanyu; Chen,Wen; An,Yuehui; Hu,Bicheng; Wu,Xufeng; Wu,Xinxing

doi:10.3892/or.2016.4686

BLCAP arrests G1/S checkpoint and induces apoptosis through downregulation of pRb1 in HeLa cells

Authors:
- Min Zhao
- Li Zhang
- Xiaoping Qiu
- Fanyu Zeng
- Wen Chen
- Yuehui An
- Bicheng Hu
- Xufeng Wu
- Xinxing Wu
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Affiliations: State Key Laboratory of Virology, Institute of Medical Virology, School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei 430071, P.R. China, Department of Gynecology, Hubei Maternity and Child Care Hospital, Wuhan, Hubei 430070, P.R. China
Published online on: March 17, 2016 https://doi.org/10.3892/or.2016.4686
Pages: 3050-3058

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Abstract

BLCAP (bladder cancer-associated protein) gene exhibited tumor suppressor function in different tumors and is regarded as a candidate tumor suppressor gene; however, the mechanism by which BLCAP exerts its function remains elusive. This study investigated the functional association between BLCAP and proliferation or apoptosis in cervical cancer cells, to identify the functional motifs of BLCAP. The BLCAP-shRNA expression vector based on pRNA-U6.1/Hygro plasmid was used to specifically inhibit BLCAP activity in HeLa cells. The optimal shRNA plasmid was selected to knock down BLCAP expression and the biological effects were investigated. The effects on cell cycle and apoptosis were detected by flow cytometric or Annexin V-FITC staining analysis. The gene expression profiles of HeLa cells transfected with blcap-wt and BLCAP-shRNA were analyzed using human signal pathway gene Oligochips. The levels of protein expression and interaction of BLCAP with Rb1 proteins were determined by western blotting and Co-IP assays. The site-specific mutagenesis assay was used to identify amino acid residues important for BLCAP. Significantly differentially expressed genes were found by gene Oligo chips analysis. These genes were all correlated with proliferation, cell cycle and apoptosis. The results of western blotting and Co-IP assays confirmed that overexpression of BLCAP could interact with Rb1 and inhibit Rb1 phosphorylation. Further investigation revealed that SAXX mutation in the key regions of BLCAP suppressed the function of BLCAP and significantly increased the level of phosphorylated Rb1 protein. Here our findings suggested that the functional association of BLCAP and Rb1 might play important roles in proliferation and apoptosis of HeLa cells. It suggested that BLCAP could be a novel therapeutic target for cervical cancer.

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