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.

中文翻译：

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BLCAP阻止G₁/ S检查点，并通过下调HeLa细胞中pRb1的表达诱导凋亡。

BLCAP（膀胱癌相关蛋白）基因在不同的肿瘤中具有抑癌功能，被认为是候选的抑癌基因。但是，BLCAP发挥其功能的机制仍然难以捉摸。这项研究调查了BLCAP与宫颈癌细胞增殖或凋亡之间的功能关联，以鉴定BLCAP的功能基序。基于pRNA-U6.1 / Hygro质粒的BLCAP-shRNA表达载体被用于特异性抑制HeLa细胞中的BLCAP活性。选择了最佳shRNA质粒来敲低BLCAP的表达，并对其生物学效应进行了研究。通过流式细胞术或膜联蛋白V-FITC染色分析来检测对细胞周期和细胞凋亡的影响。使用人类信号通路基因Oligochips分析了blcap-wt和BLCAP-shRNA转染的HeLa细胞的基因表达谱。蛋白质表达水平和BLCAP与Rb1蛋白质的相互作用是通过蛋白质印迹和Co-IP分析确定的。该位点特异性诱变测定用于鉴定对BLCAP重要的氨基酸残基。通过基因寡核苷酸芯片分析发现显着差异表达的基因。这些基因都与增殖，细胞周期和细胞凋亡相关。Western blotting和Co-IP分析的结果证实BLCAP的过表达可以与Rb1相互作用并抑制Rb1磷酸化。进一步的研究表明，BLCAP关键区域的SAXX突变抑制了BLCAP的功能，并显着增加了磷酸化Rb1蛋白的水平。在这里，我们的发现表明BLCAP和Rb1的功能结合可能在HeLa细胞的增殖和凋亡中起重要作用。提示BLCAP可能是宫颈癌的新型治疗靶点。