Suppressive Roles And Mechanism For A Differentially Expressed Gene In Human Cervical Carcinoma

Cervical cancer is the major cancer among women's health hazards. Although the high-risk type of human papillomavirous infection is a significant risk factor leading to cervical cancer, the occurrence and development of cervical cancer is a multi-element, multi-step complex process like other tumors. The incidence of etiopathogenisis and concreted mechanisms of cervical cancer are not totally clear.Using gene chip screening, our previous study demonstrates that the bladder cancer-related protein gene (BLCAP) is one of the down-regulated expression genes in cervical carcinoma tissues. BLCAP is located at 20q11.2-q12 chromosomes, and encodes 87 amino acids. BLCAP gene was highly conservative in the evolution. It suggests that this gene may play an important role in the genome. However, the research on its functions and mechanisms is limited. In order to investigate the role of BLCAP in cervical carcinoma, and explore the possibility using it as a tumor-suppressor gene candidate, we stably transfected BLCAP gene into HeLa cell----one of the cervical cell lines, and study its suppressing function on tumor. Furthermore, we seek the possible mechanisms of down-regulated expression by methylation special PCR (MSP).In this study, we tested 54 cases cervical carcinoma tissues and 25 cases of normal cervical tissue from several hospitals. We found that BLCAP gene expression rate in normal cervical tissues is 100% (25/25), but in cervical specimens it obviously decreased, or even deficiencies, deficiencies rate of 40.7% (22/54);Using semi-quantitative RT-PCR, we analyzed the expression of BLCAP in cervical carcinoma tissues and the normal tissues, the semi-quantitative values were 0.207±0.265 and 0.650±0.113 respectively. BLCAP in cervical carcinoma tissues markedly lower than normal tissues (t=-7.985. P<0.01)。These results and previous studies done by the gene chips are identical. The study also found that the unusual expression of BLCAP is obviously related to pathological stage, lymph node infiltrated and metabasis of cervical cancer, but having nothing to do with age and HPV infection etc. (P>0.05)In order to further investigate the suppressor function of the BLCAP gene, we constructed the pCMV-Tag-BLCAP expression vector, after stably transfected the recombined vector into HeLa cell, through the cytomorphology observation, we found that non-transfected HeLa cells and transfected by vector grows well, but the growth of the HeLa cells transfected by BLCAP gene was obviously suppressed. The changes are very distinct when they were cultured for six days. The soft agar colony forms assay indicate that, the colony in soft agar of the HeLa cell transfected by BLCAP gene reduces in size and the number, the colony forming efficiency is 5.6±1.20%, (non-transferred and transfected by blank vector is 33.0±1.36% and 32.4±1.18% respectively); The apoptotic cell death was determined by fluorescent microscopic analysis of cell DNA staining patterns with Hoechst 33342: after transfected by BLCAP, the changes in HeLa cell morphology were characterized by typical chromatin condensation and nuclear fragmentation, with disruption of plasma membrane integrity.Flow cytometry analysis shows that HeLa cells transfected by BLCAP gene increase in the number of cells in the G1 phase, S phase shortened, and cell cycle obviously arrested.Inactivation of the tumor suppressor genes appears to be causal events for the majority of carcinomas. And unusually high methylation on gene regulation region is one of the important mechanisms for down-regulated or missing expression of gene. We use methylation specificity of PCR (MS-PCR) methods to explore the expression of BLCAP gene in the cells and tissues. 59 cases of cervical carcinoma tissues and 25 cases of cervical normal tissues were examined; BLCAP upstream CpG-rich areas and the exon CpG island methylation were studied. The results showed that the methylation extent of the upstream CpG-rich region in cancer tissues does not change significantly, compared with that of the normal cervical tissues, whereas methylation-positive rate is increased in the exon region CpG island, the methylation-positive rate of 71.2%, the methylation-positive rate of normal cervical tissues in this region was 0.0%, there are significant differences (P <0.01). Methylation in the exon CpG island is the possible mechanisms which induce expression of BLCAP to decrease.In this study, we explored the function and mechanism of BLCAP. One of reasons of BLCAP gene being suppressed was the abnormal hyper-methylation on CpG island of exon.Our study suggests that BLCAP gene is a potential tumor-associated suppressor gene of the cervical carcinoma.We will further investigate the pathway of this anti-tumor function of BLCAP.