BLCAP基因及编码蛋白在宫颈癌发病机制中的作用

Bladder cancer associated protein (BLCAP) is a potential antioncogene involved in several cellular processes, including proliferation, apoptosis and RNA editing. Deregulating (Lost/down regulate) of BLCAP expression is reported to be a critical phenomenon in the development of human tumors, including cervical cancer, invasive bladder cancer and osteosarcoma. The former works of our group have confirmed that this gene is a significant difference expressed in normal cervical tissue and cervical cancer, and more over it is likely to be a potential antioncogene for cervical carcinoma. And it regulated cell proliferation and apoptosis in a new way which is independent of P53 or NF-κB. Objective: To clear the role that BLCAP play in the signal transduction of cell cycle and determine the target molecules which could interact with, then illustrate the molecular mechanisms of BLCAP as a tumor suppressor gene. Furthermore, to explore BLCAP as a possibly new therapeutic target of clinical oncology, provided the experimental evidences for the development of cervical carcinoma. Methods: 1. Analysis three types of the HeLa cell which were wild type, transfected BLCAP gene and silenced BLCAP gene after transfection by microchip . 2. Co-Immunoprecipitation and Immunoblotting were applied to detect the interaction between BLCAP and other biological macromolecules, and even identify which molecule it is. Results: 1. We designed two groups to do the microchip analysis. One is wild type and BLCAP transfected HeLa cell, the other is BLCAP transfected HeLa cell and silenced BLCAP gene in HeLa cell after transfection. Compared these two groups, we got 27 genes up-regulated in both group. CCNB1, CDC2, SMAD4, RB1, PRKAR1A, CCNH and PCNA, the expression of these 7 genes were significantly changed. After analysis by Bioinformatics, we found they belong to the pathway of HS_G1_to_S_Cell_cycle_R. 2. RT-PCR results showed that BLCAP over expressed at mRNA level, coincident, the RB1's mRNA level got significantly improved. Western Blot confirmed, at protein level, RB1's were also higher than control, while BLCAP's over expression, but there is no significant difference in the level of phosphorylated RB protein. 3. Co-IP and IB identified that BLCAP could combined with RB1, so as pRB, in physiological. Conclusion: The study showed that over-expression of BLCAP can up-regulate the RB1, which indicated, BLCAP maybe participate in the regulation of cell cycle via this pathway. All the results suggested that BLCAP most likely inhibit the cell getting through the G1/S checkpoint by effecting the RB1, which performed its function to inhibit cell growth and induce apoptosis. These were proved by co-IP and IB, which following show us that BLCAP can bind with both RB and pRB. According the above, we deduct that BLCAP most likely is involved in the Rb pathway, via which to regulate the cell cycle, even by which to function as antioncogene. Meanwhile, we also got purified BLCAP fusion protein, which will be applied in subsequent works to correspond with our deduction.