The Experimental Study Of Relationship Between Gene Beclin 1 And Cervical Cancer

Background & Objective Cervical cancer is regarded as one of the mostcommon cancers in the world, accounting for approximately 500 000 newcases per year, and almost 80% of cases occur in developing countries.Despite all therapeutic efforts, the five-year survival of the advanced-stagecases is almost 50%. Biochemotherapy is an important method in the therapyof carcinoma, and the inducement of apoptosis is its main means, whileautophagic cell death may provide a new target.Programmed cell death (PCD) is an essential and highly orchestratedprocess that plays a major role in morphogenesis and tissue homeostasisduring development. Two major types of PCD have been distinguished:apoptosis and autophagy. Autophagy is the bulk degradation of proteins andorganelles, a process essential for cellular maintenance, cell viability,differentiation and development in mammals, and may be defective in tumourcells. Autophagy in mammalian cells is important both for cellularremodeling during differentiation and for the negative regulation of tumorcell growth.The mammalian gene encoding Beclin 1, a novel Bcl-2-interactingprotein, has structural similarity to the yeast autophagy gene, apg6/vps30, and is mono-allelically deleted in 40-75% of sporadic human breast cancers andovarian cancers. Beclin 1 is a mammalian autophagy gene that can inhibittumorigenesis and is a candidate tumor suppressor. In the study,immunohistochemistry was employed to determine the expression of Beclin1 and explored its clinical significance in cervical cell carcinoma. Weconstructed the eukaryotic expression vector pcDNA3.1(+)-Beclinl andshRNA expression vector pSUPER-Beclinl, transfected into HeLa cells, tofred the effect of Beclin 1 overexpression and downexpression on thegrowth, autophagy and apoptosis of cervical cancer cell line HeLa in vitroand vivo, and explore the feasibility of induce of autophagy in treatment ofcervical carcinoma.Methods1. The expression of Beclin 1 was detected with SP immunohistochemistryin specimens of cervical cell carcinoma(n=81), cervical intraeoithelialneoplasm (n=20), and normal cervix (n=20). Correlations of expressionof Beclin 1 gene to clinicopathologic factors of cervical carcinoma werestatistically analyzed.2. The eukaryotic expression vector pcDNA3.1(+)-Beclinl and shRNAexpression vectors pSUPER-Beclinl were constructed.3. The vectors were transfected into HeLa cells via lipofectamine. Theexpression levels of Beclin 1 and caspase-9 mRNA and protein weredetected by real-time RT-PCR, western blot analysis in transfected cells.The cell cycles, autophagy, apoptosis and cell proliferations of HeLacells were measured with flow cytometry and MTT method aftertransfection. The ultrastructural analysis was under the electron microscope, and the formation of autophagic vacuoles was measured byMDC dyeing.4. Human cervical cancer HeLa cell was transfected by vectorpcDNA3.1-Beclinl and pSUPER-Beclinl, and was seededhypopercutaneously on nude mice. The in vivo carcinogenic and growthactivities of cancer cell were observed, and Beclinl protein expression intumor tissues was detected by immunohistochemisty.Results1. Beclinl protein expression in tumor cells were down-regulation incervical invasive carcinoma, respectively, significantly lower than thosein normal cervix, and cervical intraeoithelial neoplasm (P＜0.05), thereno significant difference between the normal cervix, and cervicalintraeoithelial neoplasm (P＞0.05).2. Expression of Beclin 1 has no correlation with FIGO stage, age, depth ofcervical infiltration, tumor size, and gross type of cervical lesion(P＞0.05),and correlation with pelvic lymph nodes metastases and tumorhistological grade (P＜0.05). The neoadjuvant chemotherapy had noinfluence on the expression ofBeclin 1 (P＞0.05).3. PCR analysis and DNA sequencing confirmed that the eukaryoticexpression vector pcDNA3.1 (+)-Beclin 1 and shRNA expression vectorspSUPER-Beclinl were constructed successfully.4. The eukaryotic expression vector pcDNA3.1(+)-Beclinl significantlyimproved the expressin of mRNA and protein of Beclinl and caspase-9 inHeLa cells (P＜0.05), the ratios of G1 phase of HeLa cell were increasedand the ratios of S phase were decreased significantly, and the cell proliferations of HeLa cell were inhibited, while more apoptosis cells andmore autophagy cells were identified in these cells.5. The shRNA expression vectors pSUPER-Beclinl significantly inhibitedthe expressin of mRNA and protein of Beclinl and caspase-9 in HeLacells (P＜0.05), the ratios of G1 phase of HeLa cell were decreased andthe ratios of S phase were increased significantly, and the cellproliferations of HeLa cell were improved, while less apoptosis cells andless autophagy cells were identified.6. After transfected by vector pcDNA3.1(+)—Beclin 1, the carcinogenicactivity of HeLa cell was decreased in nude mice, the volume and theweigh of tumors were reduced, and Beclin 1 protein level in tumor tissueswas improved. After transfected by vector pSUPER--Beclin 1, thecarcinogenic activity of HeLa cell was increased in nude mice, thevolume and the weigh of tumors were improved, and Beclin 1 proteinlevel in tumor tissues was reduced.Conclusions Autophagy and apoptosis are two types of programmed celldeath, autophagy gene Beclin 1 play an important role in these two types, anddefect of autophagy and apoptosis may be important in tumor genesis. Thelevels of Beclin 1 expression reduced in cervical invasive carcinoma tissues,and this may be related with the occurrence and development of cervicalcancer. Beclin 1 transfection can inhibit the growth of HeLa cells in vitro andvivo. Beclin 1 may play an important role in generation and development ofcervical cancers, and it might be one of the ideal strategies for gene therapyof cervical cancer to improve the ability of autophagy and apoptosissimultaneously.