Roles Of DRR1in The Occurrence And Evolution In HPV16-related Cervical Cancer

Background Cervical cancer is a common malignancy cancer in women, Infection of carcinogenic human papillomavirus (HPV) is the essential basis for occurrence and evolution of cervical cancer. Persistent expression of E7contribute to HR-HPVs carcinogenesis. E7lead to abnormal cell proliferation by interacting with and degrade pRb. Through gene expression profile chip analysis, our research team found that the expression of renal cell carcinoma downregulated gene was upregulated3to11times after silencing E7. DRR1is widely expressed in normal tissues, but in many cancer cell lines and primary tumors, the expression was dramatically reduced or even undetectable. As a tumor suppressor gene, DRR1may play an important role in tumorigenesis.Objectives Taken HPV16E7-positive and-negative cervical cancer cell line,normal cervical squamous epithelium tissues, cervical intraepithelial neoplasia tissues and cervical squamous cell carcinoma tissue as the research objects, It attempts to clarify the role of DRR1in HPV16-related cervical cancer and enrich the carcinogenic mechanisms of HPV16E7, in order to provide experimental and theoretical basis for the treatment of HPV16related cancers.Methods1. Taken CaSki cells, SiHa cells (HPV16-positive) and C33-A cells (HPV16-negative) as the research objects, the expression of E7and DRR1were detected by Western blotting. The relationship between HPV16E7and DRR1was analyzed.2. Taken normal cervical squamous epithelium tissues, cervical intraepithelial neoplasia tissues and cervical squamous cell carcinoma tissues as the research objects, detecting the expression of E7and DRR1by immunohistochemical, analyzing the relationship between HPV16E7and DRR1.3. Conventionally culture C33-A cells to logarithmic phase, extraction of total RNA, Reverse transcription synthesis of the first strand cDNA in vitro, amplify DRRl gene coding sequence by PCR, construction and identification of eukaryotic expression plasmid pcDNA3.1-DRRl, recombinant plasmid were transfected into logarithmic phase CaSki cells via Liposome. The proliferation and apoptosis of CaSki cells were detected by drawing growth curve, colony formation rate, cell cycle analysis and Annexin V-FITC/PI.Results1. The results of western blot showed:①HPV16E7was not expressed in C33-A cells, but expressed in SiHa and CaSki cells.②The expression of DRRl in C33-A cells was significantly higher than SiHa and CaSki cells (P<0.01). The expression of DRRl in SiHa cells and CaSki cells was no significant difference (P>0.05).The results of immunohistochemistry showed:①The expression of DRRl in HPV16E7-positive normal cervical squamous epithelium tissues, CIN tissues and cervical squamous cell carcinoma tissues were significantly lower than in HPV16E7-negative tissues. Correlation coefficients were-0.452,-0.485and-0.444(P<0.01), respectively.2. The result of MTT indicated that:①The growth rate of CaSki-DRRl cells were significantly slowed (P<0.01), and doubling time was significantly prolonged (P<0.01), compared with CaSki-vect cells.②The result of clone formation assay showed that:The mean of clone formation in CaSki-DRR1cells were significantly decreased and volume was significantly small, compared with CaSki-vect cells (P<0.01).③Flow cytometric analysis indicated that:the G1stage of CaSki-DRR1cells were blocked (P<0.05), the mean percentages of S and G2/M stage cells were significantly reduced (P<0.01).④The result of Annexin V-FITC/PI and Flow cytometry indicated that:The mean of apoptotic cells in CaSki-DRRl cells were significantly high (P<0.01), compared with CaSki-vect cells.Conclusions 1. In HPV16-related cervical cancer cells and tissues, the expression of E7are negatively correlated with DRRl.2. The stable expression of DRRl can significantly inhibit the ability of proliferation and promote apoptosis in HPV16-related cervical carcinoma cells.