Investigation On The Cross-talk Between HPV E7 And CIP2A And Its Prognostic Value Assessment In Cervical Cancer

BackgroundCervical cancer is one of the most common diseases among women.It is the second most common cancer to breast cancer in women worldwide. In developed countries, the incidence and mortality of cervical cancer decreased obviously thanks to the popularity of cancer screening, in recent decades. But in developing countries, the incidence and mortality of cervical cancer arises quickly. About 150 thousand women develop cervical cancer and 20 thousand women die from the disease in China each year. Although a series of factors have been found to be associated with the prognosis of cervical cancer patients, there is no objective prognostic indicators so far.The molecular biology research has confirmed that the infection with high-risk types of human papillomavirus (HPV) is strongly-associated with the development of cervical cancer. HPVs are small non-enveloped double-stranded DNA virus. Now more than 100 different viral types are found and they are divided into two kinds:low risk HPV (LR-HPV) and high-risk HPV (HR-HPV). Persistent infection by high risk-human papillomavirus (HR-HPV) is an essential factor leading to multi-step carcinogenesis, especially HR-HPV 16 and 18 genotypes. The viral genome integrates into the host cell genome and encodes transforming oncoproteins E6 and E7. E6 and E7 play a key role in driving cells from the precancerous to invasive cancer stage. However, HPV E6 and E7 overexpression alone can not induce the transformation of the host cells. HPV infection is only the first hit during the carcinogenesis of cervical cancer. The detailed changes of the molecular signal pathways induced by HPV are still needed to be elucidated. Cancerous inhibitor of protein phosphatase 2A is a new oncoprotein identified in 2007. CIP2A can interact directly with the oncogenic transcription factor c-Myc, which can inhibit PP2A activity to c-Myc serine 62 (S62) and thereby prevent c-Myc proteolytic degradation and stabilize c-MYC in tumor cells. It has bee reported that CIP2A is overexpressed in many cancers, such as HNSCC, gastric cancer, colon cancer and breast cancer et.al. This suggests that CIP2A plays an important role in the carcinogenesis of human cancer, however its carcinogenic mechanism is still unclear.Our previous work in 2011 confirmed that CIP2A was overexpressed in cervical cancer. It promoted the proliferation of cervical cancer cells, enhanced its ability of colony formation and soft agar colony formation. Interestingly, we found that HPV E7 expression was tightly associated with that of CIP2A by immunohistochemstry method in cervical cancer tissues. However, whether HPV E7 can regulate CIP2A and by what mechanism it regulates CIP2A gene expression in cervical cancer is still unknown.ObjectiveTo explore whether HPV E7 can regulate CIP2A and by what mechanism it regulates CIP2A gene expression in cervical cancer, and to assess the prognostic significance of the related proteins in this signal pathway in cervical cancer.Methods1) Knock-down the expression of HPV E7 in HeLa and SiHa cells by transfecting the specific siRNA into cells; Detect the protein and mRNA levels of HPV E7 and CIP2A by Quantitive Real-time PCR and Western Blot method after transfection; Compare the expression level of CIP2A in PHK-E7 and PHK cells by Quantitive Real-time PCR and Western Blot method.2) Previously we have established the PHK cell line with the mutated HPV E7 (E7 without pRb binding site). To explore whether HPV E7 regulate CIP2A through the pRb-dependent way, Western blot method was used to compare the ability that HPV E7 regulate CIP2A in wild type PHK-E7 cells and PHK cells with mutant E7.3) Biology Softwares were used to predicted the transcription factors in CIP2A promoter region which can be combined with HPV E7. Real-time PCR was used to find out the most probable transcription factor X by in HeLa and SiHa cells.4) To reveal the cross-talk between E7,CIP2A and transcription factor X, we altered their expression levels separately by transfecting the specific siRNA and plasmid into HeLa and SiHa cells. Quantitive Real-time PCR and Western Blot method were used to detect the changes of gene expression.5) Immunofluorescence staining and laser confocal microscopy were used to detect the subcellular localization of CIP2A and transcription factor X in HeLa and SiHa cells.6) Immunohistochemistry(IHC) was used to detect CIP2A and transcription factor X expression in 184 cervical cancer tissues. SPSS software was used to analyze the staining result. The relationship between two protein expression level with the clinical pathologic parameters was analyzed by Chi square test. The effect of CIP2A and transcription factor X on specific survival was assessed by KM survival analysis and Cox regression analyses.Results1) CIP2A mRNA and protein were decreased significantly in HeLa and SiHa cells after knocking down the HPV E7 gene level. The CIP2A gene expression was significantly higher in PHK-E7 cells compared with PHK cells. Furthermore, the regulatory effect of HR-HPV E7 on CIP2A was stronger than that of LR-HPV E7. Thus HPV E7 could up-regulate the CIP2A gene expression in transcriptional level.2) When the binding site of E7 to pRb was mutated, the regulation effect of E7 on CIP2A expression was decreased greatly. It indicated that E7 regulated the CIP2A expression in a pRb-dependent manner.3) By the literature review and software prediction, we focused on several transcription factors (E2F1、ETS1 and Elk1) which might bind to the promoter region of CIP2A. By real-time PCR screening, we finally target the most probable transcription factor E2F1.4) We demonstrated that HPV E7 could upregulate CIP2A expression via E2F1, while CIP2A could also upregulate E2F1 expression in transcriptional level. Thus our results revealed a feedback loop between E2F1 and CIP2A in HeLa and SiHa cells, and the crosstalk between E2F1 and CIP2A was in transcriptional level.5) By immunofluorescence staining and laser confocal microscopy, we confirmed the subcellular co-expression between E2F1 and CIP2A in HeLa and SiHa cells.6) A significant correlation was found between E2F1 and CIP2A protein in cervical cancer tissues. Patients with CIP2A and E2F1 co-expression have poor overall survival and disease-free survival rate. CIP2A and E2F1 expression could be an independent prognostic factor for cervical cancer patients.Conclusions1) HPV E7 upregulated CIP2A expression in a pRb dependent manner. HPV E7 could upregulate CIP2A expression in transcriptional level via E2F1, while CIP2A could also upregulate E2F1 expression in transcriptional level. Thus our results revealed a feedback loop between E2F1 and CIP2A in cervical cancer cells.2) A significant correlation was found between E2F1 and CIP2A protein in cervical cancer tissues. CIP2A and E2F1 co-expression could be an independent prognostic factor for cervical cancer patients.