The Regulatory Mechanisms Of Oct4and PinX1Expression In Cervical Cancer Cells

Background: Cervical cancer is the malignant disease seriously threatening the healthof women. The infection of high risk human papillomavirus (HPV) and the expression ofHPV E6and E7oncoproteins are key factors for carcinogenesis and development of cervicalcancer. Besides HPV, the abnormal expression and dysfunction of many other molecules arealso involved in the occurrence of cervical cancer. Oct4, an important transcriptional factor,maintains the self-renewal and pluripotency of stem cells. Overexpression of Oct4contributesto the transformation of normal cells into cancer stem-like cells and to the initiation ofneoplasia. It has been reported that Oct4is highly expressed in several tumors. In addition, theactivation of human telomerase promotes the proliferation of cancer cells. Human telomerasereverse transcriptase (hTERT) is the key component of telomerase. The Pin2/TRF1-interacting protein X1(PinX1) can inhibit the activity of telomerase by either directlyinhibiting hTERT activity or suppressing the transcription of hTERT. Although both Oct4andPinX1are correlated with the initiation and propagation of various cancers, it is unkownabout their expression profiles in cervical cancer cells as well as their regulatory mechanisms.Objective:1. To investigate the expression of Oct4and PinX1in HPV positive andnegative cervical cancer cells, which may provide scientific evidences for exploring therelationship between these two molecules and cervical cancer.2. To elucidate the epigeneticregulatory mechanisms of Oct4expression and HPV16E6-mediated regulation of PinX1expression and the relevant mechanisms in cervical cancer cells, which may lay a theoreticfoundation for developing novel therapeutic strategies to cervical cancer by targeting Oct4and PinX1.Methods:1. The clone-formation abilities of HeLa (HPV18positive), CaSki (HPV16positive) and C-33A (HPV negative) cells were examined by soft agar clone forming assay.The expression of Oct4, DNA methyltransferases (DNMTs) including DNMT1, DNMT3Aand DNMT3B, and histone deacetylase1(HDAC1) were analyzed by RT-PCR, Real time PCR, Western blot and Laser confocal-based immunofluorescence. The DNA methylationin the key regulatory regions of Oct4gene was detected by bisulfite genomic sequencing(BGS), and the whole level of genomic DNA methylation in above three cervical cancercell lines was separately analyzed by reversed-phase high-pressure liquid chromatography(RP-HPLC). After the treatment of cervical cancer cells with a HDAC1inhibitor (valproicacid, VPA), the changes of Oct4were assayed. It was investigated whether HDAC1andDNMT3A existed in a common complex by co-immunoprecipitation (Co-IP) assay, andwhether the complex was affected by VPA.2. SiRNA against HPV16E6was designedand synthesized, and HPV16E6expression vector (pEGFP-HPV16E6) was constructed.Subsequently, the siRNA was transfected into CaSki cells, and pEGFP-HPV16E6wastransfected into C-33A cells, so that the cervical cancer cell models with HPV16E6knockdown or ectopic expression were obtained. Then the model cells were used toexamine the expression of PinX1, NF-κB p50and p65by RT-PCR, Real-time PCR andWestern blot. Following the treatment of the model cells with an inhibitor of IκBαphosphorylation (BAY11-7082), which resulted in the suppression of NF-κB activity, thechanges of PinX1expression were detected. The influence of NF-κB on transcriptionalactivity of PinX1promoter was evaluated by luciferase report assay. It was explored whetherNF-κB could bind to the specific elements in5′regulatory region of PinX1gene byelectrophoretic mobility shift assay (EMSA). The telomerase activities of the model cellswere assayed by telomeric repeat amplification protocol (TRAP). The proliferations of themodel cells were examined by [3H]TdR incorporation assay.Results:1. The mRNA and protein levels of Oct4in HPV positive cervical cancercells(HeLa and CaSki cells) were higher than that in HPV negative cervical cancercells(C-33A cells), which was paralleled with the colony-forming ability of the differentcell lines. The levels of DNMT3A and HDAC1in C-33A cells were higher than that inHeLa and CaSki cells. However, DNMT3A could neither regulate the expression of Oct4through catalyzing the DNA methylation of regulatory regions of Oct4gene, nor affect thewhole level of genomic DNA methylation. DNMT3A and HDAC1existed in a commoncomplex, and the level of the complex was inversely correlated with the expression of Oct4.Inhibition of HDAC1activity by VPA could significantly increase the expression of Oct4incervical cancer cells.2. HPV16E6could upregulate NF-κB p65expression and downregulate PinX1expression. Repression of IκBα phosphorylation could efficientlyattenuate HPV16E6-mediated suppression of PinX1expression. Reporter assay showed thatNF-κB could reduce the transcriptional activity of PinX1gene promoter. EMSA showedthat NF-κB could bind the site-909～-898in5′regulatory region of PinX1gene. HPV16E6was positively correlated with the telomerase activity and the proliferation of cervicalcancer cells, which could be efficiently alleviated by the inhibition of IκBαphosphorylation.Conclusion:1. HPV infection was positively correlated with Oct4expression, butinversely correlated with DNMT3A and HDAC1levels. Oct4promoted the colon-formingability of cervical cancer cells. DNMT3A could neither affect the DNA methylation of Oct4gene nor affect the degree of genomic DNA methylation. DNMT3A and HDAC1existed ina common complex which might suppress Oct4expression. The above results suggest thatthe infection of high risk HPV might decrease the level of DNMT3A/HDAC1-containingcomplex by regulating the HDAC1activity, which led to the upregulation of Oct4expression.2. NF-κB could negatively regulate the expression of PinX1, which suggest thatHPV16E6might suppress PinX1expression through activating NF-κB, leading to theattenuation of PinX1-mediated inhibition of telomerase activity, and eventually resulting inthe increase of telomerase activity and proliferation of cervical cancer cells.3. Oct4andPinX1might serve as potential novel targets for the treatment of cervical cancer.