Mechanisms Of HPV E6/E7 Regulating Biological Function Of Cervical Epithelial Cells Through IFI16/p53 Pathway

Background:Cervical cancer is the second most common female malignancy worldwide.There are more than 500,000 new cases each year,of which more than 260,000 patients die of cervical cancer.It is estimated that there are about 570,000 new cases in 2018.The occurrence and development of cervical cancer is a complex biological process involving a variety of factors and steps.High-risk human papillomavirus(HPV),such as HPV 16,18,33,45 and 31,is main cause.HPV16 and 18 are the most common and most carcinogenic high-risk HPV,resulting in more than 75%of cervical cancer cases.The carcinogenicity of HPV is mainly attributed to the immortalization and transformation properties of HPV oncoproteins E6 and E7,which are selectively retained and expressed at various stages of carcinogenesis.Uncontrolled expression of the high-risk HPV E6 and E7 genes,functionally equivalent to p53 and pRb gene mutations,leads to cell immortalization,genetic instability,accumulation of genetic mutations,and consequent malignant transformation.The changes in cell biological function caused by the expression of E6 and E7 oncoproteins in infected cervical epithelial cells and the specific molecular mechanisms involved have been the focus of researchers.IF116 is a member of the HIN200 family.The IFI16 protein has a structural motif shared by family members,the N-terminal PYRIN domain(PYD domain)and two relatively conserved HIN domains of approximately 200 amino acids contained in C-terminus.PYD is commonly found in cell death-associated proteins such as PYRIN,ASC and zebrafish caspase,also known as the PAAD/DAPIN domain.PYD has been found at the N-terminus of IFI16,suggesting that IFI16 may play a key role in the regulation of apoptosis.PYD in IFI16 protein is a homologous protein-protein interaction domain that allows IFI16 to interact with other proteins,including BRCA1,pRb,p53,E2F,and NF-κB.The HIN domain contains two linked oligonucleotides/oligosaccharide-binding folds(OB-folds)that allow IFI16 to bind to single or double stranded DNA.In addition,the HIN domain also contains a pRb binding region and a p53 binding region.Compared with other family members,IFI 16 has a wider range of functions:1)IFI 16 can act as a double-stranded DNA receptor,causing an immune response against certain pathogens or broken cells;2)participating in damaged tissues or Angiogenesis during tumor formation;3)acting as a pro-inflammatory protein in autoimmune diseases;4)involved in cell proliferation,apoptosis,cell senescence,and cell cycle regulation.At the same time,evidence of IFI16 related to cancer is accumulating.At present,IFI16 has been found to play a role in nine human solid tumors such as hepatocellular carcinoma,head and neck cancer,breast cancer and oral squamous cell carcinoma;however,few studies have reported that IFI16 is associated with cervical cancer and HPV infection.Studies have shown that IFI61 is down-regulated in breast cancer,and its expression is also significantly negatively correlated with the grade of head and neck squamous cell carcinoma(HNSCC),and is closely related to HNSCC growth,apoptosis,blood vessels,inflammation.In addition,studies have shown that histone deacetylase-dependent transcriptional silencing of the IFI16 gene in prostate epithelial cells is beneficial to the development of prostate cancer.Contrary to the anti-cancer effect of IFI16 in breast cancer,HNSCC and prostate cancer,some researchers found that the expression of IFI16 protein was up-regulated in testicular cancer and chemoresistant epithelial ovarian cancer tissues,but there is no related function and mechanism.In addition,the interaction between IFI16 and p53 continues to receive the attention of researchers.Studies have shown that IFI16 can activate p53 expression under DNA damage,ionizing radiation or oxidative stress,affect the expression of p53 downstream target protein and p53-mediated apoptosis and cycle regulation,thus affecting tumorigenesis and development.Interestingly,it has been established that high-risk HPV E6 protein induces cervical cancer by affecting p53 expression and regulating p53-mediated cell cycle and apoptosis.Studies have shown that in the complex with E6,the ubiquitin ligase E6AP targets p53,which is ubiquitinated and degraded,resulting in HPV-induced cervical cancer.E6 functions as a powerful activator of E6AP with an unknown mechanism.Although studies have shown that the LxxLL motif of E6AP binds to E6,causing a conformational change that enables E6 to bind to p53.However,the specific mechanism of action between E6 protein and p53 remains unclear.Part I:The expression level and significance of IFI16 in cervical cancer tissuesObjective:To study the relationship between expression level and prognosis of IFI16 in cervical cancerMethods:1 Using the GEPIA online tool to analyze the RNA sequencing expression data of 9,736 tumors and 8,587 normal samples in the TCGA and GTEx libraries,and analyze the expression level of IFI16 in pan-tumor and its relationship with prognosis.The current status of research on IFI16 in PUBMED database in each tumor was searched.2 Immunohistochemistry:Immunohistochemistry was performed using cervical cancer tissue microarray to detect the expression level of IFI16 in cervical cancer and adjacent tissues,and the correlation between IFI16 and various clinical pathological features.Results:1 GEPIA analysis showed that IFI16 was up-regulated in 11 tumors of CHOL,DLBC,GBM,HNSC,KIRC,LAML,LGG,PAAD,SARC,SKCM,STAD in 31 tumors in the database,in KICH,PRAD,UCEC,UCS4 tumors were down-regulated.Survival analysis found that patients with low expression of IFI16 in KIRC,KIRP,and LGG had a good prognosis(P<0.05),and we found that IFI16 was the highest in CESC among the 31 tumor data provided by the database.2 In PUBMED,we searched the literature related to IFI16 tumor research.The results showed that IFI16 plays a role in tumor suppressor genes in prostate cells and medullary thyroid carcinoma cells,breast cancer,and squamous cell carcinoma of the head and neck in vitro.Overexpression of IFI16 in lymphoma,Wilms renal cell carcinoma,and hepatocellular carcinoma promotes tumor cell proliferation.Few studies on IFI16 in CC have been reported.3 Immunohistochemistry results showed that the positive expression rate of IFI16 was 67.7%in cancer tissues,while the positive expression rate of IFI16 was only 12.9%in adjacent tissues.Further statistical analysis of pathological features showed that there was no statistically significant correlation between the expression level of IFI16 and tumor size and age(p>0.05),but it was associated with differentiation of tumors.In low differentiated tumors,IFI16 showed a high expression trend.Conclusion:In TCGA and GTEx pools,IFI16 is up-regulated in 11 tumors such as CHOL,and expressed in 4 tumors such as KICH.Down.Among all tumors,IFI16 was the highest expressed in CC.In the literature,IFI6 was closed related to many tumors.In cervical cancer tissues,IFI16 is highly expressed in cancer tissues compared with adjacent tissues,and the expression level is related to differentiation of tumors.Part II:HPV-E6E7 affects the biological function of H8 cells by regulating the expression of IFI16Objective:H8 stably transfected cells expressing HPV-E6E7 protein were constructed,and HPV E6/E7 was investigated to inhibit the biological function of H8 cells by inhibiting the expression of IFI16.Methods:1 The HPVI8 E6 and E7 genes were amplified from the pcDNA3.1-HPV18-E6 and pcDNA3.1-HPV18-E7 plasmids respectively,and the E6,E7 gene OVERLAP was PCR-ligated and ligated into the shuttle plasmid pLVX-mCMV-ZsGreenl.On the Puro,the empty vector was used as a control,and the recombinant plasmid was used for lentiviral packaging.After lentivirus infection of H8 cells,the H8-E6E7 stably transfected cells were screened using puromycin.Two methods were used to identify stable transfectants by fluorescence observation and PCR detection.2 Using CCK8 assay,clone formation assay,Transwell assay and flow apoptosis assay to detect the effect of E6E7 expression on cell proliferation,colony forming ability,migration,invasion and apoptosis.3 Western blot analysis of the effect of overexpression of HPV-E6E7 on the expression of IFI16 protein in H8 cells.4 H8-WT cells and H8-E6E7 cells up-regulated the expression of IFI16.The effects of E6E7 on the proliferation of H8 cells after IFI16 rescue were detected by CCK8 assay and colony formation assay.The effect of knockdown of the target gene on apoptosis was detected by flow cytometry and western blot,and whether HPV-E6E7 regulates IFI16 was investigated.Expression affects the biological function of H8 cells.Results:1 By fluorescence observation and PCR detection,H8 stably transfected cells stably expressing HPV-E6E7 protein were successfully screened.2 The results of CCK8 showed that the proliferation rate of H8-E6E7 cells was significantly higher than that of control group H8-WT cells.After 72 h of culture,the OD value of H8-E6E7 cells was significantly higher than that of H8-WT cells(P<0.05).The results of cell cloning experiments showed that the number of clones and the size of clones of H8-E6E7 cells were significantly higher than those of control group H8-WT cells.3 Transwell experiments showed that the number of cells that migrated and invaded H8-E6E7 cells was significantly higher than that of the control group H8-WT cells at the same time.The results of statistical analysis showed that the number of cells that migrated in H8-WT cells was 63± 4,and the number of cells that completed invasion was 73±6;while the number of cells in which H8-E6E7 cells completed migration was 140± 14,completing the invasion.The number of cells was 168 ± 13;the difference was statistically significant(P<0.05).4 Flow cytometry results showed that the expression of E6E7 inhibited the apoptosis of H8 cells.The apoptosis rate of H8-WT cells in the control group was 7.65%±0.95%,while the apoptosis rate of H8-E6E7 cells was significantly decreased,only 1.86%±0.33%(P<0.05).Western blot analysis showed that the expression of E6E7 in H8 cells promoted the expression of anti-apoptotic protein Bcl2 and inhibited the expression of proapoptotic proteins Bax and Active Caspase-3.50verexpression of HPV E6E7 in H8 cells down-regulated intracellular IFI16 expression.The expression level of IFI16 mRNA in H8-WT cells was significantly higher than that in H8-E6E7 cells,which was more than 20 times that of H8-E6E7 cells.The protein expression trend was consistent with the mRNA expression trend:the protein expression level of IFI16 in H8-WT cells was significantly higher than that of H8-E6E7 cells,which was about 5 times that of H8-E6E7 cells.6 CCK8 experimental results show that overexpression of IFI16 can inhibit the proliferation of H8 cells.At the same time,overexpression of IFI16 partially offset the effect of E6E7 protein expression on cell proliferation.In the four experimental groups,the cell proliferation rate was:H8-E6E7>H8-E6E7+IFI16>H8-WT>H8-WT+IFI16,The experimental results were significantly different and statistically significant(P<0.05).In the colony formation experiment,the results of each group were consistent with the results of CCK8 experiments.The number of clone formation in the four groups of cells was:H8-E6E7>H8-E6E7+IFI16>H8-WT>H8-WT+IFI16,the difference was statistically significant..7 Overexpression of IFI16 can affect the expression of apoptosis-related proteins in cells.Overexpression of IFI16 in H8 cells inhibited the expression of the apoptosis inhibitory protein Bcl2 and up-regulated the expression of the proapoptotic proteins Bax and Active Caspase3.Up-regulation of IFI16 expression in E6E7 overexpressing H8 cells attenuated the anti-apoptotic effect of E6E7.Conclusion:In H8 cells,overexpression of E6E7 can promote cell proliferation and clonality,enhance cell transfer ability,and increase the anti-apoptotic ability of cells.The expression of E6E7 protein enables immortalized cervical epithelial cells to obtain tumor cells.Characteristics.Upregulation of E6E7 inhibits the expression of IFI16 mRNA and protein.Up-regulation of IFI16 in H8 cells attenuates E6E7-induced cell proliferation and anti-apoptosis.IFI16 acts as a tumor suppressor gene.Part Ⅲ:IFI16 participates in the regulation of p53 expression in H8 cellsObjective:To explore the molecular mechanism of IFI16 involved in the regulation of H8 cell proliferation and apoptosis.Methods:1 The protein binding prediction tool STRING was used to predict the binding protein of IFI16 online,and the P53 protein was selected for subsequent validation.2 The pcDN A3.1 IFI 16-FLAG and pcDNA3.1-TP53 plasmids were co-transfected into H8 cells.After 48 hours,the total protein was collected and subjected to protein immunoprecipitation to detect the binding of IFI16 and P53 proteins.The expression of IFI16 gene was overexpressed in 3 H8 cells,and the effect of FIF16 overexpression on the expression of P53 protein was detected by Western blot.4 Overexpression of IFI16 in H8 cells,cycloheximide was added to the experimental group and the control group to inhibit the synthesis of new proteins.Proteins were collected at 0,1,2,4,and 6 h,and the expression of P53 was detected by Western blot.The effect of IFI16 expression on the stability of P53 protein.Results:1 The results of immunoprecipitation experiments showed that IFI16 and P53 proteins overexpressed in H8 cells could bind to each other.2 Westernblot results showed that in H8,E6E7 could decrease the expression of P53 protein,and up-regulate IFI16,the protein expression of P53 increased.3 The results of protein degradation experiments showed that the expression of IFI16 was beneficial to the stability of P53 protein.Conclusion:In H8 cells,IFI16 enhances its stability by binding to the tumor suppressor gene P53 protein,thereby acting as a tumor suppressor gene.Significance:In this study,HPV-E6E7 stably and highly expressed H8 cell line was successfully constructed by lentiviral infection,which enhanced the cell proliferation,metastasis and anti-apoptosis ability,and made the cells have the characteristics of tumor cells.The results of subsequent experiments demonstrated that IFI16 can bind to the tumor suppressor gene P53 protein and play a role as a tumor suppressor gene by increasing its stability.The cancer-promoting effect of HPV-E6E7 can be achieved by down-regulating the expression of IFI16-P53 protein.In this study,we found a correlation between IFI16 and HPV infection,cervical epithelial cell carcinogenesis and p53 expression,revealing a new molecular mechanism of cervical cancer,and providing a new idea and theoretical basis for the prevention and treatment of cervical cancer.