Effects Of HnRNP K On Cervical Carcinogenesis Through Mediating HPV16 E2/E6 Expression

Objective:Cervical cancer is the fourth most common cancer among women globally,and presents a serious threat to women’s physical and mental health.Persistent HPV16 infection is a leading cause of cervical carcinogenesis.Furthermore,integration of HPV 16 DNA into the human genome is a key point in the development of cervical cancer.The tumorigenesis-related genes mainly include HPV16 E2 and and E6.E6 is a viral oncogene which participates in immortalization and malignant transformation of cervical cells,and E2 is one of HPV16 key integrative genes and can inhibit E6 gene expression.Heterogeneous nuclear ribonucleoprotein K(hnRNP K)is a nucleic acid binding protein and has special molecular architectures which could bind to HPV16 DNA.It regulates diverse cellular processes involving in transcription,mRNA splicing,mRNA stability,polyadenylation,and mRNA translation.hnRNP K is closely related to the occurrence and development of tumor.However,the effects of hnRNP K on cervical carcinogenesis and the expression regulation of HPV16 E2 and E6 genes remain unclear.An epidemiological study,in vivo cell experiments,and bioinformatics analysis of public databases for gene expression were all performed.The aim of this study was to explore the roles and potential molecular mechanisms of hnRNP K through mediating HPV16 E2/E6 expression in the progression of cervical carcinogenesis,and the results may afford the theoretical basis for biological mechanisms of cervical canceration and provide new insights into the diagnosis and treatment of cervical cancer.Methods:1.Epidemiological study:257 participants were involved in this study and formed the multistep natural populations of cervical cancer development,including 67 normal cervix(NC),69 CIN(cervical intraepithelial neoplasia)I,68 CINⅡ/Ⅲ,and 53 cervical squamous cell carcinoma(SCC).They were new cases by pathological diagnosis from the community(Jiexiu City of Shanxi Province)and hospital(The Tumor Hospital of Shanxi Province and The Second Hospital of Shanxi Medical University)cohorts that were previously established from June 2014 to June 2015.A structured questionnaire was used to collect relevant information of cervical cancer including socio-demographic characteristics,obsterical history,gynecological disease history,tumor familial history,and personal hygiene behavior.Cervical exfoliated cells and cervical tissues from biopsy or surgery were selected.HPV genotyping was detected by flow-through hybridization and gene chip.The protein expression levels of hnRNP K were evaluated by immunohistochemistry(IHC)and Western blotting.The protein expression levels of HPV16 E2 and E6 were tested by Western blotting.2.In vivo cell experiment:HPV16-positive SiHa cell lines(cervical squamous cell carcinoma)were used to in vivo study.Down-regulation and up-regulation of hnRNP K expression in SiHa cells were performed by shRNA interference and over-expression plasmid transfection techniques.Cell proliferation ability was assessed by cell counting kit-8(CCK8)assay.Cell cycle and apoptosis were detected by flow cytometry.The mRNA and protein expression levels of hnRNP K,HPV16 E2,HPV16 E6,c-Myc,and β-actin were measured by real-time PCR and Western-blot.Chromatin Immunoprecipitation followed by Sequencing(ChIP-Seq)was conducted to detected hnRNP K DNA-binding sites.Real-time PCR was further used to validate whether hnRNP K directly binds to regulatory elements of HPV16 genome and the promoter region of c-Myc.3.Bioinformatics analysis of public cancer database for gene expression:three Gene expression Omnibus(GEO)publically available data sets(GSE6791,GSE7803,and GSE63514)were selected to evaluate the hnRNP K mRNA expression levels in different cervical lesions groups.Gene Set Enrichment Analysis(GSEA)in The Cancer Genome Atlas(TCGA)database of 308 cervical cancer cases was performed to discover the biological functions which hnRNP K involved in during the cervical carcinogenesis.Additionally,the relationship between hnRNP K and c-Myc mRNA expression was analyzed in this TCGA database.4.Double data entry and data verification were performed by Epidata3.1 software.Statistical analysis and drawing diagrams were done by SPSS 23.0 and Graphpad prism 6.0 softwares.Hypothesis tests were conducted usingX2 test,X2 trend test,and one-way ANOVA.Multiple comparison was completed by the Bonferroni method.Moreover,multifactor analysis and calculation of the odds ratios(ORs)were performed using the unconditional logistic regression models.The correlation analysis of two quantitative variables was conducted by the Spearman’s rank correlation.Two-way interactions were analyzed using additive model and related indicators(RERI,AP,and S).Results:1.Relationship between HPVs infection and cervical lesions:HPVs infection rates in different cervical lesions groups were 19.4%(NC),40.6%(CIN I),60.3%(CINⅡ/Ⅲ),and 84.9%(SCC),respectively(P<0.001).UPV16 infection rates were 10.4%(NC),14.5%(CIN Ⅰ),41.2%(CIN Ⅱ/Ⅲ),and 66.0%(SCC),respectively(P<0.001).Moreover,the infection rates of HPVs and HPV16 were gradually elevated with the increasing severity of cervical lesions(x2trend=55.88,P<0.001;x2 trend-51.13,P<0.001).2.Relationship between HPV E2,E6 and cervical lesions:there were significant differences in the protein expression of HPV16 E2 and E6 during different cervical lesions groups(H=26.35,P<0.001;H=21.10,P<0.001).HPV16 E2 protein expression in SCC and CIN Ⅱ/Ⅲ groups was significantly lower than that in CIN I and NC groups,and HPV16 E6 protein expression in SCC and CIN Ⅱ/Ⅲ groups was significantly higher than that in CIN Ⅰ and NC groups(P<0.0083).Protein expression of HPV16 E2 and E6 was increased with the increasing severity of cervical lesions(x2trend= 15.51,P<0.001;x2trend= 12.33,P<0.001).The median of the E2/E6 protein ratios in NC,CIN Ⅰ,CIN Ⅱ/Ⅲ,and SCC groups were 8.09,9.41,1.98,1.27,respectively(P<0.001).The E2/E6 protein ratios in SCC and CIN Ⅱ/Ⅲ groups was significantly lower than those in CIN Ⅰ and NC groups(P<0.0083).3.Relationship between hnRNP K and cervical lesions:the immunohistochemical results indicated hnRNP K protein was mainly expressed in the cell nucleus of cervical epithelial cells,and a small amount was expressed in the cytoplasm.In addition,hnRNP K protein expression in the cell nucleus and cytoplasm was all increased with the development of cervical lesions.The results from Western blot showed hnRNP K protein expression was significantly different in NC,CIN I,CIN II/III,and SCC groups(H=58.43,P<0.001),and revealed an increased trend with the severity of cervical lesions(x2trend=29.84,P=0.001).Furthermore,the hnRNP K protein expression in CIN I group was significantly higher than that in NC group(P<0.0083),and its protein expression in SCC and CIN Ⅱ/Ⅲ groups was all higher than that in CIN I and NC groups(P<0.0083).4.Interaction of hnRNP K and HPV16 infection in cervical lesions:there were additive effects between high expression of hnRNP K protein and HPV16 infection in both CIN II/III and SCC groups compared with NC group.In contrast,the additive effect between them in CIN I group did not exist.5.Correlation between hnRNP K,HPV16 E2 and E6 in cervical lesions:the results by Spearman’s rank correlation test showed a positive correlation between the protein expression of hnRNP K and HPV16 E6(rs=0.254,P=0.023),and negative correlations between hnRNP K and HPV16 E2(rs=-0.280,P=0.012),between hnRNP K and HPV16 E2/E6(r,=-0.243,P=0.030),between HPV16 E2 and E6(rs=-0.377,P=0.001).6.Effects of hnRNP K on biogical functions in SiHa cells:down-regulation of hnRNP K by shRNA interference in SiHa cells inhibited cell proliferation,induced cell cycle arrest at G0/G1 phase,decreased the percentage of SiHa cells in S and G2/M phases,up-regulated the late and total apoptosis ratios(P<0.05).On the contrary,up-regulation of hnRNP K promoted cell proliferation,decreased the cell population in the G0/G1 stage and increased the cell population in the S and G2/M stages,reduced the late and total apoptosis ratios(P<0.05).7.Effects of hnRNP K on the expression of HPV16 E2 and E6:hnRNP K down-regulation in SiHa cells decreased the mRNA expression levels of HPV16 E2 and E6(P<0.05),and further reduced the protein expression levels of HPV16 E6(P<0.05).Meanwhile,hnRNP K up-regulation in SiHa cells increased the mRNA expression levels of HPV16 E2 and E6(P<0.05),and then elevated the protein expression levels of HPV16 E6(P<0.05).HPV16 E2 protein expression in all the intervention groups was not detected by Western blotting.8.Analysis of the hnRNP K-binding sites in HPV16 and the human genome:according to the results of ChIP real-time PCR,hnRNP K bound to the gene regulation regions of HPV16(555F/7681R,7854F/65R)specifically(P<0.05).ChIP-Seq analysis demonstrated there were 1321 human hnRNPK-bound genes in SiHa cells and the enrichment of hnRNP K peaks in the promoter region of c-Myc was much higher.Real-time PCR results further validated hnRNP K directly binds to the promoter region of c-Myc(P<0.05).9.Effects of hnRNP K on the c-Myc expression:hnRNP K down-regulation in SiHa cells decreased the mRNA and protein expression levels of oncogene c-Myc(P<0.05).In contrast,hnRNP K up-regulation in SiHa cells increased the mRNA and protein expression levels of oncogene c-Myc(P<0.05).10.Bioinformatics analysis of hnRNP K mRNA expression in cervical lesions:GSE6791 data showed hnRNP K mRNA expression in cervical cancer group was higher than that in NC group(P<0.05).GSE7803 data indicated hnRNP K mRNA expression in both SCC and HSIL groups was higher than that in NC group(P<0.0167).GSE63514 data demonstrated hnRNP K mRNA expression in SCC and CIN3 groups was all higher than that in NC and CIN1/2 groups(P<0.0083).11.Prediction of hnRNP K biological functions in cervical cancer:GSEA analysis in TCGA database of cervical cancer revealed 194 gene sets were up-regulated and 7 gene sets were significantly enriched in hnRNP K over-expression group(P<0.05).These significant enrichment pathways were cell cycle,RNA degradation,G1 pathway,basal transcription factors,ubiquitin mediated proteolysis,ATM signaling pathway,and nucleotide excision repair.12.Relationship between hnRNP K and c-Myc in cervical cancer:the results from TCGA database of cervical cancer showed that the mRNA expression levels of c-Myc in hnRNP K high-expression group were higher than those in hnRNP K low-expression group(P<0.05).Moreover,the results by Spearman’s rank correlation test showed that there was a positive correlation between the mRNA expression of hnRNP K and c-Myc(rs=0.190,P=0.001).Conclusions:1.HPVs infection,especially HPV16 infection was a risk factor of cervical carcinogenesis.Early HPV testing,HPV vaccination,and control of HPV infection could help to decrease the risk of cervical cancer.HPV16 E6 over-expression and HPV16 E2 low-expression were closely related to the increased risk of cervical cancer.The protein ratio of HPV16 E2/E6 might become a potential biomarker of high-grade squamous intraepithelial lesion and cervical cancer.2.Over-expression of hnRNP K was associated with the increased risk of cervical cancer and hnRNP K may be viewed as a potential biomarker for early detection of cervical carcinogenesis.Additionally,over-expression of hnRNP K may facilitate cell proliferation,enhance the cell cycle switch to G2/M phase,and suppress cell apoptosis.hnRNP K might be acted as a potential therapeutic target of cervical cancer3.There may be the synergistic effects of hnRNP K overexpression and HPV16 infection on the progress of CEST Ⅱ/Ⅲ and cervical cancer.hnRNP K might up-regulate the mRNA expression of HPV 16 E2 and E6 through binding to gene regulatory elements of HPV16,increase the protein expression of HPV16 E6,and promote cervical carcinogenesis.4.hnRNP K may increase its transcriptional activity via binding to the promoter region of c-Myc,up-regulate the expression of oncogene c-Myc,and promote cervical carcinogenesis.5.hnRNP K might be closely associated with these biological pathways including RNA degradation,basal transcription factors,ubiquitin mediated proteolysis,ATM signaling pathway,and nucleotide excision repair.In-depth studies on the underlying mechanism of hnRNP K are needed.