Association Between HnRNP E2 And Cervical Carcinogenesis And The Function Of HnRNP E2 Binding Genes

Objective:Repeated or persistent HPV infection is the main but not the only pathological factor in the occurrence of cervical cancer,and the search for other carcinogenic factors or carcinogenic factors that cooperate with HPV has become the focus of attention.HnRNP E2 as an RNA binding protein,contains a unique KH domain with binding sites with DNA and RNA,which provides a prerequisite for undergoing a series of biological functions,such as cell signal transduction,gene transcriptional and mRNA stability regulation,and so on.Studies have shown that hnRNP E2 is abnormally highly expressed in many tumors,but its expression in cervical cancer has not been determined.HnRNP E2 can interact with DNA,RNA and proteins,whereas,the binding of hnRNP E2 to whole genome DNA during Cervical Carcinogenesis is still unclear.The aim of this study is to explore the association between hnRNP E2 and cervical carcinogenesis by population study and vitro experiment,moreover,and to reveal the binding site of hnRNP E2 in the whole genome and possible biological functions by using ChIP-Seq which combines chromatin immunoprecipitation（ChIP）with second-generation sequencing technology.It will provide the evidence for the further research on the etiology and mechanism of cervical cancer and open up novel thought to find the biological targets for cervical cancer prevention and treatment.Methods:The participants were from the cervical lesions cohort established during June 2014 to September 2014 in Jiexiu City,Shanxi Province,who were selected through cervical cytology thin film cytology（TCT）screening,colposcopy,and pathological diagnosis,including 67 women with normal cervical（NC）,69 with low cervical intraepithelial neoplasia（CIN I）,68 with high cervical intraepithelial neoplasia（CIN II/III）,and 53patients with cervical squamous cell carcinoma（SCC）The information related to cervical lesions were collected using structured questionnaire,simultaneously the infection of HPV16 in cervical exfoliated cells was detected by flow-through hybridization,the protein expression of hnRNP E2 was detected by Western blot.SPSS 21.0 software was used to analysis relevant data,and the additive model was used to evaluate the interaction.At the same time,HPV16-positive Siha cell was selected as the experimental subject.Application of ChIP-Seq technology identified the binding sites of hnRNP E2 in the whole genome.The enrichment analysis of hnRNP E2 binding genes function and pathway were performed using GO and KEGG database,respectively.Results:1.The role of hnRNP E2 and HPV16 infection in cervical cancer and its interaction effects:hnRNP E2 expression from NC,CINⅠ,CINⅡ/Ⅲto SCC groups gradually reduced,statistically significant difference between groups（H=32.09,P<0.001）.The infection rate of HPV16 gradually increased with the increasing severity of cervical lesions(χ²_trend=51.13,P<0.001).The interaction analysis showed that there were additive effects between low expression of hnRNP E2 and HPV16 infection both in CIN II/III and SCC groups.2.Distribution of hnRNP E2 binding sites in whole genome:The number of DNA binding sites for hnRNP E2 in genome-wide was 646,with an average length of 163bp,440 of them were annotated as gene fragments,covering 245 genes.HnRNP E2 binding sites and genes distributed on each chromosomes,mainly concentrated on chromosomes 7,5,2 and 3.The hnRNP E2 binding sites were distributed in all five functional components,but the intergenic region（58.2%）and intron region（35.8%）were the most.3.Characterization of hnRNP E2 binding DNA sequences:Motif analysis revealed that hnRNP E2 had three motifs that were easy to bind.The contents of Cytosine（C）and Guanine（G）in these motifs were high,especially Cytosine（C）,suggesting that hnRNP E2tended to bind DNA sequences enriched with Cytosine（C）and Guanine（G）.4.GO enrichment analysis:At the molecular function aspect,the target genes were mainly enriched in cGMP inhibiting cyclic nucleotide phosphodiesterase activity（GO:0004119）,ion channel activity（GO:0005216）,exonuclease activity（GO:0004527）,transport activity（GO:0005215）,transcription cofactor activity（GO:0003712）,nucleic acid binding（GO:0003676）,DNA binding（GO:0003677）and RNA binding（GO:0003723）.Biological processes mainly included cell growth regulation（GO:0060284）,cell cycle regulation（GO:0051726）,cell adhesion（GO:0007155）,biological adhesion（GO:0022610）,mRNA processing（GO:0006396）and signal transduction（GO:0023052）.The cellular component mainly included the cell periphery（GO:0071944）and plasma membrane part（GO:0044459）and so on.5.Pathway enrichment analysis:The gene enriched in cell adhesion molecules（CAMs）（ko04514）was the most,followed by the cAMP signaling pathway（ko04024）.In addition,the biological pathways of hnRNP E2 binding genes were mainly concentrated in multiple cancer pathways such as renal cell carcinoma（ko05211）,melanoma（ko05218）,small cell lung cancer（ko05222）,non-small cell lung cancer（ko05223）,endometrial cancer（ko05213）and cancer center carbon metabolism（ko05230）.As well as MAPK signaling pathway（ko04010）,nucleotide resection and repair（ko03420）,ovarian steroid production（ko04913）,progesterone mediated oocyte maturation（ko04914）,steroid hormone biosynthetic（ko00140）and other important tumor-related pathways.Conclusion:1.HPV16 infection was a major risk factor for cervical lesions occur,hnRNP E2lower expression could increase the risk of CINII/Ⅲand SCC,and low expression of hnRNP E2 and HPV16 infection had a synergistic effect in the occurrence of cervical lesions.It was suggested that HPV16 infected women with low expression of hnRNP E2protein had a higher risk of malignant transformation of cervical lesions.Therefore,while actively preventing and controlling high-risk HPV infection,it was important to detect hnRNP E2 for the prevention and prediction of cervical cancer.2.With the severity of cervical lesions,hnRNP E2 expression gradually decreased.It was suggested that the expression of hnRNP E2 could reflect the progress of cervical lesions,and the low expression of hnRNP E2 might be an effective indicator of the aggravation of cervical lesions,and could be used as a diagnostic and early warning sign of high cervical lesions and canceration.3.The hnRNP E2 binding sites and genes were widely distributed,on all 23chromosomes.It was further found that there were many binding sites on functional elements in the intergene region and intron region,suggesting that hnRNP E2 might bind introns,regulated gene expression,and then affected the occurrence of cervical cancer.4.HnRNP E2 target genes and their products were involved in molecular functions and biological processes,including gene transcription,translation,cell cycle and apoptosis regulation,suggestting that hnRNP E2 might promote the occurrence of cervical cancer by affecting the biological function of cells.5.The hnRNP E2 binding genes were enriched in a variety of tumor pathways,the MAPK signaling pathway,and the estrogen signaling pathway,which were closely related to cervical cancer,suggestting that hnRNP E2 has a deeper molecular mechanism in the development of cervical cancer.