Relationship Between HnRNP K And Cervical Carcinogenesis And Analysis Of Whole Genomic Binding And Function Of HnRNP K In Siha Cell

ObjectiveCervical cancer is a complicated disease affected by multiple factors,of which persistent infection of high risk human papillomavirus（HPV）has been recognized as a necessary cause.However,there are other factors that contribute to its development.K homology（KH）domain of heterogeneous nuclear ribonucleoprotein K（hnRNP K）can recognize and bond DNA and RNA sequences,and participates in a variety of biological processes such as the regulation of circle,apoptosis,invasion and metastasis of cell,therefore,it is of great significance to the development of a tumor.It has been found that hnRNP K was expressed abnormally high in many tumors,but the expression of hnRNP K in cervical carcinogenesis remains unclear.The current researches on the interaction hnRNP K and DNA is common in the combination of hnRNP K with the promoter of some genes,whereas,the binding of hnRNP K with cervical cancer cell whole genome DNA is still unclear.This study aims to investigate the relationship between hnRNP K and cervical cancer,moreover,explores the binding sites in the whole genome,possible functions and pathways taking part in of hnRNP K in Siha cell comprehensively and systematically by using Ch IP-Seq which combines Ch IP with second-generation sequencing technology.Finally,it provides new clues for further research on the pathogenesis of cervical cancer and puts forward a scientific basis for finding the target of prevention and cure for cervical cancer.MethodsIn the cervical lesions cohort established by our research group from June 2014 to September 2014 in Jiexiu,Shanxi Province,the patients were randomly selected for cervical cytology thin film cytology（TCT）screening,colposcopy,and pathological diagnosis.There were 67 females with normal cervical（NC）,69 with low cervical intraepithelial neoplasia（CINI）,68 with high cervical intraepithelial neoplasia（CINII/III）,and 40 patients with cervical squamous cell carcinoma（SCC）who were diagnosed at the Shanxi Provincial Cancer Hospital.A structured questionnaire was used to investigate the relevant factors of cervical lesions and collected cervical exfoliated cells,and biopsy or surgical cervical tissue specimens in all study objects.The Infection of HPV and expression level of hnRNP K protein were detected by Diversion Hybridization and Western Blot,respectively.SPSS 21.0 software was used to perform Logistic regression analysis,Kruskal-Wallis H test,Bonferroni test,x² test and x_trend² test.At the same time,HPV16-positive Siha cell was selected as the experimental subject.Application of Ch IP and second-generation sequencing technology identified the binding sites of hnRNP K in the whole genome.The characteristics of hnRNP K-binding DNA sequences analysis,the enrichment analysis of hnRNP K binding genes function and pathway were performed using MEME software,GO database,and KEGG database,respectively.Results 1.Relationship between hnRNP K protein expression and cervical lesions: The total distribution of hnRNP K protein expression in each group of cervical lesions was statistically significant（H=42.57,P<0.001）.The expression levels of hnRNP K protein in CINI group,CINII/III group and SCC group were higher than those in NC group.Except that the difference between CINII/III group and SCC group had no statistical significance,the difference among the other groups was statistically significant.With the development of cervical lesions,the high expression rate of hnRNP K protein showed a gradually increasing trend（χ2 trend = 21.20,P<0.001）.After adjustment for related factors of cervical lesions,a high expression of hnRNP K protein increased the risk of CINII/III（OR=6.23,95%CI:2.50-13.44）and SCC（OR=5.01,95%CI:2.15-14.02）.2.Distribution of hnRNP K binding sites in whole genome: The number of DNA binding sites for hnRNP K in genome-wide was 21,963,and the average length was 150 bp.The hnRNP K binding sites were found in each chromosome,and there were more binding sites in chromosomes 1,2,5 and X.The hnRNP K binding sites were mostly in the intergenic region（31.85%）and the intron region（28.56%）,but were also distributed in the exon region,2 kb upstream of the transcription initiation site,and 2 kb downstream of the transcription initiation site.3.Characterization of hnRNP K binding DNA sequences: Motif analysis revealed that hnRNP K had four motifs that were easy to bind.The contents of Cytosine（C）and Thymine（T）in these motifs were high,especially Cytosine（C）,suggesting that hnRNP K tended to binding to DNA sequences enriched with Cytosine（C）and Thymine（T）.4.Distribution of hnRNP K binding genes in chromosomes: Of the 21,963 hnRNP K binding sites,14,968 were annotated as gene fragments covering 1,321 genes.The distribution of hnRNP K binding sites was found in each chromosome,and there were more genes distributed in chromosomes 1,2,5 and 7.5.Functional analysis of hnRNP K binding genes: GO enrichment analysis revealed that hnRNP K binding genes were mainly enriched in nucleic acid binding（GO: 0003676）at the Molecular Function level,including DNA binding（GO: 0003677）and RNA binding（GO: 0003723）.It also had transporter activity（GO:0005215）,molecular function regulator（GO:0098772）and transcription cofactor activity（GO:0003712）,etc.There were regulation of cellular process（GO: 0050794）,signal transduction（GO:0007165）,RNA processing（GO:0006396）,biological regulation（GO:0065007）and regulation of cell cycle（GO:0051726）at the level of Biological Process.At the Cellular Component level,there were mainly membrane（GO:0016020）,cytoskeleton（GO: 0005856）and so on.6.Pathway analysis of hnRNP K binding genes: Pathway enrichment analysis showed that most of the genes accumulated in Pathways in cancer（ko05200）,moreover,Breast cancer（ko05224）,Colorectal cancer（ko05210）,Endometrial cancer（ko05213）,Melanoma（ko05218）,Pancreatic cancer（ko05212）,Prostate cancer（ko05215）and other cancer pathways were found.Tumor related pathways such as TNF signaling pathway（ko04668）,Transcriptional misregulation in cancer（ko05202）and Micro RNAs in cancer（ko05206）had been diacovered as well.There were also important pathways that were enriched in RNA transport（ko03013）,p53 signaling（ko04115）,Wnt signaling（ko04310）,MAPK signaling（ko04010）,cell cycle（ko04110）,and apoptosis（ko04210）.In addition,this study specifically identified pathways that were closely related to cervical cancer,such as the Viral carcinogenesis（ko05203）and Estrogen signaling pathway（ko04915）.Conclusion 1.The high expression of hnRNP K protein can increase the risk of high grade cervical intraepithelial neoplasia and cervical cancer.The increased expression of hnRNP K protein in low grade cervical intraepithelial neoplasia suggests that hnRNP K protein can provide a biomarker for the early diagnosis of cervical cancer.2.The hnRNP K protein is widely combined with genomic DNA,and there are binding sites and binding genes in each chromosome.The binding sites are mainly distributed in the intergenic and intron region of the human genome,while they are not in promoter,which suggests that hnRNP K may have new regulatory mechanisms in cervical cancer and it might be more complex.3.hnRNP K possesses the functions including controlling gene transcription and translation,regulating cell processes including cell cycle,apoptosis,migration,adhesion,as well as division,which plays an important role in signal transduction,biological regulation and so on,suggesting it may promote the occurrence and development of cervical cancer by affecting the biological behavior of cell.4.hnRNP K binding genes are mainly enriched in tumor related biological pathways and participate in several important signaling pathways.It is noteworthy that hnRNP K is involved in several pathways closely associated to cervical cancer,such as Viral carcinogenesis and Estrogen signaling pathway,suggesting that hnRNP K is of great significance in the development of cervical cancer.