| Cervical cancer is one of the leading causes of cancer death in women around the world, the incidence of which is the second among women. The high-risk HPV (Human papilloma virus) is the main risk factor for cervical cancer. The DNA of HPV can be detected in over 90% of cervical squamous cell carcinoma and over 50% of cervical adenocarcinoma. HPV is a double-stranded circular DNA virus. High-risk HPV-encoded viral proteins E6 and E7 play an important role in the development of cervical cancer and disrupt the process of cell cycle progression and apoptosis.Although multivalent HPV prophylactic vaccines have been successfully developed and are currently available in Mainland China, their effectiveness is limited to people who are already infected or have immune deficiency. Therefore, fully understand the mechanism by which high-risk HPV induces cancer is still very important.In order to explore the pathogenic mechanism of high risk HPV, we performed a bioinformatics analysis of cervical cancer samples though TCGA public database, a list of high-expression genes in cervical cancer and a DNA methylation status score table of related gene promoters was established and analyzed. Though liquid chromatography-tandem mass spectrometry (LC-MS/MS), E7-expressing cells and vector control cells were detected by non-labeled quantitative analysis, the differentially expressed protein group was found in E7 stably expressed cells and contral cells. We cross analyzed the datas from TCGA database , LC-MS/MS datas of E7 expression cells and control cells, as well as our former RNA-seq datas. DAVID database was applied for GO (Gene Ontology) analysis and KEGG pathway analysis.and the common upregulated genes were analyzed by online database STRING for network signaling pathway. There were only three genes which were upregulated in all the three data sets we metioned above, CENPE,CDK1 and UHRF1. We analyzed the correlation between the UHRF1 protein and cervical cancer though the TCGA and GEPIA (Gene Expression Profiling Interactive Analysis) database and carried out a follow-up study.By further analysis of the RNA-seq results, we found that there were significant differences in the expression of 237 genes between the cells expressing HPV E7 and the control cells. Among these genes, UHRF1 (coding UHRF1 protein, also known as NP95 protein or ICBP90 protein) was upregulated in HPV-16 E7 expression cells, while UBE2L6 (coding ubiquitin / ISG15- binding enzyme E2 L6, UbcH8 protein) was downregulated. As a double E2 enzyme in Ubiqutination and ISGylation, UbcH8 involves apoptosis and other biological activities. We use clinical cervical cancer tissue samples, the E7expression cell line and control cell line, and cervical cancer cell line to further verify the expression of UHRF1 and UBE2L6 in mRNA and protein levels. We found that the expression of UHRF1 was negatively correlated with UBE2L6 gene, UHRF1 regulated methylation of UBE2L6 gene promoter epigeneticly, thus affecting the expression of its encoding protein UbcH8. Functional studies have shown that UHRF1 affects apoptosis by regulating UBE2L6/UbcH8.This study confirmed our recent RNA-seq results, that is, UHRF1 is up-regulated in HPV16 E7 expression cells, and UbcH8 is down-regulated. In cervical cancer cells,we found that UHRF1 regulates the gene UBE2L6 through promoter methylation. In addition, their role in cell apoptosis is also confirmed. These results suggest that UBE2L6 can be used as a new target for mediating apoptosis of UHRF1. Therefore,our study reveals the mechanism that UHRF1 inhibits the apoptosis of cervical cancer cells and provides potential candidate drug targets for the treatment of cervical cancer.Part I Bioinformatics analysis of cervical cancer and the relationship of UHRF1 and cervical cancerMethods1. Use TCGA-Assembler to download data of cervical cancer in the TCGA database: mRNA expression data in the CESC (cervical squamous cell carcinoma and cervical adenocarcinoma) sub-database and DNA methylation data collected on the IlluminaInfiniumHumanMethylation450 bead platform.2. Detect the differentially expressed genes at the protein level in E7-expressing and control cells by tandem mass spectrometry (LC-MS / MS). The RPE1 cells (human retinal pigment epithelial cells expressing human telomerase reverse transcriptase) stably expressing HPV E7 gene and corresponding control cells(constructed by our laboratory) were selected for nuclear protein extraction and differential proteomic analysis.3. The data obtained from TCGA database and tandem mass spectrometry were processed and analyzed to obtain differentially expressed genomes, annotated with GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) cell pathways.4. Cross-analysis of TCGA data and differentially expressed proteomic data of E7 cells by mass spectrometry, as well as the RNA-seq data of E7 cell differentially expressed genes obtained earlier in our research group, a common high expression gene set was obtained and and analysised by STRING Protein network.5. The correlation of UHRF1 and cervical cancer was analysed by TCGA and GEPIA public database.Results1. Through screening and analysis of differentially expressed genes in cervical cancer in TCGA database, we found 2043 genes were significantly differentially expressed, of which 779 genes were up-regulated and 1264 genes were down-regulated when compared with normal control tissues.2. We performed a functional and pathway enrichment analysis of 779 up-regulated genes and 1264 down-regulated genes, respectively. The analysis of GO enrichment showed that in the biological process subset, the up-regulated genes were mainly concentrated in cell division and mitosis. The down-regulated genes were mainly involved in signal transduction, RNA polymerase II promoter transcription positive regulation, cell adhesion related genes and other biological processes. In the subset of cellular components, up-regulated genes were mainly concentrated in the cytoplasm, nucleus, cytosol, etc.; down-regulated genes were mainly concentrated in the plasma membrane, extracellular regions and other cellular components. In the molecular subset, the up-regulated genes were mainly enriched in genes related to protein binding, genes related to ATP binding, and genes related to homologous protein binding. Down-regulated genes were mainly enriched in genes associated with metal ions, related genes that bind to calcium ions, genes that bind to zinc ions, etc. KEGG pathway analysis showed that up-regulated genes were predominantly enriched in the cell cycle and cancer-associated signaling pathways.3. By comparing 779 genes that were significantly up-regulated in cervical cancer in TCGA database and 150 genes that were significantly up-regulated in E7-expressing cells in the preliminary RNA-seq results of this project group, and we obtained a total of 76 genes that were common in both up-regulated gene sets.4. We obtained the average DNA methylation values of 20764 promoter regions by DNA methylation analysis of 50 random samples of cervical cancer samples downloaded from the TCGA database CESC subset. Using the VLOOKUP function to sort out the promoter methylation values corresponding to the top 50 differentially expressed genes of TCGA cervical cancer from the above-mentioned 20764 values to obtain the high expression in cervical cancer with a low degree of methylation of the promoter or Low expression and high degree of promoter methylation gene list, the list of genes for methylation control candidate gene.5. Through the analysis of the correlation between UHRF1 and cervical cancer,we found that the gene copy number variation, expression level and mutation of UHRF1 have different degrees of impact on each cancer and, of which the impact of cervical cancer ranked the fourth in each cancer. The survival rate was lower in high UHRF1 expression group.6. We used mass spectrometry to analyze nucleoproteome that were differentially expressed between E7-expressing and control cells. A total of 374 proteins were found to be significantly expressed between E7 and control cells, of which 191 were up-regulated in E7-expressing cells and 183 were down-regulated in E7-expressing cells.7. Gene Ontology (GO) and cell pathway analysis (KEGG) showed that most of the genes that were upregulated in E7-expressing cells focused on biological processes such as rRNA process, viral transcription, initiation of transcription and regulation, whereas in E7-expressing cells down-regulated genes focused on cell adhesion, mRNA cleavage and other biological processes. Pathway analysis also showed that upregulated proteins in E7-expressing cells were more abundant in the ribosomal pathway and downregulated proteins were more abundant in focal adhesion and other pathways.8. Pathway analysis of the differentially expressed proteins revealed that the up-regulated nuclear proteins in E7-expressing cells were concentrated in the viral processes and mRNA metabolism pathway, the expression was also found to be enriched in the ribosomal pathway, the spliceosome pathway, the Parkinson's disease pathway, and the Huntington's disease pathway. Down-regulated nuclear proteins were concentrated in the subunit pathways of cell components and protein complexes,and in molecular function, they are enriched in the pathways of RNA binding, cytoskeletal protein binding and recruitment of cellular components.9. We cross-analyzed of the three groups of differential expression data to obtain a common up-regulated protein set. Among them, seven up-regulated proteins, DEK, CENPE, RFC3, LBR, CDK1, CDK2 and UHRF1, were common in the two groups of data (RNA-seq and mass spectrometry). There are three proteins cross-linked to these seven proteins in the TCGA database for cervical cancer, including CENPE, CDK1 and UHRF1.Part ? UHRF1 epigenetically down-regulates UbcH8 to inhibit apoptosis in cervical cancer cellsMethods1. The paraffin samples of cervical cancer and normal cervical tissue were collected to detect the expression of the highly expressed protein UHRF1 and low expression protein UbcH8 in cervical cancer and normal cervical tissue by immunohistochemistry (IHC).2. Real-time quantitative PCR and Western blot analysis validated the differentially expressed proteins.3. The effect of knockdown and overexpression of UHRF1 on UbcH8 expression was examined by transfecting siRNAs and plasmids of UHRF1 in cervical cancer cell lines.4. Chromatin immunoprecipitation (ChIP) determination of binding of UHRF1 to the UBE2L6 (encoding UbcH8) promoter.5. The methylation status of UBE2L6 promoter was detected by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP).6. The effect of UHRF1 and UbcH8 on apoptosis was detected by fluorescence double staining and V-FITC / PI flow cytometry.Results1. Through the immunohistochemical evaluation of the expression of UHRF1 and UbcH8, the expression of UHRF1 in cervical cancer tissues was significantly higher than that in normal cervical tissues, while UbcH8 was expressed in both cervical cancer tissues and normal cervical tissues. Nuclear staining in normal tissues was more pronounced.2. The knockdown of UHRF1 in cervical cancer cell line HeLa increased the expression of UbcH8, while over-expressed UHRF1 down-regulated the expression of UbcH8. Similar result was obtained in the breast cancer cell line MCF7 cells.3. Chromatin immunoprecipitation (ChIP) analysis confirmed that UHRF1 bind to two predicted CCAAT-containing domains in the UBE2L6 (encoding UbcH8) promoters. It indicates that UHRF1 plays a role in epigenetic regulation by binding to the promoter of UBE2L6.4. Methylation specific PCR (MSP) showed that the UBE2L6 promoter was hypermethylated in E7-expressing cells, and the degree of UBE2L6 promoter methylation was reduced after knocking down UHRF1. Among the 14 loci detected by BSP in HeLa cells, the methylation of loci 3-7, 9 and 12-14 reduced after knocking down UHRF1 indicating that UHRF1 apparently regulates the UBE2L6 gene by promoter methylation in cervical cancer cells.5. UHRF1 inhibits apoptosis through the regulation of UBE2L6 expression.Knockdown of UHRF1 in E7-expressing cells, apoptosis was increased by V-FITC / PI measurements. Similarly, transfection of UbcH8 expression plasmids in E7-expressing cells increased apoptosis as a target of UHRF1. Similar results were obtained in HPV E7-positive HeLa cells.Conclusions1. Through analysis of TCGA database, we found the relevant signaling pathway of differentially expressed proteins in cervical cancer and the biology progress and molecular function pathways related to UHRF1 are also found.2. Differentially expressed proteins were found in HPV E7-expressing cells and control cells and the protein-related pathways were also found.3. In cervical cancer cells, the expression of UHRF1 and UbcH8 was negatively correlated and verified in clinical tissue samples.4. In cervical cancer cells, it is confirmed that UHRF1 regulated the expression of UbcH8 by methylation of UBE2L6 promoter.5. We found that UbcH8 as a new target of UHRF1 which regulated the apoptotic function of UHRF1.Significance and innovation1. We established a method of using public database and cross analysis of big data to screen related genes.2. We obtained cervical cancer differential expression protein group and the related pathways, providing ideas for further research.3. The negative correlation between the expression of UHRF1 and UbcH8 in cervical cancer cells was found for the first time and was verified in the clinical tissue samples, which provided a candidate target for the detection of cervical cancer.4. It is confirmed that UHRF1 regulated the expression of UbcH8 by methylation of UBE2L6 promoter in cervical cancer cells for the first time. Our research shows that UHRF1 / UbcH8 are potential targets for drug development in treating cervical cancer. |
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