| Part One To reveal potential molecular markers for cervical squamous cell carcinoma by analysis of differentially expressed and methylated genes based on bioinformatics analysisObjective: To screen potential molecular markers of CESC,based on integrated bioinformatics analysis.Methods:1.The Gene Expression Omnibus(GEO)database contains highthroughput gene expression data submitted by research institutions around the world,including genome methylation,chromatin structure and genomeprotein interaction data.In this study,the GEO database was used to screen out the m RNA datasets and methylation datasets of cervical squamous cell carcinoma and normal control tissues that met the inclusion criteria.2.The meta MA package was used to analyze RNA-seq gene expression data in cervical squamous cell carcinoma and normal tissues to identify differentially expressed mrnas(dem Rnas).3.The KEGG database contained 17 sub-databases in 4 categories.The genome information was stored in the gene database.Gene function information is stored in the advanced functional PATHWAY database,which deals with the functional interactions of biomolecules(including proteins)and contains graphically detailed 4794 biochemical processes(i.e.,pathways,sub-pathways,signaling,cell cycle,etc.)in human cells.The GO database was used to describe the function of gene products.In this study,various biological information databases such as KEGG and GO and analysis tools such as R were used to perform GO and KEGG enrichment analysis of differentially expressed mrnas.4.The Bio GRID database is a database of gene and protein interactions in humans and model species,which mainly manages and stores information on protein,genetic and drug interactions.In this study,the protein interaction data were downloaded from Bio GRID database,and Cytoscape software was used to search the top 50 up-regulated differentially expressed mrnas and the top 50 down-regulated differentially expressed mrnas.After excluding the non-differentially expressed genes,the interaction network of differentially expressed m RNA proteins was drawn.5.Quantile standardization between methylation data sets,using R package COHCAP differences for the identification of patients with cervical squamous carcinoma and normal tissues methylation site.The identified differentially methylated sites were clustered among the samples to screen the differentially methylated regions.Using David6.8 to GO different methylation m RNA enrichment and enrichment of KEGG function analysis.The differentially methylated regions,differentially methylated sites and differentially expressed genes were further correlated.Genes with hypomethylation and high expression or hypermethylation and low expression were selected for functional enrichment analysis.6.Prognostic information of CESC patients was downloaded from The Cancer Genome Atlas(TCGA)database to construct differential methylated genes(DMGs)prognostic risk model.7.Kaplan-Meier Plotter is an online database based on gene chip and RNA-seq data from GEO,EGA and TCGA,which can evaluate the impact of more than 54,000 genes on the survival prognosis of 21 common malignant tumors.This study used this database to analyze the 1-year,3-year,and 5-year survival rates of CESC patients,and to evaluate the predictive efficiency of the risk score for 1-year,3-year,and 5-year survival rates.l.8.Based on the risk model,the patients who met the search requirements in TCGA were divided into high and low risk groups,and the Kaplan-Meier method was used to evaluate the overall survival curves of the high-risk and low-risk risk groups.A time-dependent receiver operating characteristic(ROC)curve was used to evaluate the diagnostic accuracy of the prognostic model.An AUC greater than 0.65 was considered an ideal model.9.THPA database was used to verify the protein expression levels of candidate genes,and real-time quantitative polymerase chain reaction(RT-PCR)was used to verify the expression levels of candidate genes in clinical samples..Results:1.A total of 1438 DEm RNAs(723 up-regulated and 715 down-regulated)were obtained by differential expression analysis based on GEO database.2.A total of 342,355 methylation sites and 1669 DMS(corresponding to844 mrnas)were screened.In addition,46 differentially methylated Cp G islands were obtained,including 42 hypermethylated regions and 4 hypomethylated regions.A total of 79 methylation sites(35 hypermethylation sites and 44 hypomethylation sites)associated with differentially expressed genes were identified,and a total of 53 differentially methylated genes were identified.Among the differentially expressed genes,17 were up-regulated and 36 were down-regulated.3.12 low-expressed hypermethylated genes and 16 high-expressed hypomethylated genes were selected for functional enrichment analysis,and String database was used to construct PPI network,which contained 22 interacting gene pairs.The highest interaction scores were MX2(hypomethylated with higher expression)and IRF8(hypermethylated with lower expression).4.Five DMGs(HHEX,VCAM1,ZCWPW1,ZIK1 and ZNRF2)were found to be associated with poor prognosis of cervical squamous cell carcinoma.The established risk score model suggested that the mortality rate in the high-risk group was higher than that in the low-risk group,and the high risk score and higher stage of tumor lymph node metastasis(TNM)were associated with poor prognosis.Multivariate cox analysis showed that the five gene methylation risk model and TNM stage could be used as independent prognostic indicators,and the risk score could be used as an independent prognostic factor in patients with cervical cancer.5.Diagnostic analysis in TCGA database showed that the AUC of ZCWPW1 was 0.461,which was not suitable as a diagnostic marker.The AUC values of HHEX,VCAM1,ZIK1 and ZNRF2 were all greater than 0.7.6.Based on THPA database,the protein expression levels of HHEX,VCAM1,ZNRF2,MX2 and IRF8 were up-regulated,while ZIK1 was down-regulated in CESC tissues.The q RT-PCR results showed that the m RNA expression levels of HHEX,VCAM1,ZNRF2,MX2 and IRF8 were up-regulated,while ZIK1 was down-regulated,which was consistent with the results of bioinformatics analysis.Conclusions:1.It suggests that differentially methylated genes and differentially expressed genes may play an important role in the progression of cervical squamous cell carcinoma.2.The survival prognosis model based on HHEX,VCAM1,ZCWPW1,ZIK1 and ZNRF2 can accurately predict the prognosis of cervical squamous cell carcinoma.3.HHEX,VCAM1,ZIK1 and ZNRF2 are not only differentially expressed genes but also differentially methylated genes,which may be potential molecular markers for the diagnosis and prognosis evaluation of CESC.Part Two Expression level and clinical significance of VCAM1 in cerv-ical squamous cell carcinomaObjective: To determine the expression level and clinical significance of VCAM1 in cervical squamous cell carcinomatumor(CESC).1.Study subjects:A total of 39 patients with cervical intraepithelial neoplasia grade III(CIN group)and 54 patients with cervical squamous cell carcinoma(cervical cancer group)who underwent surgery in Hebei Provincial People’s Hospital from March 2019 to November 2020 were selected,and 30 patients with benign uterine lesions who underwent surgery during the same period were selected as the control group.All patients underwent transvaginal color Doppler ultrasound(TVCDU)examination before operation.2.Specimen collection:Cervical cancer group and control group: about 200 mg of fresh normal cervical tissue and cervical squamous cell carcinoma tissue samples were collected in the operating room.The tissue samples were washed with 0.9%saline immediately after isolation and divided into two parts.One part was immediately stored in liquid nitrogen,and the other was fixed in 10%formaldehyde and embedded in paraffin.CIN group: postoperative paraffin specimens.Methods:1.The differential expression of VCAM1 in cervical squamous cell carcinoma and normal cervical tissues were measured by RT-PCR.2.To analyze the correlation between the m RNA expression level of VCAM1 in cervical squamous cell carcinoma and clinicopathologic features of cervical squamous cell carcinoma.3.To investigate the correlation between the expression level of VCAM1 and the ultrasonic blood flow parameters in the three groups.1)Using the American GE-Voluson E8 color Doppler ultrasound diagnostic system,transvaginal color Doppler ultrasound was performed on the three groups of patients.The uterus and bilateral adnexa were routinely detected,the size,echo and blood flow of the cervix were observed,and PSV and RI were recorded.For those with cervical lesions,the size,boundary and internal echo of the lesion were observed,and the longest diameter of the lesion was recorded.The long diameter,PSV and RI were measured three times and averaged at last.2)The expression levels of VCAM1 in cervical squamous cell carcinoma tissues,normal cervical tissues and cervical intraepithelial neoplasia grade III tissues were detected by immunohistochemistry,and the correlation between VCAM1 expression and ultrasonic blood flow parameters was analyzed.Results:1.RT-PCR showed the expression level of VCAM1 in cervical squamous cell carcinoma and normal cervical tissues,and the expression level of VCAM1 in CESC cancer tissues was significantly higher than that in normal tissues(P<0.05).2.Differential expression of VCAM1 in cervical squamous cell carcinoma is correlated with the degree of differentiation of cervical squamous cell carcinoma(P<0.05),correlated with lymph node metastasis(P< 0.05),and had no clear correlation with age,BMI,tumor size or FIGO stage(P>0.05).3.The results showed that blood flow parameters PSV and RI of TVCDU in patients in the control group,CIN group and cervical squamous cell carcinoma group were statistically significant(P<0.05),PSV in cervical cancer group was significantly higher than that in control group and CIN group,PSV in CIN group was significantly higher than that in control group,RI in cervical cancer group was significantly lower than that in control group,RI in CIN group was significantly lower than that in control group,and the differences were statistically significan(P<0.05).4.Immunohistochemical analysis showed that that the positive rates of VCAM-1 expression in stage I,II and III patients were significantly higher than those in the control group and CIN group,the positive rates of VCAM-1expression in CIN group were significantly higher than those in the control group.All the differences were statistically significant(P <0.05).However,there was no significant difference in the positive rate of VCAM1 expression in stage I,II and III cervical cancer patients(P <0.05).5.Spearman correlation analysis showed that positive expression of VCAM-1 was significantly positively correlated with PSV(r>0,P<0.05);The positive expression of VCAM1 was negatively correlated with RI(r>0,P<0.05).Conclusions:1.VCAM1 is highly expressed in cervical squamous cell carcinoma,and is positively correlated with lymph node metastasis and malignant degree of cervical squamous cell carcinoma.It suggests that VCAM1 is closely involved in the progression of cervical squamous cell carcinoma and may be an important target for the treatment and prognosis evaluation of cervical squamous cell carcinoma.2.The expression level of VCAM1 and the blood flow parameters PSV and RI of TVCDU are closely related to the development of cervical lesions,which can reflect the degree of cervical lesions from different angles,and provide a new method for early diagnosis and early prognosis evaluation of cervical cancer.Part Three Effects of VCAM1 on proliferation,migration,invasion and apoptosis of cervical squamous cell cells and the related mechanismsObjective: To investigate the effect of VCAM1 on proliferation,migration,invasion and apoptosis of cervical squamous cell cells and the related mechanisms.Methods:1.Western blot were used to detect the expression level of VCAM1 in Caski and Siha cells of cervical squamous cell carcinoma.2.Si-VCAM1 and blank control NC were designed and synthesized,and transfected into Caski and Siha cells.The expression of VCAM1 in Caski and Siha cells was detected by RT-PCR.3.CCK-8 assay was used to detect the effect of knockdown of VCAM1 on the proliferation of cervical squamous cell carcinoma cells.4.Colony Formation Assays were used to detect the effect of knockdown of VCAM1 on the colony formation ability of cervical squamous cell carcinoma cells5.Flow cytometry(FCM)was used to detect the effect of knockdown of VCAM1 on the apoptosis of cervical squamous cell carcinoma cells.6.Flow cytometry(FCM)was used to detect the effect of knockdown of VCAM1 on cell cycle of cervical squamous cell carcinoma cells.7.The effect of knockdown of VCAM1 on the migration ability of cervical squamous cell carcinoma cells was detected by Transwell chamber migration assay.8.Transwell chamber invasion assay was used to detect the effect of knockdown of VCAM1 on the invasion ability of cervical squamous cell carcinoma cells.9.Wound healing assay was used to detect the effect of knockdown of VCAM1 on the migration ability of cervical squamous cell carcinoma cells.10.Western blot was used to detect the effect of knockdown of VCAM1 on the expression of Caspase-3 and Caspase-8 in cervical squamous cell carcinoma cells.11.Western blot was used to detect the effect of knockdown of VCAM1 on the expression of Ki-67 and PCNA in cervical squamous cell carcinoma cells.12.Western blot was used to detect the effect of knockdown of VCAM1 on the expressions of N-cadherin,E-cadherin and Vimentin in cervical squamous cell carcinoma cells.13.Western blot was used to detect the effect of knockdown of VCAM1 on the expression of NF-κB signaling pathway proteins in cervical squamous cell carcinoma cells.Results:1.Western blot was used to detect the expression level of VCAM1 protein in two types of cervical squamous cell lines.The expression level of VCAM1 protein in Caski and Siha cells was relatively high,so it was possible to interfere with the expression of VCAM1 in Caski and Siha cells for follow-up studies.2.After transfection with different VCAM1 si RNA,q RT-PCR analysis showed that the expression of VCAM1 m RNA in Caski and Siha was decreased.3.The results of cell proliferation showed that the cell proliferation ability of VCAM1 knockdown group was significantly lower than that of control group(P<0.05).4.Colony formation assay showed that the colony formation ability of VCAM1 knockdown cervical squamous cell carcinoma cells was significantly decreased compared with the control group(P<0.01).5.Flow cytometry showed that the apoptosis level of cervical squamous cell carcinoma cells in VCAM1 knockdown group was significantly higher than that in the control group(P<0.05).6.Flow cytometry analysis showed that the proportion of S phase of cervical squamous cell carcinoma cells in VCAM1 knockdown group was significantly higher than that in control group(P<0.01).7.The cell scratch assay and Transwell cell migration assay showed that,compared with the control group,the migration ability of cervical squamous cell carcinoma cells decreased significantly after VCAM1 knockdown(P<0.05).8.Transwell cell invasion assay showed that the invasion ability of cervical squamous cell carcinoma cells decreased significantly after VCAM1 knockdown compared with the control group(P<0.05).9.Western blot results showed that the expression levels of Caspase3 and Caspase8 in knockdown of VCAM1 group were significantly lower than those in control group(P<0.05).10.Western blot results showed that,compared with the control group,the expression levels of Ki-67 and PCNA in cervical squamous cell carcinoma cells were significantly decreased after VCAM1 knockdown(P<0.01).11.Western blot results showed that the expression level of E-cadherin in the VCAM1 knockdown group was significantly higher than that in the control group(P<0.01),while the expression levels of N-cadherin and Vimentin were significantly lower than those in the control group(P<0.01).12.Western detection results showed that compared with the control group,the protein expression levels of p-IκBα and p-NF-κB in cervical squamous cell carcinoma cells in the knockdown of VCAM1 group were significantly different(P<0.05).Conclusions:1.Transfection of si-VCAM1 can effectively reduce the expression level of VCAM1 in cervical squamous cell carcinoma cells.2.Knockdown of VCAM1 can significantly inhibit the proliferation and colony formation ability of cervical squamous cell carcinoma cells,and promote the apoptosis of cervical squamous cell carcinoma cells.3.Knockdown of VCAM1 expression can significantly inhibit the migration and invasion ability of cervical squamous cell carcinoma cells.4.VCAM1 may regulate the apoptosis of cervical squamous cell carcinoma cells by inhibiting the expression of Caspase3 and Caspase8 proteins.5.VCAM1 may mediate the migration and invasion of cervical squamous cell carcinoma cells by regulating EMT pathway.6.VCAM1 may be involved in the progression of cervical squamous cell carcinoma by targeting the NF-κB signaling pathway. |
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