| Hepatocellular carcinoma(HCC)is one of the most common malignant tumors worldwide.China has a high incidence of HCC,accounting for 55%of the world's incidence.Due to the long-term infection of hepatitis B virus,most patient in China developed into HCC after chronic hepatitis and cirrhosis.HCC is characterized by insidious onset,difficult early diagnosis,poor prognosis and rapid progression.The mortality rate ranks second in tumors,so it is crucial to find a new and effective biomarker for the diagnosis,prognosis and treatment of HCC.There is growing evidence that the immune system plays a decisive role in the development and progression of cancer.Immune cells not only regulate the body's anti-tumor immunity,but also play an important role in regulating the proliferation and metastasis of tumor cells.Immunotherapy has received more and more attention in recent years,especially with the clinical effect of CTLA-4 and PD-1 antibodies.For example,as an anti-PD-1 monoclonal antibody,Nivolumab can block PD-1 by interfering with signal pathways and restore the body's anti-cancer immune response.In HCC,Immunotherapy can provide a safe,effective and promising treatment for HCC.However,the liver's immune tolerance and the heterogeneity of the tumor itself are still the main problems.It is of great value to find new immune target genes that affect prognosis and immunotherapy.The research on immune-related genes is still limited at home and abroad.Despite the continuous improvement of sequencing technology and microarray technology in HCC research,there are still a large number of differentially expressed immune-related genes in HCC,and the molecular mechanism of functional regulation is unclear.So far,no immune-related genes have been screened for clinical diagnosis,prognosis or therapeutic targets.DCK(deoxycytidine kinase)belongs to the DCK/DGK family and is essential for the phosphorylation of several deoxynucleotides and their analogues.DCK is also a key enzyme in DNA repair,which plays a vital role in keeping normal DNA metabolism.Current studies have shown that the lack of DCK is related to the resistance of anti-virus and anti-cancer chemotherapy drugs.At the same time,studies have reported that DCK plays an important role in the occurrence and development of breast cancer,cervical cancer,esophageal cancer and other tumors.Through bioinformatics analysis,we noted that DCK was highly expressed in HCC,which was associated with the survival of HCC patients.The abnormal high expression of DCK indicated poor prognosis.However,the specific mechanism of DCK in HCC has not been reported,and needs to be further explored.Exploring and discovering new immune-related genes and its specific regulatory mechanism can provide a new theoretical basis for the study of pathogenesis of HCC,and provide a theoretical basis for the discovery of new therapeutic targets.This paper takes this as the starting point,and the research contents are as follows:Part I,Based on the TCGA database and the ImmPort database,differentially expressed immune-related genes between HCC tissues and adjacent tissue were screened by using bioinformatics analysis.We further excavated immune-related genes that have prognostic value for patients with HCC,and identified immune-related genes that play a key role in the development of HCC.Part II,several open databases were established to identify the expression of DCK in HCC.We focused on the relationship between DCK and immune microenvironment.Then we detected the expression of DCK in HBV-related HCC and adjacent tissues,and investigated the relevance between DCK and clinicopathological characteristics.Part ?,By silencing the expression of DCK in HepG2 and SKHEP1,the effects of DCK on various malignant biological behaviors of hepatoma cell lines were studied.The effects of DCK on the tumorigenicity of HepG2 cells in vivo were evaluated by nude mouse tumorigenesis experiment.The molecular mechanism of DCK regulating the occurrence and progress of hepatoma was preliminarily explored.Part ? Screening of candidate immune-related genes for HCC Objectives:Based on the TCGA database and ImmPort database,the bioinformatics analysis was used to screen out differentially expressed immune-related genes between HCC tissues and adjacent tissues,and then excavate immune-related genes that have prognostic value for patients with HCC.Methods:1.Screening differentially expressed immune-related genes in HCC tissues using TCGA database and ImmPort database.Gene expression data and clinical data of HCC patients were downloaded from TCGA database.Then we downloaded 1811 immune-related genes in the ImmPort database.In R software,the Wilcoxon test was used to detect differentially expressed immune-related genes in HCC tissues and adjacent tissues.The potential biological functions of differentially expressed immune-related genes were evaluated.2.Screening for differentially expressed immune-related genes related to survival.Survival analysis was performed only in patients with HCC who were followed up for less than 2,000 days.In R software,Kaplan-Meier survival analysis method and Cox method were used to search for differentially expressed immune-related genes related to survival,respectively,and P value less than 0.01 was used as the filtration criteria.After searching by the two methods,the co-existence of differentially expressed immune-related genes with prognostic value was retained.3.Screening of independent prognostic factors.Multivariable Cox analysis was performed for each gene obtained in the previous step along with clinical data(age,grade,T stage,etc.),and a P value of less than 0.01 was used as a screening condition to screen independent prognostic immune-related genes.4.Predicting analysis of transcription factors that regulate immune-related genes for independent prognosis of HCC.In order to study the regulatory mechanism of immune-related genes,we extracted 318 transcription factors from the Cistrome cancer database for further research.We used TCGA HCC data to find differentially expressed transcription factors and analyze their survival.Then we used contest function in R to get the correlation between survival related TFs and prognostic immune-related genes.The filtering criteria were correlation coefficients greater than 0.5 and P values less than 0.05.Cytoscape software was applied to build and visualize regulatory networks.Results:1.Results of differentially expressed immune-related genes in HCC tissues.The pre-processed RNA-Seq-FPKM data of 374 cases of HCC and 50 cases of adjacent tissues were downloaded from the TCGA database for further analysis.A total of 329 immune-related genes(267 up-regulated and 62 down-regulated)were differentially expressed in HCC tissues compared with adjacent tissues.The biological functions of 329 differentially expressed immune-related genes were further analyzed by GO and KEGG analysis.2.Screening of survival-related differentially expressed immune-related genes.We used the 329 differentially expressed immune-related genes from the differential analysis for survival analysis.According to the original data,a total of 337 HCC patients who were followed up for less than 2000 days were found.Univariate Cox analysis was used to detect the expression of 329 differential immune-related genes in 337 HCC tissues,and a total of 64 survival-related immune-related genes were found.The Kaplan-Meier method was used to analyze 58 survival-related immune-related genes.Using the Venn diagram,32 coexisting survival-related immune-related genes were found,all of which were high-risk genes.3.Results of screening independent prognosis of immune-related genes.Independent prognosis analysis was performed on the 32 immune-related genes obtained above,and 12 immune-related genes were obtained as independent prognostic factors.Among them,DCK was abnormally expressed in a variety of malignant tumors and participated in the initiation and progression of tumors.However,the specific mechanism of DCK in HCC has not been elucidated,which needs to be further explored.4.Construction of regulatory network of transcription factors and independent prognostic immune-related genes.Compared with adjacent tissues,a total of 117 transcription factors(108 up-regulated and 9 down-regulated)were differentially expressed in HCC tissues.Using univariate Cox analysis and Kaplan-Meier method analysis,27 survival-related transcription factors were obtained.According to the filtering criteria,a total of 25 survival related transcription factors and nine prognostic immune-related genes were identified to establish a network.Among them,HCFC1 regulated most immune-related genes.We predicted that the most important transcription factor upstream of DCK was HCFC1.Conclusions:1.Through the analysis of TCGA and ImmPort database,we found that 12 differentially expressed immune-related genes were closely related to the prognosis of HCC,and could independently predict the prognosis.Among them,the biological role of DCK in HCC is not clear,and DCK may be the key immune-related gene of HCC.2.It was predicted that 25 transcription factors could regulate one or more immune-related genes,among which HCFC1 played an important regulatory role in HCC,and it was preliminarily confirmed that HCFC1 was the most important upstream regulatory transcription factor of DCK.Part ? The expression of DCK in hepatocellular carcinoma and its correlation with pathological parameters in HBV-related hepatocellular carcinomaObjectives:The expression of DCK in HCC was determined by several open databases.We explored the relationship between DCK and immune microenvironment,and predicted the related pathways of DCK regulation in HCC.We detected the expression of DCK in HBV-related hepatocellular carcinoma.Then we studied the relationship between DCK expression and the clinicopathological characteristics of HBV-related hepatocellular carcinoma.The expression levels of DCK mRNA and protein in different human hepatoma cell lines(HepG2,SKHEP1)and normal hepatocytes HL-7702 were detected.Methods:1.We queried the expression of DCK in HCC tissues and corresponding normal tissues through the oncomine database,GEPIA website and ICGC database.2.We used TIMER website to investigate the relationship between DCK and tumor immune microenvironment in TCGA HCC patients;Then we used GSEA method to predict the related pathways of DCK regulation in HCC.3. QRT-PCR was used to detect the expression of DCK in 53 cases of HBV-related HCC tissues and adjacent tissues,and the relevance between DCK and clinicopathological parameters was analyzed.4.QRT-PCR and Western blot were used to detect the expression of DCK in different human hepatoma cell lines(HepG2,SKHEP1)and normal liver cells HL-7702.Results:1.The results of ICGC,oncomine and GEPIA suggested that DCK expression was higher in HCC tissues.2.DCK expression was positively correlated with CD4+ T cells,neutrophils,dendritic cells,B cells,macrophages and CD8+ T cells in the tumor immune microenvironment.GSEA shows that DCK can positively regulate a variety of pathways to affect the occurrence and development of HCC, including WNT signaling pathway and ERBB signaling pathway.3.QRT-PCR detection showed that the expression level of DCK in HBV-related hepatocellular carcinoma tissues were significantly higher than that in adjacent tissues.The expression of DCK was not correlated with age and gender,but correlated with tumor size,Edmondson grade,microvascular invasion.4.The expression levels of DCK mRNA and protein in HepG2 and SKHEP1 were higher than those in normal hepatocytes.Conclusions:DCK was highly expressed in HCC tissues.The expression level of DCK was positively correlated with immune ceils in immune microenvironment,indicating that DCK could reflect the status of immune microenvironment.DCK was highly expressed in HBV-related hepatocellular carcinoma tissues,and associated with a variety of clinicopathological parameters.DCK was highly expressed in HepG2 and SKHEP1,which can be used in subsequent experiments.Part ? The malignant biological characteristics of hepatoma cells promoted by DCKObjectives:By interfering with the expression of DCK in HepG2 and SKHEP1 cells,we detected the effect of DCK on the proliferation,apoptosis,invasion and other malignant biological behaviors of hepatoma cells,and fiirther verified it by nude mice tumor formation experiment.We preliminarily discussed the molecular mechanism of DCK promoting the occurrence and progress of HCC.Methods:QRT-PCR and Western blot were used to screen the most efficient sequence.Transwell invasion test,wound healing test,CCK-8 proliferation test and flow cytometry were used to examine the migration,invasion,proliferation and apoptosis of HCC cells after low expression of DCK gene.Nude mice were subcutaneously injected with HepG2 of siRNA DCK group and negative control group.After 4 weeks,the nude mice were killed,and the tumor was stripped and weighed.Western blot and cellular immunofluorescence were used to detect the effect of DCK on Wnt-I ?-catenin and c-myc.Results:One siRNA was successfully screened for subsequent experiments.CCK-8 assay showed that the proliferation of HepG2 and SKHEP1 cells was significantly decreased.Cell scratch test and Transwell invasion assay showed that HepG2 and SKHEP1 cells with silenced expression of DCK exhibited significantly attenuated migration and invasion.Flow cytometry analysis showed that DCK down-regulation could promote the apoptosis of hepatoma cells.The results of animal experiments showed that the growth rate of tumor in DCK interference group was slow,and the mass and volume of tumor decreased significantly.After inhibiting the expression of DCK,the protein expression of WNT signaling pathway decreased.This suggested that DCK abnormally activated WNT signaling pathway,which enables increased the expression of key signaling molecules Wnt-1,?-catenin and c,myc,and promoted the malignant process of hepatocellular carcinoma.Conclusions:In vitro,silencing DCK gene inhibited the proliferation,migration and invasion of hepatoma cells HepG2 and SKHEP1,and promoted the apoptosis of hepatoma cells.Animal experiments showed that,silencing DCK significantly inhibited the tumorigenicity in nude mice.DCK may afifect the malignant biological behavior of HCC by activating WNT signaling pathway.DCK is expected to be a new target for Ihe treatment of HCC. |
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