Screening And Prelimilary Resech Of Aberrantly Expressed RNA In HBV-related Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma is ranked as the fifth most common neoplasm and the second leading cause of cancer related death.And China alone accounting for about 50%of the total number of new cases and deaths.Hepatocellular carcinoma accounts for about 90%of the primary liver cancer and is the main type of primary liver cancer.The vast majority of hepatocellular carcinoma is associated with known cause of potential risk factors.The main risk factors include chronic viral hepatitis especially chronic hepatitis B,alcohol-related liver disease,non-alcoholic fatty liver disease,obesity,diabetes,aflatoxin exposure and cirrhosis caused by any factors.While in China,chronic hepatitis B is the main risk factors for the prevalence of hepatitis B virus infection.Hepatocellular carcinoma is a highly aggressive malignant tumor with poor prognosis.Hepatic resection and liver transplantation are regarded as the most radical way of therapy for an early stage of hepatocellular carcinoma.Unfortunately,tumor recurrence,including true recurrence due to dissemination and de novo tumors with the oncogenic liver,complicates 70%of cases ant 5 years.Advanced hepatocellular carcinoma has even poorer prognosis with a median survival time less than one year.Until now,the pathogenesis in hepatocellular carcinoma tumorigenesis,tumor invasion and tumor metastasis are unclear.Searching for novel cancer biomarkers and exploring the pathogenesis in hepatitis B related hepatocellular carcinoma tumorigenesis,tumor invasion and tumor metastasis is the critical and difficult aspect of primary liver cancer studies.In recent years,tumor genomics based on high-throughput biological technology has been widely used in cancer researches.Screening specific tumor biomarkers by using genomics or high-throughput genetic sequencing may find effective specific tumor biomarkers associated with some special clinical role such as tendency of tumor metastasis and sensitivity or resistance to specific treatment,and even offering potential therapeutic target for the treatment of neoplasm.ObjectionThe main aim of the study was to explore the genetic and gene expression changes in the hepatitis B related hepatocellular carcinoma and paired normal liver tissue through bioinformatics analysis.And try to find effective novel tumor biomarkers that can be used for the diagnosis and treatment of hepatitis B related hepatocellular carcinoma.Methods and materialsThe study includes two parts.In the first part,we download RNA-seq and clinical data of HCC datasets from The Cancer Genome Atlas(TCGA)。Extracting of data from chronic hepatitis B related hepatocellular carcinoma and paired normal liver sample.Comparative analysis was perform by R and GraphPad Prism 6.We subjected 3 pairs of fresh tissue samples to quantitative real-time polymerase chain reaction and microarray in 4 paired Hepatocelullar carcinoma and normal liver tissues analyses to investigate the expression of CLEC4G、SLITRK3、CHST4、SPSB4、WNT2、ITLN1、COL6A6、CLEC1B.In the second part,We subjected 132 HBV-related hepatocellular carcinoma RNA-seq data from TCGA to determine the clinical significance of CLEC4G expression in HBV-related HCC.To further investigate the functional associations of CLEC4G-related genes,Go analysis were performed by the online bioinformatic tool,The Database for Annotation,Visualization and Integrated Discavery(DAVID)v6.8。We subjected 33 pairs of fresh tissue samples to quantitative real-time polymerase chain reaction and subjected 38 pairs of paraffin tissue sample to immunohistochemistry technology to detect the different expression of CLEC4G.We also subjected liver cell and five different hepatoma cells to western bolting analyses to investigate the expression of CLEC4G.ResultsIn the first part,RAN-seq and clinical data of 132 cases of HBV-related hepatocellular carcinoma sample and 49 normal sample extracted from TCGA.eight candidates with CLEC4G,SLITRK3,CHST4,SPSB4,WNT2,ITLN1,COL6A6 and CLEC1B were selected.except for SLITRK3,the result of RT-PCR has significant difference agree with the analysis results of analysis of gene differential expression.Combined with the results microarray in 4 paired Hepatocelullar carcinoma and normal liver tissues analyses,CLEC4G was selected as the objective gene for the next study.In the second part,CLEC4G has a higher expression in HBV-related HCC with none microvascular invasion,neoplasm grade of G1 and G2,pathology T1 and TNM stage I and the patient which AFP<20ng/ml.And has a lower expression in the early recurrence with 2 years.Go analysis shows CLEC4G-related genes were significantly represented by the GO biological categories of "response to radiation,positive regulation of inflammatory response and angiogenesis".The results of RT-PCR in paired tissue shows that CLEC4G has a lower expression in cancer.And the IHC shows that CLEC4G are presented at the normal hepatic hemostasis and vascular endothelium of highly differentiated HCC,while cannot be observed at the middle and poor differentiated HCC.And the expression of CLEC4G in the analysis of WB in hepatic cell and hepatoma cell has no difference.ConclusionThis study provides useful information for the molecular mechanism of the development of hepatocellular carcinoma in HBV-related hepatocellular carcinoma.And CLEC4G may be used as a potential biomarker to predict the prognosis of HBV-related hepatocellular carcinoma.And we believe that CLEC4G may provide a potential new therapeutic target for hepatocellular carcinoma in HBV-related hepatocellular carcinoma.However,more clinical samples and studies and need to verify this conclusion.