Transcriptome Profiling Of HBV-related Cirrhotic Hepatocyte By RNA-sequencing

Background and aims In the treatment of liver diseases,antiviral therapy and anti-fibrosis treatment are both very important.With the popularity of nucleotide analogues and the era of DAA,patients suffering from CHB and CHC had gotten excellent treatment.Hence,the treatment of liver cirrhosis has become more and more urgent.Along with the new generation sequencing technology,an increasing number of studies focused on transcriptomics,nevertheless,researches on liver cirrhosis,especially cellular levels were rare.Primary human hepatocytes account for 80% of liver,play an very important role on the development of liver cirrhosis.Our study aims to find some new transcriptome profile from normal hepatocyte and HBV-related cirrhotic hepatocyte by RNA-sequencing,in order to obtain some cirrhosis specific m RNA and lnc RNA,to discover new therapic targets for liver cirrhosis.Methods Using modified four-step collagenase perfusion in combination with low-speed centrifugation,density gradient centrifugation to isolate primary human hepatocytes from normal and cirrhotic liver tissues abandoned by hepatectomy or liver transplantation.After collagenase digestion,hepatocytes were purified by density centrifugation and Percoll density gradient centrifugation.To evaluate the effect of patients’ age,warm ischemic time,alanine aminotransferase,total bilirubin on cell viability and amount.We enrolled 5 normal hepatocyte as normal group and 6 HBV-related cirrhotic hepatocyte as cirrhotic group from part I.Cells were subjected to RNA extraction using Trizol reagent,RNA purity,concentration and RNA integrity number were identified by Agilent 2100.We analyzed the expression of lnc RNA,m RNA and differentially splicing genes by RNA-seq,with vacano plot and cluster analysis of lnc RNA,m RNA.KEGG and GO were used to analyze differentially expressed m RNA and lnc RNA co-expressed m RNA,differentially expressed lnc RNA-m RNA interaction analysis to predict possible process.According to the result of bioinformatics and literature review to find new potential markers related with liver cirrhosis for furture study.Results Forty-one liver tissue were isolated,finally only 20 were successful,12 with cirrhosis and 8 were normal.The average age of patients were 49±13.75 years old,tissue weight was 63.9±17.42 g,warm ischemic time was 3.31±1.57 hours,alanine aminotransferase were 142.41±217.46 U/L,total bilirubin were 58.35±90.30umol/l.The average viability and amount were 24.4±13.83 × 105/g and 83.3%±8.44.Compared with normal group,cirrhotic group has longer warm ischemic time and higher total bilirubin（p≤0.05）,however,patients’ age,tissue weight and alanine aminotransferase showed no significant differences.When warm ischemic time≤3h,tissue weight≤60g,total bilirubin≤30umol/l,cell viability and amount showed no significant change,nevertheless,beyond the range,cell amount and viability become worse.By RNA-seq,we found 190 differentially expressed m RNA,188 were significantly down-regulated,2 were significantly up-regulated;87 differentially expressed lnc RNA,32 were significantly down-regulated,55 were significantly up-regulated;72489 differentially splicing genes.GO and KEGG classification and enrichment analysis for differentially expressed m RNA and lnc RNA,we found differentially expressed m RNA were associated with may biological process,such as metabolism,acute inflammation response,regulation of enzyme activity.Virus infectious disaes pathway enrich 14 m RNA,they are FGL2、CHEK1、RANBP3L、FGB、WDR72、PTTG3P、ANKRD37、CYCSP52、IL18、C3P1、AZGP1、POLR3GL、TBPL1、novel_G001217,previous studies show that FGL2、AZGP1 were associated with liver cirrhosis.Differentially expressed lnc RNA co-expressed m RNA enriched in virus infectious disaes pathway were AKT1、KLHDC2、SH3BP4、RANBP1、TRAF5、WNT2B.Significantly enriched pathway were alcoholism and virus carcinogenesis.SRC was associated with HBV carcinogenesis.lnc RNA-m RNA interaction analysis find 6 lnc RNA and 13 m RNA,XLOC₀58660 regulate 11 m RNA and UGT1A1 was regulated by 4 lnc RNA.Gene accepted more than two times of alternative splicing were PZP,AFM,RNF121,ATF3,DNAH14,BHLHE40,ELF3,GMDS-AS1,L3MBTL4,LOC728040,PPOX,WASH3 P.Conclusions Our study successfully isolated primary human hepatocyte from normal and cirrhotic liver tissue with modified four-step EGTA/collagenase perfusion.Reducing warm ischemic time and repairing liver tissue into appropriate shape and weight to improvecell amount and viability.Our research established differentially expressed m RNA and lnc RNA profiles associated with liver cirrhosis in the level of primary human hepatocyte.Providing the basis for screening therapeutic target related with liver cirrhosis.Explore the function and signal pathways in which m RNA and lnc RNA may be involved.Predict m RNA which may be regulated by lnc RNA.Find some differentially splicing genes may be related with liver cirrhosis.