Expression And Role Of Tregs Subgroup And CD40LG And COX2 In Jaundice Disease

Background:Jaundice is one of the major symptoms in hepatobiliary surgery,and the common causes of jaundice include bile duct stones,cholangiocarcinoma and primary hepatocellular carcinoma,whereas early surgical treatment of such diseases has a large impact on patient outcomes.At present,the preoperative etiological diagnosis of jaundice patients mainly relies on clinical manifestations,blood biochemical tests,tumor marker determination,and imaging examination,but the early clinical symptoms of some jaundice diseases are not typical,especially malignant tumor diseases,which progress rapidly and are aggressive,and some patients lose the opportunity of early surgical treatment because of unclear diagnosis.Therefore,early diagnosis of patients with jaundice is very important.In recent years,with the development of high-throughput sequencing technology,there are more and more applications of transcriptome high-throughput sequencing in clinical research,but it is still relatively rare in the pathogenesis and differential diagnosis of surgical jaundice,so we used high-throughput sequencing technology to genetically test peripheral blood mononuclear cell（PBMC）in surgical jaundice patients,It is desirable to find out whether there are differences in gene expression profiles of surgical jaundice caused by different etiologies.Meanwhile,the findings indicated that compared with healthy people,patients with jaundice had reduced immune function,which may be related to the reduced number and functional suppression of immune cells,especially regulatory T cells（Tregs）.Whereas Triple^hiTregs are a powerful Tregs cell subset that can recognize self antigens to maintain hyperplasia and suppress inflammatory responses in skin and peripheral lymph nodes,removing Triple^hiTregs can induce massive inflammatory cell infiltration and cause severe inflammatory responses.In the present study,we wanted to identify early surgical jaundice etiologies by flow cytometric analysis of changes in the expression of subpopulations of Triple^hiTregs in the peripheral blood of jaundiced patients,because Triple^hiTregs have been shown to play an important role in immune rejection after liver transplantation and rheumatoid arthritis,but little is known about their role in jaundice.Objectives:The etiology of surgical jaundice was identified by PBMC gene expression profile detection,differential gene screening and the proportion of Tregs and its subsets in peripheral blood.Methods:PBMC from patients with cholangiolithiasis,cholangiocarcinoma,and primary hepatocellular carcinoma and healthy individuals were collected from a Hospital between January 2021 and may 2021,and the isolated cells were frozen in liquid nitrogen tanks,m RNA was extracted within 2 weeks and subjected to high-throughput detection.Differential gene analysis was performed using DESeq2software;At the same time,the proportion of each subset of B cells,T cells,NK cells and the proportion of Tregs with high expression of Tregs and GITR,PD-1,and CD25 among the lymphocytes isolated from peripheral blood were analyzed by flow cytometry.The statistical software SPSS 25.0 was used to analyze the data.And the quantitative data were expressed using mean±standard error（x^<sub>±S）.For continuous variables with normal distribution and homogeneity of variance,LSD-T test was used.For continuous variables with normal distribution and uneven variances,Tamhane T2 test was adopted and statistically significant was defined as P<0.05.Results:The first part Differential gene screening of gene expression profile in the PBMC of patients with surgical jaundice1.Compared with the healthy group,there were 711 up-regulated genes and 829 down regulated genes in the bile duct stone group（P≤0.05）.According to the criteria of meantpm value≥5,absolute value of log2foldchange>1 and Q（corrected P value）<0.05 in at least one sample of the two groups,there were8 up-regulated genes,namely CLEC4F,RBM47,NUSAP1,FGD4,AC215522.2,CLASP1,HDAC9 and INPP4A,and 8 down-regulated genes AL928654.4,RPSAP47,Trav1-2,Trav9-2,CYP2S1,LINC02446,CD40LG and TMEM25;In the cholangiocarcinoma group,there were 2734 up-regulated genes and 2174down regulated genes（P≤0.05）.According to the criteria of meantpm value≥5,absolute value of log2foldchange>1 and Q（corrected P value）<0.05 in at least one sample of the two groups,there were388 up-regulated genes,among which the top 10 genes were LINC02224,SLC4A1,HBB,METTL7B,SELENBP1,HBA1,SNORD83B,HBA2,CA1 and AHSP,and 265 down-regulated genes,among which the top ten genes were BAIAP3,CD79A,RNVU1-15,MIR4539,AC245014.3,LCN10,VPREB3,TRDV2,SNORA6 and XIST;There were 2104 up-regulated genes and 1902 down regulated genes in the primary hepatocellular carcinoma group（P≤0.05）.75 up-regulated genes were screened according to the criteria of meantpm value≥5,absolute value of log2foldchange>1 and Q（corrected P value）<0.05 in at least one sample of the two groups,among which the top 10 genes were ALAS2,HBB,SNORA17,SNORD36A,SLC4A1,HBG1,HBA1,HBG2,CA1 and HBA2,and 77 down-regulated genes,the top ten genes were TCL1A,HLA-DQB1-AS1,PRSS21,AC245014.3,U1,VPREB3,RNVU1-19,RNVU1-15,ZBED6 and AL713999.1.Compared with the healthy group,KEGG enrichment analysis showed that CD40LG was enriched in T cell receptor signaling pathway and NF-Kappa B signaling pathway in the biliary calculi group compared with the healthy group.Differentially expressed genes were mainly enriched in T cell receptor,B cell receptor and NF-Kappa B signaling pathways in the cholangiocarcinoma group.The significant difference genes in HCC group were mainly enriched in T cell receptor and B cell receptor signaling pathway.2.Comparison between patients with cholangiocarcinoma and cholangiolithiasis:Compared with the bile duct stone group,there were 1508 up-regulated genes and 655 down regulated genes in the cholangiocarcinoma group（P≤0.05）.127 up-regulated genes were screened according to the criteria of meantpm value≥5,absolute value of log2foldchange>1 and Q（corrected P value）<0.05 in at least one sample of the two groups,among which the top 10 genes were INO80b-WBP1,SLC4A1,HBB,HBA1,TMOD1,AHSP,METTL7B,HBD,AL928654.4,CA1,five down-regulated genes were ZNF331,AC006042.3,TRDV2,XIST,Bloc1S5-TXNDC5.KEGG enrichment analysis showed that compared with the cholangiolithiasis group,the significant difference genes in the cholangiocarcinoma group were mainly enriched in TNF and NF-Kappa B signaling pathways,and the inflammation-related high expression genes COX1 and IL1B and immunoregulation related low expression genes played an important role.3.Comparison between primary hepatocellular carcinoma and bile duct calculi group:Compared with the bile duct stone group,there were 1054 up-regulated genes and 712 down regulated genes in the primary hepatocellular carcinoma group（P≤0.05）.13 up-regulated genes were screened according to the criteria of meantpm value≥5,absolute value of log2foldchange>1 and Q（corrected P value）<0.05 in at least one sample of the two groups,INO80b-WBP1,HBB,SLC4A1,HBA1,HBA2,AL928654.4,CA1,HBD,AQP10,PVALB,HPSE,VCL and ZDHHC20 respectively.Five down-regulated genes were TBC1D27,IGHJ5,NSRP1,GUCD1 and INPP4A.KEGG enrichment analysis compared with the cholangiolithiasis group,the highly expressed gene VCL was significantly enriched in the leukocyte migration signaling pathway in the primary hepatocellular carcinoma group.4.Comparison between the primary hepatocellular carcinoma and cholangiocarcinoma groups:Compared with the cholangiocarcinoma group,there were 427 up-regulated genes and 453 down regulated genes in the primary hepatocellular carcinoma group（P≤0.05）.According to the criteria of meantpm value≥5,absolute value of log2foldchange>1 and Q（corrected P value）<0.05,only one up-regulated gene was GSE1,and only two down-regulated genes were IGKV3D-20 and HRA-DQB1-AS1,indicating that the PBMC gene expression profiles of patients with these two malignant tumors were roughly the same.The second part The changes of Tregs and Triple^hiTregs in surgical jaundice diseases1.The proportion of Tregs in CD4⁺T cell subsets in peripheral blood of patients with cholangiocarcinoma was significantly higher than that of healthy control group,and the difference was statistically significant（P<0.05）.The proportion of GITR subgroup in Tregs of patients with cholangiocarcinoma was significantly higher than that of healthy control group,and the difference was statistically significant（P<0.05）.The percentage of PD-1 subgroup expressed in Tregs in peripheral blood of patients with cholangiocarcinoma,patients with cholangiolithiasis and patients with primary hepatocellular carcinoma was higher than that of healthy control group,and the difference was statistically significant（P<0.05）.The proportion of Triple^hiTregs subgroup in the cholangiocarcinoma group,the cholangiolithiasis group and the primary hepatocyte group was higher than that in the healthy control group,and the differences between the cholangiocarcinoma group and the primary hepatocellular carcinoma group and the healthy group were statistically significant（P<0.05）.2.Comparison between the three surgical jaundice groups:the proportion of Triple^hiTregs subgroup in peripheral blood Tregs of patients with surgical jaundice in the three groups was the highest in the cholangiocarcinoma group,the second in the primary hepatocellular carcinoma group,and the lowest in the cholangiolithiasis group.Therefore,the proportion of Triple^hiTregs subgroup in peripheral blood tregs of patients with surgical jaundice may be used as an auxiliary supplement for the etiological diagnosis of patients with surgical jaundice,but the reasons and mechanism of the increased proportion of Triple^hiTregs subgroup in Tregs need to be further discussed.Conclusion:1.Cholangiocarcinoma can be distinguished from bile duct stones by the high expression of IL-1B and COX2 genes in PBMC,but it is difficult to distinguish cholangiocarcinoma and hepatocellular carcinoma by high-throughput differential gene analysis in patients’PBMC.2.The proportion of Triple^hiTregs subgroup in peripheral blood was the highest in cholangiocarcinoma group,the second in primary hepatocellular carcinoma group and the lowest in bile duct stone group.The different proportion can well distinguish these three kinds of surgical jaundice.