Related Genomics Research On Spontaneous Rupture Of HCC

Background The main point of view is that the occurrence and development of HCC(hepatocellular carcinoma)is the result of the dual effects of environmental factors and genetic factors.The development process is also a multi-factor synergistic and multi-step process.Among many environmental factors,such as HBV(Hepatitis B virus)infection,HCV(Hepatitis C virus)infection,food pollution containing aflatoxin B1,water pollution and alcoholism are all important factors that significantly increase the incidence of HCC.In terms of genetic factors,liver cancer is a disease in which genetic material has changed.Some causes such as HBV/HCV infection,aflatoxin B1 food contamination and alcohol damage the normal liver cell genome,which leads to form an irreversible damage and inactivation of tumor suppressor genes or activation of proto-oncogenes.It leads to abnormal changes in the law of normal liver cell proliferation,differentiation and apoptosis,which promotes the occurrence and development of tumors.Spontaneous rupture of HCC is one of the most serious and fatal complications of HCC.If the complication cannot be treated correctly and promptly,the one month fatality rate can be as high as 25%.The ultimate clinical treatment goal is to prolong the survival time of patients with HCC.In recent years,with the development of genomics,transcriptomics,proteomics and metabolomics,research on tumor systems biology has gradually developed.Under the guidance of tumor systems biology theory,the relevant information of gene expression and translation,structural and functional protein expression and interaction,and the metabolic pathways of various products in the later stage are fully integrated,providing different types and different levels study on tumor diseases.In this study,a multi-level study from the clinical characteristics of RHCC patients,genetic susceptibility and gene expression profiles,were conducted to improve the overall understanding of RHCC.The screening and diagnosis of high-risk groups,the design of RHCC treatment plans,and the improvement of prognosis and survival of patients with rupture and bleeding of HCC provide certain theoretical support.2 Materials and methods2.1 Subjects The subjects of the study were from 1543 patients diagnosed with HCC in our hospital from 2002 to 2020.Among them,186 patients suffered from spontaneous rupture of HCC.Definition for the rupture group.1357 patients with no rupture of HCC were defined as the non-ruptured group(NHCC).2.2 Methods2.2.1 Clinical data research The clinical data include: age,gender,liver cirrhosis,hypertension,diabetes,smoking,drinking,hemoglobin,white blood cell,platelets,creatinine,blood urea nitrogen,total bilirubin,albumin,aspartate aminotransferase,alanine aminotransferase,prothrombin time,international normalizd ratio,Hepatitis B surface antigen,Hepatitis C virus antibody,alpha fetoprotein,maximum tumor diameter and number of tumors,etc.Then contact patients and their relatives to obtain follow-up information through the contact information recorded on the first page of the medical record in the clinical case data.The clinical influencing factors of two groups of patients were analyzed by univariate,and the multivariate Logistic regression model was used to explore independent risk factors affecting the occurrence of spontaneous rupture of HCC.Cox regression model for survival analysis.2.2.2 Genome-Wide Association Studies The main steps are as follows: DNA extraction kits are used to extract DNA from liver tumor tissues and normal liver tissues adjacent to cancer.After quality inspection is passed,a DNA library is established.After library quality inspection,the two next-generation sequencing platform performs on-machine sequencing.A standardized process is used to compare and analyze the data,and finally screen out high-frequency mutation genes and mutation types related to spontaneous rupture of HCC.2.2.3 Transcriptomics sequencing The main steps are as follows: RNA extraction kits are used to extract RNA from liver tumor tissues and normal tissues adjacent to cancer,and after quality inspection,spectrophotometer(OD260/280?1.9)are used to detect RNA concentration and purity.Fragmentation of m RNA is reverse transcribed into double-stranded c DNA and amplified to construct a sequencing library.Perform PCR amplification on the constructed library to generate DNA clusters,linearize the DNA amplicons into single strands,complete DNA cluster amplification.After high-throughput sequencing to obtain fastq data,transcriptome data analysis was performed.2.2.4 Statistical analysis The database of patients with HCC was set up with Excel 2007.SPSS 23.0 was used for statistical analysis.Two independent sample t test,chi-square test or Fisher exact probability method,and Wilcoxon rank sum test were used.Multivariate Logistic regression model was used to determine the independent risk factors leading to spontaneous rupture of HCC.To compare the differences in overall survival(OS) and recurrence-free survival(RFS)between the two groups,Log-rank method,Kaplan-Meier Curve with Graph Pad Prism 8.2.1 and Cox regression model were used to analyze the survival time.P value is less than 0.05 and the difference is considered to be statistically significant.3 Results 3.1 Retrospective analysis of clinical data of patients with HCC3.1.1 Single factor analysis of clinical indicators In RHCC patients,the plasma ALB and HGB values were significantly lower than those of NHCC patients,while the WBC,PLT,TBIL,AST,ALT,PT and INR values were significantly higher than those of NHCC patients.3.1.2 Multivariate Logistic regression analysis A multivariate Logistic regression analysis results showed that the main factors affecting spontaneous rupture of HCC were gender,hypertension,Maximum tumor diameter,WBC,ALB.3.1.3 Survival analysis and multivariate Cox analysis The OS rates of patients in the ruptured group were 75.7%,54.2%,and 35.6%,after surgery for 1-year,2-year,and 3-year,respectively.The 1-year,2-year,3-year,OS of patients in the non-ruptured group were 85.3%,76.5%,and 67.6%,respectively.The difference in OS between the two groups was statistically significant.The RFS rates of patients in the ruptured group after surgery for 1-year,2-year,and 3-year,were 70.7%,47.6%,23.8%,respectively.The 1-year,2-year,3-year,RFS of patients in the non-ruptured group were 74.5%,57.1%,and 42.7%,respectively.The difference in RFS between the two groups was statistically significant.Multivariate analysis of Cox proportional hazards model showed that these parameters inclucing rupture,PLT,ALB,and AST were risk factors in patients with RHCC.3.2 Whole genome exome sequencing results Genome-wide association analysis was performed on the typing data of(Single nucleotide polymorphism,SNP)of 123 RHCC group samples and 1052 NHCC group control samples,and 1 susceptibility locus/gene(17q25.3/TBC1D16)was confirmed that significantly associated with spontaneous rupture of HCC.Genome-wide association analysis result showed that the mutation of TBC1D16 gene(17q25.3)is related the RHCC.TBC1D16 is mainly manifested in the process of possibly participating in the transport of membrane proteins and the phagocytosis of Immune complex(IC)into the cytoplasm of macrophages for degradation,leading to further impairment of the binding efficiency of macrophages to IC.It causes the deposition of IC on the walls of small blood vessels,and the body damages the blood vessels through local inflammatory reaction,causing spontaneous rupture and bleeding of the tumor.Imputation analysis of GWAS data by using a large sample of MHC deep sequencing database as a reference pannelrevealed that HLA genetic variation may be related to spontaneous rupture of HCC.3.3 Transcriptomics sequencing results Through transcriptome sequencing of 15 cases of spontaneous rupture of HCC and 18 cases of non-ruptured HCC control samples,it was found that differential genes were enriched in biological processes or pathways such as tumor metabolism and immune regulation,and the expression profile of the susceptible gene TBC1D16 in genomics Up-regulation may be related to impaired macrophage function.4 Conclusion(1)The analysis of clinical data of patients with HCC found that the levels of ALB and HGB in the RHCC group were significantly lower than those in the NHCC group.The levels of WBC,PLT,TBIL,AST,ALT,PT and INR in the RHCC group were significantly higher than those in the NHCC group.(2)The main factors affecting the spontaneous rupture of HCC are gender,hypertension,maximum tumor diameter,WBC,and ALB.(3)Rupture,age,PLT,ALB,AST are important factors that affect the survival risk of patients with HCC.(4)TBC1D16(17q25.3)is a gene that is susceptible to rupture of HCC.Mutations in this gene may lead to further impairment of the binding efficiency of macrophages to IC,which is related to the occurrence of spontaneous rupture of HCC.(5)It is found that HLA genetic variation may be related to spontaneous rupture of HCC.(6)Transcriptome sequencing results indicated that the expression profile of the susceptibility gene TBC1D16 was up-regulated.It may be related to the impaired phagocytic function of macrophages.