| Hepatocellular carcinoma(HCC)is a kind of malignant tumor produced by hepatocytes.It is one of the most common cancers and the second leading cause of cancer death.The incidence of HCC in the world varies widely.About 70% of liver cancer occurs in Asia each year,of which China accounts for nearly half.Most of the causes include hepatitis B virus(HBV)infection,ingestion of food contaminated by fungi,excessive drinking,and cirrhosis.Apart from external factors,little is known about the role of intrinsic factors in the progression of liver cancer,such as genetic mutations.It has been reported in the literature that the role of different genetic polymorphisms is involved in the development of cancer,which provides new ideas for the treatment and prevention of liver cancer.With the development of the genome-wide association study(GWAS)in recent years,a large number of cancer-associated SNP sites have been discovered.Among these sites,rs9272105 located between HLA-DQA1 and HLA-DQB1 and rs9275319 located in the HLA-DQ region have been reported to be involved in the development of HCC.However,in different races and different groups,the results of research on the relationship between these two sites and HCC have caused great controversy.Human leukocyte antigen(HLA)plays an important role in immune function and also participates in the occurrence and development of liver cancer.Therefore,HLA genetic polymorphisms may play a important role in immune-mediated diseases.Inanother GWAS study,the occurrence of rs4678680 at the GLB1 gene was also associated with the occurrence of HCC.The GLB1 gene is involved in the encoding of a glycosyl hydrolase that catalyzes galactose residues and some glycosyl complexes.More and more research shows that GLB1 is involved in the aging of cells and the occurrence of cancer.However,little is known about the relationship between rs4678680 polymorphism and HCC.The risk of death from HCC is extremely high,and most patients choose to undergo hepatectomy.However,the prognosis is not ideal.In previous studies,the prognostic factors of HCC patients were often derived from some clinical parameters.However,the impact of genetic factors on the prognosis of patients was rarely reported.Therefore,based on the study of the relationship between rs9272105,rs9275319,rs4678680 polymorphisms and susceptibility to HCC,the relationship between polymorphism and survival time was also studied in this experiment.At the same time,we also included a number of clinical indicators for comprehensive analysis to explore the role of these three loci polymorphism in the development of liver cancer and prognosis of HCC patients.Methods 1 Blood sample collection In this experiment,a total of 1109 blood samples were collected.548 healthy control samples including 394 males and 154 females;561 cases of HCC patients diagnosed by pathological diagnosis,including 459 males and 102 females.All blood samples were collected from the First Affiliated Hospital of Zhengzhou University.This study was permited by the Ethics Committee of Zhengzhou University,and all subjects have informed consent for this study.The whole process of collection of blood samples is performed according to the requirements of standardization.2 Genotyping A total of three SNP loci were selected for genotyping.The three loci were rs9272105,rs9272319 and rs4678680.According to the requirements of the human blood genomic DNA kit,whole genomic DNA was extracted from the collected blood samples,and the extracted DNA was subjected to content determination.After the design of the primers and the amplification of the DNA target fragment,the genotypes of these three sites were confirmed by sequencing.3 Determination of clinical indicators The clinical indicators of all blood samples were detected by the automatic biochemical analyzer.4 Survival investigation of HCC patients From February 2013 to May 2016,291 patients diagnosed with HCC diagnosis at the First Affiliated Hospital of Zhengzhou University were included in this study of survival.The patient's clinical data was organized and preserved.All patients included in this study had undergone liver resection or radical radiofrequency ablation and recorded the time of surgery.5 Follow-up The follow-up of HCC patients in the survival time was followed.Follow-up methods include telephone follow-up,home follow-up and outpatient follow-up.The last follow-up time for this investigation was December 30,2017.The postoperative survival period was defined as the time from liver cancer resection or radiofrequency ablation to the time that the patient death for HCC or the last follow-up.The unit of survival is day.6 Statistical methods Statistical analysis was performed on the data using software SPSS 17.0.Clinical indicators did not meet the normal distribution,so non-parametric tests were used.A binary Logistic regression model was used to analyze the association between SNP locis and HCC.Kaplan-Meier method was used for survival analysis,and Log-rank test was used for univariate analysis.The relationship between multivariate survival analysis and gene polymorphism and prognosis was analyzed by COX proportional hazard model.All analyses were performed using a two-sided test,P<0.05 indicating statistical significance.Results 1 Association of HLA,GLB1 genetic polymorphisms with susceptibility to hepatocellular carcinoma and clinical indicators1.1 Effect of HLA,GLB1 genetic polymorphisms on susceptibility to Hepatocellular Carcinoma Healthy people as control,the A/G and G/G genotypes of people significantly reduced the risk of HCC compared with those rs9272105 A/A genotype(A/G:OR=0.49,95%CI=0.33-0.74,P=6.71*10-4;G/G:OR=0.57,95%CI=0.37-0.87,P=0.009).Compared with those who did not carry the G allele,the risk of HCC was significantly lower in those who carried the G allele(OR=0.52,95%CI=0.36-0.77,P=9.75*10-4);Those with the A/A genotype in rs9275319 had a significantly increased risk of HCC compared with those who carry the G allele(OR=1.71,95%CI=1.18-2.48,P=0.004).At the same time,the frequency of allele A in the HCC group was significantly higher than that of the healthy control group,indicating that allele A increased the risk of HCC(OR=1.5,95%CI=1.1-2.04,P=0.011);The rs4678680 gene polymorphisms was not associated with HCC susceptibility.1.2 Effect of HLA,GLB1 genetic polymorphisms on clinical parameters In the healthy control group,the thrombin time(TT)levels in the G/G genotype population and total protein(TP)levels in the A/G genotype population were significantly lower than those in the population with the rs9272105 A/A genotype(P<0.05),The fibrinogen(FIB)levels in A/G and G/G genotype population was significantly increased compare with the population with rs9272105 A/A genotype(P<0.05);the prothrombin time(PT)levels in the G/A genotype population and the fibrinogen(FIB)levels in the G/G genotype population was significantly higher compare with the population with A/A genotype in rs9275319(P<0.05);The total bile acid(TBA)levels in the rs4678680 with T/T genotype population were significantly higher than those in the T/G genotype population.In the HCC group,the level of globulin(GLB)at the people whose rs9275319 locus with G/A genotype was significantly higher than that with the A/A genotype people(P<0.05).2.Effect of HLA,GLB1 genetic polymorphism on the prognosis of HCC patients2.1 Univariate and multivariate analysis of survival in patients with HCC Kaplan-Meier method was used to analyze the clinical data of 291 patients with HCC.The results showed that ALT,AST,GGT,ALB,TP,TBIL,PT,D-DT,FIB,and AFP were prognostic factors in HCC patients(P<0.05).The univariate analysis results were included in the COX multivariate risk regression model to obtain ALT(HR=1.997,95%CI=1.212-3.291,P=0.007),GGT(HR=2.235,95%CI=1.366-3.657,P= 0.001),ALB(HR=0.529,95%CI=0.338-0.829,P=0.005),D-DT(HR=2.046,95%CI=1.277-3.278,P=0.003),and AFP(HR=2.041,95%CI = 1.331-3.128,P =0.001)were independent factor that influences the prognosis of HCC patients.2.2 Effect of HLA,GLB1 genetic polymorphism on the survival of HCC patients The risk of death was approximately 2.4 times higher in patients with HCC at the rs9272105 A/G genotype than in patients of the A/A genotype.(HR=2.37,95%CI=1.29-4.35,P=0.005).HCC patients carrying the G allele had a 2.1-fold increased risk of death compared with HCC patients who did not carry the G allele(HR=2.11,95%CI=1.17-3.78,P=0.013).The genetic polymorphisms at rs9275319 and rs4678680 had no significant effect on the survival of HCC patients.However,compared with HCC patients carrying the G allele at rs9275319,who did not carry the G allele had a reduced risk of death(HR=0.70,95%CI=0.43-1.14,P=0.15).At the same time,further analysis by Kaplan-Meier survival curve showed(Fig 5)that there may be a certain correlation between rs9275319 gene polymorphism and survival time of HCC patients(PLog-rank=0.071). 2.3 The influence of HLA polymorphisms combined with prognostic risk factors on the prognosis of HCC patients The five independent factors that affect the prognosis of HCC patients obtained from multivariate analysis were combined with different genotypes of rs9272105 and rs9275319 to assess whether there was a combined effect.The results showed that in this two polymorphic loci,The risk of death was significantly higher in patients with G allele meanwhile ALT,GGT,D-DT,and AFP in high-level groups than in those without G allele meanwhile low levels of ALT,GGT,D-DT,and AFP.Those who carry the G allele meanwhile with a low ALB level group have a significantly higher risk of death than non-G alleles meanwhile with a high ALB group.And with the increase of prognostic risk factors(carrying G alleles,high levels of ALT,GGT,D-DT,AFP or low levels of ALB)in HCC patients,the risk of death is on the rise.A further analysis of Kaplan-Meier survival curves showed that deaths significantly advanced as HCC patients carried more risk factors.2.4 Effect of HLA genetic polymorphisms on the prognosis of HCC patients in each subgroup A stratified analysis of age,sex,smoking history,drinking history,and serum AFP levels found that in patients younger than 55 years old and those with serum AFP levels less than 400 ng/ml,who with the G allele at rs9272105 have a significantly greater risk of death than patients with A/A genes.In patients older than55 years old,non-drinking patients,and those with serum AFP levels less than 400ng/ml,who with a rs9275319 A/A locus were more likely to have a significantly lower risk of death than those with the G allele.2.5 Effect of gene accumulation on survival of patients with HCC A combined analysis of the two polymorphisms sites rs9272105 and rs9275319 in HCC patients was conducted to investigate whether the risk allele G in these two SNPs had a cumulative effect on the prognosis of HCC patients.The results showed that the risk of death increased significantly with the increase in the number of risk alleles(Ptrend = 0.004).Kaplan-Meier survival curve analysis showed that with the increase in the number of risk alleles in HCC patients,the median survival time was significantly shorter(PLog-rank = 0.016).Conclusions 1.The rs9272105 and rs9275319 polymorphisms were associated with susceptibility of HCC,and carrying the G allele reduces the risk of HCC.The rs4678680 gene polymorphisms was not associated with HCC susceptibility.2.ALT,GGT,ALB,D-DT,and AFP are independent factors influencing the prognosis of HCC patients.3.Carrying the G allele in loci rs9272105 and rs9275319 increases the risk of death in HCC patients.The rs4678680 gene polymorphism was not associated with HCC survival.4.rs9272105 and rs9275319 genetic polymorphisms combined with prognostic risk factors have a significant impact on the survival of HCC patients.With the increase of prognostic risk factors in HCC patients,the risk of death is on the rise.5.With the increase in the number of risk alleles,the rs9272105 and rs9275319 loci also showed a significant increase in the risk of death. |
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