Correlation Between Thiol Oxidase 1 And HBV-associated Liver Disease And Its Role In The Growth Of Hepatocellular Carcinoma

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Association of QSOX1 serum levels and gene polymorphisms with HBV-associated hepatitis,HBV-liver cirrhosis and hepatocellular carcinomaObjective:Sulfhydryl oxidase 1(QSOX1)is an enzyme that oxidizes thiols during protein folding and reduces molecular oxygen to hydrogen peroxide.Up to the present,many studies have found that the high expression of QSOX1 promotes the occurrence and development of solid tumors such as breast cancer,lung cancer and pancreatic cancer.However,the study on the genetic susceptibility of QSOX1 and hepatocellular carcinoma(HCC)at the genetic level have not been reported.In this study,we examined QSOX1 serum levels and single nucleotide polymorphisms(SNPs)at the rs11589519 and rs7597 sites of the QSOX1 gene to investigate the association between QSOX1 polymorphisms and risk of hepatitis B virus-related liver disease(CHB,LC,HCC)and their impact on QSOX1 serum levels.Methods:A total of 425 patients and 146 healthy subjects were enrolled in the study.Among them,the case group included 138 cases of CHB,137 cases of LC and 150 cases of HCC.The polymorphisms of rs11589519 and rs7597 of QSOX1 gene were determined by SNa Pshot technique,and the serum QSOX1 level was determined using an ELISA kit.In addition,demographic data and laboratory findings of the subjects were obtained by retrieving in the hospital electronic medical records database.Logistic regression was used to calculate the ratio ratio(OR)and the corresponding 95% confidence interval,and to correct the confounding factors such as age and gender.The Kruskal-Wallis H test was used to analyze the effect of the rs11589519 and rs7597 polymorphisms of the QSOX1 gene on the QSOX1 serum levels.Results:1.The genotype distribution of the rs11589519 and rs7597 SNP loci in the QSOX1 gene was consistent with the HWE equilibrium law,indicating the genetic balance of the population in our study.2.There are three genotypes of TT,TC and CC in the rs11589519 locus of QSOX1 gene.Using the wild homozygous TT genotype as a reference,the risk of HBV-HCC in individuals carrying the CC genotype could be significantly increased to 3.38-fold(95% CI: 1.23-9.33,P = 0.018).Similarly,CC genotype was relative to T allele carriers increased the risk of HBV-HCC to 2.00-fold(95% CI: 1.26-3.19,P = 0.004).Using the T allele as a reference,the C allele increased the risk of HBV-HCC to 1.80-fold(95% CI: 1.24-2.61,P = 0.002).3.No genetic model of rs7597 was observed to be associated with the risk of CHB,HBV-LC,and HBV-HCC(P > 0.05).4.There is no linkage disequilibrium between the QSOX1 gene rs11589519 and rs7597 sites,indicating that the two SNPs form a haplotype completely randomly in the genetic process.The haplotype analysis found that the Crs11589519Trs7597 haplotype significantly increased the risk of HBV-HCC(OR 1.53,95% CI: 1.092-2.383,P = 0.0147),while the TT haplotype significantly reduced the risk of HBV-HCC(OR 0.36,95% CI: 0.216-0.601,P < 0.001).5.The serum levels of QSOX1 in patients with HBV-associated liver disease were significantly higher than those in healthy controls(P < 0.05),but there was no significant difference in the expression level of QSOX1 between CHB,HBV-LC and HBV-HCC(P > 0.05).Furthermore,the rs11589519 and rs7597 polymorphisms were not associated with elevated serum levels of QSOX1 in patients with HBV-associated liver disease.Conclusion:The mutation of rs11589519 in QSOX1 gene can increase the risk of HBV-HCC.Serum QSOX1 levels are elevated in patients with HBV-related liver disease,but the polymorphisms of rs11589519 and rs7597 have no effect on the serum level of QSOX1 in patients with CHB,HBV-LC and HBV-HCC.Part ? Tumor-associated macrophages overexpressing QSOX1 promotes the growth of xenografted tumors in nude miceObjective:Tumor-associated macrophages(TAMs),which affect the growth process of hepatocellular carcinoma(HCC)through interaction with stromal cells and tumor cells,are the primary immune cells infiltrating in the tumor microenvironment(TME).Secreted proteins in TME are important mediators for interaction and action between cells.In this study,we established human hepatocellular carcinoma xenograft in nude mice to explore the effect of TAMs exocrine sulfhydryl oxidase 1(QSOX1)on the biological function of SMMC-7721 cells,and to explore whether QSOX1 affects the development of HCC by regulating the polarization state of TAMs.Methods:1.The QSOX1 overexpressing gene infected THP-1 cells carrying lentiviral virus(Lv QSOX1 group),was compared by transfected empty viral vector(Lv Nega group).The expression of QSOX1 in THP-1 cells was detected by q RT-PCR.2.Using phorbol ester(PMA)and SMMC-7721 hepatocellular carcinoma conditioned medium to induce THP-1 cells differentiation into TAMs,and the polarization index of TAMs was detected by q RT-PCR.3.TAMs and SMMC-7721 cells were injected into the skin of nude mice to establish a model of subcutaneous hepatocellular carcinoma in nude mice.Tumor volume was measured and immunohistochemistry(IHC)SP two-step method was used to detect hepatocellular carcinoma proliferation,invasion-related indicators(PCNA,MMP-9)and M2 macrophage phenotypic markers(CD206).Results:1.The mRNA expression levels of QSOX1 gene in Lv QSOX1 group were significantly higher than those in Lv Nega group,indicating that stable transfected THP-1 cell lines overexpressing QSOX1 gene were successfully established(P < 0.05).2.Compared with Lv Nega group,the expression of M1 macrophage polarization markers TNF-? and IL-6 were significantly decreased in Lv QSOX1 group,and the M2 macrophages polarization markers IL-10,TGF-?1 and CCL22 were increased(P < 0.05).3.Tumor-bearing nude mice experiment showed that the tumor grew rapidly in Lv QSOX1 group,and the average tumor volume was higher than that in Lv Nega group.IHC staining showed a significant increase in the expression of CD206,PCNA and MMP-9 in the Lv QSOX1 group compared to the Lv Nega group(P < 0.05).Conclusion:The TAMs of QSOX1 over-expression can enhance the proliferation and invasion ability of hepatocellular carcinoma cells in vivo and promote the growth of subcutaneous hepatocellular carcinoma transplantation tumor in nude mice.This may be related to TAMs over-expression QSOX1 promoting TAMs trend towards M2-type polarization.