To evaluate the influence of tumor necrosis factor - alpha (TNF-α - 308 G>A) polymorphism on hepatocellular carcinoma risk. Subjects and methods: 102 hepatocellular carcinoma patients with HBsAg (+) and 102 healthy blood donors were enrolled in the study.
Journal of military pharmaco-medicine no5-2019 THE EFFECT OF TNF-α - 308 G>A POLYMORPHISM ALTERS THE RISK OF HEPATOCELLULAR CARCINOMA Phan Thi Hien Luong1; Nguyen Ba Vuong2; Tran Viet Tu2; Luong Thi Lan Anh3 SUMMARY Objectives: To evaluate the influence of tumor necrosis factor - alpha (TNF-α - 308 G>A) polymorphism on hepatocellular carcinoma risk Subjects and methods: 102 hepatocellular carcinoma patients with HBsAg (+) and 102 healthy blood donors were enrolled in the study Polymorphisms of TNF-α – 308 G>A gene were determined using the sequence specific prim - PCR To analyze the association between TNF-α – 308 G>A polymorphisms and the risk of hepatocellular carcinoma, which were estimated by odds ratios and their 95%CI Results: Using the GG genotype as reference genotype, GA was significantly associated with increased risk of hepatocellular carcinoma (OR=2.721, 95%CI: 1.258 - 5.888)), similarly AG + AA genotype showed 2.83 fold increased hepatocellular carcinoma risk Furthermore, we found A allele was significantly associated with increased risk of hepatocellular carcinoma, compared G allele (OR = 2.676; 95%CI: 1.290 - 5.555) Conclusion: The present study showed that TNF-α - 308 G>A polymorphism was associated with increased hepatocellular carcinoma risk Further prospective studies on large will be necessary to confirm our findings * Keywords: Hepatocellular carcinoma; HBV; TNF-α - 308 polymorphism INTRODUCTION Hepatocellular carcinoma (HCC) is one of the common malignant tumors globally, which is the sixth most prevalent cancer in the worldwide, the most common causes of cancer-related deaths in Vietnam Although chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, aflatoxin B1, alcohol and nonalcoholic steatohepatitis, HBV is regarded as the main carcinogenic mechanism In fact, only a few of patients with these risk factors developed HCC during their lifetime, suggesting genetic factors may contribute to the carcinogenic mechanism [1] TNF-α is a potent pleiotropic proinflammatory cytokine and plays critical roles in the pathogenesis of inflammatory autoimmune and malignant diseases It affects the growth, differentiation, cellular function and survival of all cells In chronic HBV infection, persistent hepatic inflammation is a hallmark In response to live injury, hepatic Kupffer cells activate nuclear factor-κB (NF-κB) to produce pro-inflammatory cytokines including the TNFα TNFα plays an important role in hepatic Bachmai Hospital 103 Military Hospital Hanoi Medical University Corresponding author: Phan Thi Hien Luong (phanthihienluong@gmail.com) Date received: 19/03/2019 Date accepted: 22/05/2019 170 Journal of military pharmaco-medicine no5-2019 fibrogenesis and progression of fibrosis in chronic liver disease Circulating TNF-α concentration was elevated in subjects with acute and chronic HBV infection and HCC It has been correlated with severity of hepatic inflammation, fibrosis, and tissue injury [2] Otherwise, HBV X protein (HBx) is a key factor in HBVinduced HCC Ruchi Shukla (2011) had evidence that through NF-κB signaling activator, TNF-α induces the accumulation of HBx in cells by increasing protein stability due to reduced proteasomal degradation, which may account for HBV- mediated liver carcinogenesis [3] TNF-α gene is located on the human chromosome 6p21.3 Several single nucleotide polymorphisms (SNPs) have been identified in the TNF-α promoter region, which are through to affect TNF-α production The best documented SNPs is at position - 308 of the TNF-α gene promoter It involves in the substitution of a guanine (G) by an adenine (A) and is associated with an increase in TNF-α expression levels and risk of HCC [4] Growing evidence suggests that TNF-α gene plays role in HCC development There is overexpression of TNF-α mRNA in HCC tissue HCC cells produce TNF-α protein, resulting in elevation of circulating TNF-α concentration which is decreased after anticancer therapy [2] Understanding the risk factor for HCC development in patients is thus of great importance for refinement of treatment strategy healthcare delivery and improve the scientific basis for preventive interventions The present case control study was performed: To assess the association of HCC risk and TNF-α - 308 G>A polymorphism in a Vietnamese population SUBJECTS AND METHODS This study was carried out at Bachmai Hospital and Hanoi Medical University between January, 2016 to January, 2018 Subjects * Patients: All 102 patients (8 women and 94 men) with the diagnosis of HCC to the recommendation for diagnosis of HCC of Vietnam Ministry of Health in 2012, had HBsAg (+) [5] Patients with anti-HCV (+), HIV (+) had other liver disease and metastasis from other organs to liver were excluded * Healthy subject population: 102 blood donors (sex - matched with patients) with no evidence liver diseases, had HBsAg (-), anti-HCV (-), HIV (-) served as controls Methods * Clinical assessment: Clinical characteristics data as well as related factors: gender, age, α-FP level, HBV-DNA, size of tumour, portal vein thrombosis, metastasis * DNA extraction and polymorphism genotyping: Blood samples were extracted DNA by PathogenFree DNA Isolation PCR kit (Gene Proof) and amplified wanted gen by PCR We determined the TNF-α-308 promoter polymorphism by using 5′-AGGCAACAATTCTTGAGGGCCAT-3′ and 5′TCCTGGTG -GCATAACTAAATTGC-3′ as forward and reverse primers respectively At the end µL of the products were loaded into 1% agarose gel containing ethidium bromide for electrophoresis The PCR product size was 331 bp 171 Journal of military pharmaco-medicine no5-2019 Sequences were determined using a ABI 3500 Genetic Analyzer and Variant Reporter version 2.0 (Thermo Fisher) and BioEdit The polymorphism was divided into AA homozygote, G/G homozygote and A/G heterozygote types AA homozygote GA heterozygote GG homozygote * Statistical analysis: All statistical analyses were performed using the statistical package for social science (SPSS) software, version 17.0 The test was used to assess differences between cases and controls with regard to clinical characteristics Analyze the association between TNF-α - 308 G>A polymorphism and the risk of HCC, which were estimated by odds ratios (OR) and their 95% confidence intervals (95%CI) The significance levels of all tests were set at p < 0.05 RESULTS 102 HCC patients with HBsAg (+) and 102 healthy blood donors were investigated General characteristics of the subjects Table 1: Mean age ± SD (years) Sex (male/female) HBsAg status HBV-DNA (cp/mL) αFP (ng/ml) 172 HCC patients with HBV (n = 102) Healthy control (n = 102) 57.4 ± 9.7 24.8 ± 3.9 11.8/1 11.8/1 (+) (-) 24519299 ± 93871575.9 14525 ± 34906 Journal of military pharmaco-medicine no5-2019 Single 72.5% Multiple 27.5% Present 19.6% Absent 80.4% Positive 22.5% Negative 77.5% < cm 29.4% ≥ cm 70.6% Type of HCC Metastasis Portal vein thrombosis Size Total large diameters of tumors (cm) 10.81 ± 12.03 (1.7- 98.7) There was the same rate of sex between HCC patients and control groups The mean age of HCC patients was 57.4, and mean age of healthy control was 24.8 The association of TNF-α- 308 G>A polymorphism with HCC risk Table 2: TNF-α - 308 G>A HCC (n = 102) Healthy control (n = 102) OR (95%CI) GG 76 91 Ref GA 25 11 2.72 (1.26 - 5.89) 0.009 AG + AA 26 11 2.83 (1.31 - 6.10) 0.006 G allele 177 193 Ref A allele 27 11 2.68 (1.29 - 5.56) p 0.006 (Ref: Reference) Using the GG genotype as reference genotype, GA was significantly associated with increased risk of HCC (OR = 2.72, 95%CI: 1.26 - 5.89) Similarly, AG + GG genotype showed 2.83 fold increased HCC risk in a dominant model Furthermore, we found A allele was significantly associated with increased risk of HCC, compared with G allele (OR = 2.68, 95%CI: 1.29 - 5.56) Table 3: Effect of TNF-α - 308 G>A polymorphism on clinical characteristics TNF-α- 308 G>A Age (mean ± SD) Total large diameters of tumors (mean ± SD) AA + GA 55.88 ± 10.11 98.5 ± 70.9 GG 57.95 ± 9.85 111.36 ± 133.26 0.36 0.64 p There were no associations between TNF-α-308 G>A polymorphism with patient’s age and size of tumor 173 Journal of military pharmaco-medicine no5-2019 Table 4: Effect of TNF-α - 308 G>A polymorphism on characteristics of tumor Characteristics of tumor AA + AG GG Single 19 (25.7%) 55 (74.3%) Multiple (25%) 21 (75%) High (25%) (75%) Average (34.6%) 17 (65.4%) Low (30%) (70%) Diffuse (25%) (75%) Tumor 24 (25.5%) 70 (74.5%) < cm (20%) 24 (80%) ≥ cm 20 (27.8%) 52 (72.2%) Type p 0,944 Pathologic tumor Morphology 0,834 0,974 Size 0,411 Concerning number of focal lesions frequencies of multiple lesions were out of 30 (25%), 21 out of 30 (75%) in HCC patients with AA + AG and GG genotypes respectively while tumor size ≥ cm was present in 20 out of 72 (27.8%), 52 out of 72 (72.2%) in HCC patients with AA + AG and GG genotypes, respectively There was no significant difference in the distribution of either genotype or allelic frequency of TNF-α - 308 in HCC patients with different number and size of tumors Table 5: Effect of TNF-α- 308 G>A polymorphism on subclinical characteristics Subclinical variables HBV-DNA AFP AA + AG GG < 10,000 cp/mL 10 (29.4%) 24 (70.6%) ≥ 10,000 cp/mL 16 (23.5%) 52 (76.5%) < 400 ng/mL 16 (29.6%) 38 (70.4%) ≥ 400 ng/mL 10 (20.8%) 38 (79.2%) Positive (17.4%) 19 (82.6%) Negative 22 (27.8%) 57 (72.2%) Present (30%) 14 (70%) Absent 20 (24.4%) 62 (75.6%) 0,52 0,309 Portal vein thrombosis Metastasis p 0,311 0,606 Among 102 HCC patients with HBV infection, frequencies of portal vein thrombosis were out of 23 (17.4%) HCC patients with AA + AG genotype and 19 out of 23 (82.6%) HCC patients with GG genotype Concerning other metastasis, frequencies were out of 20 (30%) HCC patients with AA +AG genotype and 14 out of 20 (70%) HCC patients with GG genotype No significant difference was found in the distribution of either genotype or allelic frequency in HCC patients with different subclinical characteristics 174 Journal of military pharmaco-medicine no5-2019 DISCUSSION Advances in molecular and genetic epidemiology have increased our knowledge of the mechanisms underlying hepatocarcinogenesis and the relationship between susceptibility and individual genetic variations Based on the genetic information, we determine the disease etiology in terms of genetic determinant to be used for identifying the high risk individuals One of the key molecules mediating the inflammatory processes in tumor promotion is cytokine The existing evidence implicates the role of TNF-α in inflammatory pathway that increase tumorigenesis and TNF-α may be a determinant of pathogenesis and disease progression in HCC The result in the current study showed significant difference in the frequency of TNF-α - 308 genotypes and alleles between control subjects and patients We then analyzed the effects of the tested genotypes under different genetic models Using the GG genotypes as the reference genotypes, AG was significantly associated with increased risk of HCC (OR = 2.72; 95%CI: 1.26 - 5.89, p = 0009) Similarly, AA + AG genotype showed 2.83 fold increased HCC risk in a dominant model Furthermore, we found A allele was significantly associated with 2.68 fold increased risk of HCC, compared with G allele This was in line with the previous study by Hikmet Akkiz (2009) who revealed that TNF-α - 308 A allele significantly associated with HCC Our findings were also similar to Hua Feng’s study (2014), who reported in Turkish and Han populations as they reported that TNF-α-308 A allele was significantly associated with increased risk of HCC [6, 7] On the contrary, in relatively small sample size, Hai - Zhou Shi (2012) reported that inheritance of the TNF-α promoter genotype at position 308 was not associated with increased risk of HCC [8] The significance of these polymorphisms reflects their possible influence on the transcription of the TNF gene TNF-α 308 G>A polymorphism involves the substitution of a guanin (G) by an adenin (A) and is associated with an increase in TNF-α expression levels The increased risk associated with higher levels of TNF-α might be linked to the angiogenesis in the inflammation site and metastasis Several mechanisms of protumor activities of TNF-α in cancer have been suggested, such as: induction of promalignant chemokines, matrix metalloproteinases, cell adhesion molecular, angiogenic mediators, reactive oxygen intermediates [9, 10] Having these findings in mind, it is reasonable that individuals carrying the TNF-α - 308 A allele may be susceptible to HCC However, in a subsequent analysis of the association between TNF-α - 308 gene polymorphisms and clinical characteristics of HCC including tumor size, type, presence or absence of metastasis, and portal vein thrombosis, there was no significant difference in the distribution of genotype frequency within HCC patients, indicating that although 308 G>A exchange in TNF-α gene may contribute to the occurrence and development of cirrhosis 175 Journal of military pharmaco-medicine no5-2019 and HCC in patients, it had little influence on the progression of HCC This point is accepted since HCC is a multifactorial disease whose development is dependent on several genetic and environmental factors Ruchi Shukla et al cytokine TNF-α increases hepatitis B virus X protein signaling Carcinogenesis pp.978-985 Our study has some limitations Firstly, sample size of patients was relatively small Besides, the similarity of age of HCC patients and control group was not achieved Maqsood M.Elahi et al Tumor necrosis factor alpha - 308 gene locus promoter polymorphism: An analysis of association with health and disease Biochimica et Biophysica Acta 2009, 1792, pp.163-172 CONCLUSION Our study showed that TNF-α - 308 G>A polymorphism was associated with increased HCC risk However, there was no significant difference in the distribution of genotype frequency of TNF-α - 308 G>A in HCC patients with different clinical characteristics REFERENCES Bộ Y tế Hướng dẫn chẩn đoán điều trị ung thư tế bào gan nguyên phát 2012 Jemal A et al Global cancer statistics CA Cancer J Clin 2011, 61 (2), pp.69-90 Jeng J.E et al Independent and additive interactive effects among tumor necrosis factor-alpha polymorphisms, substance use habits, and chronic hepatitis B and hepatitis C virus infection on risk for hepatocellular carcinoma Medicine (Baltimore) 2009, 88 (6), pp.349-357 176 Proinflammatory the stability of through NF-κB 2011, 32 (7), Hikmet Akkiz et al G-308 A TNF-α polypmorphism is associated with an increased risk of hepatocellular carcinoma in the Turkish population: Case - control study Cancer Epidemiology 2009, 33, pp.261-264 Hua Feng et al Tumor necrosis factoralpha gene - 308G>A polymorphism alters the risk of hepatocellular carcinoma in a Han Chinese population Diagnotic Pathology 2014, (199) Hai - Zhou Shi, Peng Ren, Q.-J Lu Association between EGF, TGF-β and TNF-α gene polymorphisms and hepatocellular carcinoma Asian Pacific Journal of Cancer Prevention 2012, 13, pp.6217-6220 Xia Wang, Y Lin Tumor necrosis factor and cancer, buddies or foes? Acta Pharmacol Sin 2008, 29 (11), pp.1275-1288 10 Wu Y, B.P Zhou TNF-α/NF-κB/Snail pathway in cancer cell migration and invasion British Journal of Cancer 2010, 102 (4), pp.639-644 ... increased risk of HCC [8] The significance of these polymorphisms reflects their possible influence on the transcription of the TNF gene TNF-α 308 G>A polymorphism involves the substitution of a guanin... associated with increased risk of HCC, compared with G allele (OR = 2.68, 95%CI: 1.29 - 5.56) Table 3: Effect of TNF-α - 308 G>A polymorphism on clinical characteristics TNF- - 308 G>A Age (mean ±... frequency of TNF-α - 308 in HCC patients with different number and size of tumors Table 5: Effect of TNF- - 308 G>A polymorphism on subclinical characteristics Subclinical variables HBV-DNA AFP