The Effect Of Tnf-Α - 308 G>A Polymorphism Alters The Risk Of Hepatocellular Carcinoma - Phan Thi Hien Luong

Journal of military pharmaco-medicine n o 5-2019 170 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 pro- inflammatory 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 1. Bachmai Hospital 2. 103 Military Hospital 3. Hanoi Medical University Corresponding author: Phan Thi Hien Luong ([email protected]) Date received: 19/03/2019 Date accepted: 22/05/2019 Journal of military pharmaco-medicine n o 5-2019 171 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 HBV- induced 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. 1. 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. 2. 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 5 µL of the products were loaded into 1% agarose gel containing ethidium bromide for electrophoresis. The PCR product size was 331 bp. Journal of military pharmaco-medicine n o 5-2019 172 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. 1. General characteristics of the subjects. Table 1: HCC patients with HBV (n = 102) Healthy control (n = 102) Mean age ± SD (years) 57.4 ± 9.7 24.8 ± 3.9 Sex (male/female) 11.8/1 11.8/1 HBsAg status (+) (-) HBV-DNA (cp/mL) 24519299 ± 93871575.9 αFP (ng/ml) 14525 ± 34906 Journal of military pharmaco-medicine n o 5-2019 173 Type of HCC Single 72.5% Multiple 27.5% Metastasis Present 19.6% Absent 80.4% Portal vein thrombosis Positive 22.5% Negative 77.5% Size < 5 cm 29.4% ≥ 5 cm 70.6% 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. 2. 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) p 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) 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 p 0.36 0.64 There were no associations between TNF-α-308 G>A polymorphism with patient’s age and size of tumor. Journal of military pharmaco-medicine n o 5-2019 174 Table 4: Effect of TNF-α - 308 G>A polymorphism on characteristics of tumor. Characteristics of tumor AA + AG GG p Type Single 19 (25.7%) 55 (74.3%) 0,944 Multiple 7 (25%) 21 (75%) Pathologic tumor High 3 (25%) 9 (75%) 0,834 Average 9 (34.6%) 17 (65.4%) Low 3 (30%) 7 (70%) Morphology Diffuse 2 (25%) 6 (75%) 0,974 Tumor 24 (25.5%) 70 (74.5%) Size < 5 cm 6 (20%) 24 (80%) 0,411 ≥ 5 cm 20 (27.8%) 52 (72.2%) Concerning number of focal lesions frequencies of multiple lesions were 7 out of 30 (25%), 21 out of 30 (75%) in HCC patients with AA + AG and GG genotypes respectively while tumor size ≥ 5 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 AA + AG GG p HBV-DNA < 10,000 cp/mL 10 (29.4%) 24 (70.6%) 0,52 ≥ 10,000 cp/mL 16 (23.5%) 52 (76.5%) AFP < 400 ng/mL 16 (29.6%) 38 (70.4%) 0,309 ≥ 400 ng/mL 10 (20.8%) 38 (79.2%) Portal vein thrombosis Positive 4 (17.4%) 19 (82.6%) 0,311 Negative 22 (27.8%) 57 (72.2%) Metastasis Present 6 (30%) 14 (70%) 0,606 Absent 20 (24.4%) 62 (75.6%) Among 102 HCC patients with HBV infection, frequencies of portal vein thrombosis were 4 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 6 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. Journal of military pharmaco-medicine n o 5-2019 175 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 Journal of military pharmaco-medicine n o 5-2019 176 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. 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. CONCLUSION Our study showed that TNF-α - 308 G>A polymorphism was associated with increased HCC risk. 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