Single Nucleotide Polymorphisms in miR-149 ( rs 2292832 ) and miR-1011 ( rs 7536540 ) Are Not Associated with Hepatocellular Carcinoma in Thai Patients with Hepatitis B Virus Infection

Hepatitis B virus infection is the most common risk factor for cirrhosis and liver cancer. Liver cancer ranked the fifth most common cancer in the world (Jemal et al., 2011). Considerably, chronic hepatitis B virus can cause at least 60% of circumstance of liver cancer. Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. The highest prevalence of liver cancer can be found in East, the middle zone of Eastern Asia and in sub-Saharan Africa (El-Serag et al., 2007). HCC is the most frequent subtype of the liver cancer which accounts for 70-85% (Perz et al., 2006). Single nucleotide polymorphism (SNP) is one of many mechanisms accounted for genetic variation in human, described by a single-nucleotide substitution of one base into another in DNA sequences. The abnormal forms of SNPs were recorded that SNPs on coding region can


Introduction
Hepatitis B virus infection is the most common risk factor for cirrhosis and liver cancer.Liver cancer ranked the fifth most common cancer in the world (Jemal et al., 2011).Considerably, chronic hepatitis B virus can cause at least 60% of circumstance of liver cancer.Hepatocellular carcinoma (HCC) is the major type of primary liver cancer.The highest prevalence of liver cancer can be found in East, the middle zone of Eastern Asia and in sub-Saharan Africa (El-Serag et al., 2007).HCC is the most frequent subtype of the liver cancer which accounts for 70-85% (Perz et al., 2006).
Single nucleotide polymorphism (SNP) is one of many mechanisms accounted for genetic variation in human, described by a single-nucleotide substitution of one base into another in DNA sequences.The abnormal forms of SNPs were recorded that SNPs on coding region can result in the malformation of protein structures and affect its functions.Moreover, SNPs on regulatory regions of genome like promoter, operator and enhancer can affect gene expression while SNPs on non-protein-coding region, which encode for RNAs that regulate translation such as microRNA (miRNA) mechanism (Ramirez-Bello et al., 2013), can create alternative polyadenylation signals that lead to loss of microRNA regulation (Thomas et al., 2012).
MicroRNAs (miRNAs) are small non-coding RNA with approximately 17-22 nucleotides in length which have been shown the important role in post-transcriptional regulation of gene expression process.The mechanism of miRNAs processing are counted on the complementary interaction between miRNAs and mRNA of target genes on 3'UTR region that effect a translational repression or mRNA degradation of target genes.Normally, miRNAs are transcribed by RNA polymerase II in order to generate primary miRNAs (pri-miRNAs) before endonuclease enzyme named "Drosha" cleaves pri-miRNAs to generate a hair-pin structure which are approximately 60-80 nucleotides in size called precursor miRNAs (pre-miRNAs).After that, pre-miRNAs are transported to cytoplasm followed by it will be cleaved into mature miRNA duplexes (20-22 nucleotides) by enzyme called Dicer.The miRNAs are translocated to RNA-induced silencing complex (RISC) and then separated to single strand(s).The RISC complex moves to 3´ UTR of the target gene resulted in mRNA degradation or translational inhibition.The previous suggested that miRNAs can be either oncogenes or tumor suppressor genes (Chen et al., 2005).Therefore, the polymorphisms on miRNA sequences might be associated with a progression and development of liver cancer.

Study population
Totally, 289 Thai subjects including 104 Hepatocellular carcinoma (HCC) patients, 90 cases with chronic hepatitis B virus (CHB) infection and 95 healthy controls.Both HCC and CHB patients were received from Chulalongkorn hospital, Bangkok, Thailand whereas healthy controls were collected from National Blood Centre Thai Red Cross Society, Bangkok.HCC patients were diagnosed with CHB positive, diagnostic ultrasound or/and biopsy and serology test.CHB patients were positive for HBsAg and anti-HBc test while the healthy control subjects were negative against HBV and HCV infection and had no record for liver diseases and cancers.All data of individual subjects were obtained from case records including sex, age, HBsAg, anti-HBc, aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB) and Albumin levels.All subjects have perceived and provided their written information and consent form for this study.The protocol was approved by ethical committee at faculty of Medicine, Chulalongkorn University (IRB No. 361/56).-149 (rs2292832) and miR-101-1 (rs7536540) One hundred microliters of individual peripheral blood mononuclear cells (PBMCs) were used for DNA extraction by phenol-chloroform: isoamyl alcohol as described previously (Sopipong et al., 2013).Concentration and purity of DNA were determined by using NanoDrop 2000c spectrophotometer (Thermo scientific, USA).The SNPs on miR-149 (rs2292832) and miR-101-1 (rs7536540) were identified using a real-time PCR protocol based on the pre-validated TaqMan MGB probe for allelic discrimination assay (Applied Biosystems).The reactions carried out on a StepOne Plus Real-Time PCR System (Applied Biosystems).The PCR reaction consisted of 5 µl of 2X ABI Master mix (Applied Biosystems), 0.25 µl of 40X primers and probe mix (Applied Biosystems), 1 μl of genomic DNA (50-500 ng/μl) and nuclease-free water in a total volume of 10 µl.The thermocycling conditions were conducted according to the manufacturer's instructions.Briefly, initial denaturation at 95°C for 10 min, then followed by 40 cycles of amplification including denaturation at 92°C for 10 sec, and annealing/extension at 60°C for 1 min.Fluorescent signals (FAM and VIC) were acquired at the end of each cycle.Positive controls for each allele and negative controls were included in each experiment in order to ensure correct interpretation.Allelic discrimination was analyzed using StepOneTM software (version 2.2, Applied Biosystems).

Statistical analysis
The equation was used to calculate the sample size (Kadam et al., 2010).According to the calculation, the sample size should be at least 53 samples in each group.The association of miR-149 (rs2292832) and miR-101-1 (rs7536540) and the progression of HCC using the odd ratio (OR) with 95% confidence intervals (CIs) was calculated by MedCalc statistical software (http://www.medcalc.org/calc/odds_ratio.php).P value < 0.05 was statistical significance.

SNPs (rs2292832 and rs7536540) and their association with HBV-related HCC
The genotypes of miR-149 (rs2292832) and miR-101-1 (rs7536540) were not significantly associated (P value > 0.05) with the development of HCC when comparing with CHB group, healthy group and CHB + healthy group in Thai population.After adjusting for variables such as age and sex in the logistic regression analysis, minor allele of rs2292832 and rs7536540 were not statistically associated (P value > 0.05) with the development of HCC in Thai population.The overall odd ratio (95% CI) and P value were summarized in Table 2 and 3.

Discussion
Until now, the knowledge about SNPs in miRNAs has been studied worldwide in order to be necessary in medical treatment.There are many studies indicated associations between miRNA regulation to diseases progression including cancer (Li et al., 2010;Kim et al., 2012).The insight of this biological regulation will provide benefits of the prognosis and diagnostic in cancer development.In this research, the associations of SNPs on miR-149 and miR-101-1 genes with the risk to HCC were analyzed in HCC with CHB patients and CHB patients in Thai population.The statistical analysis showed that the number of the sample size were sufficient to calculate the reliable results.The characteristic of the subjects in each group found were matched with age (P values=0.49,>0.005).Considerably, the data suggested that gender was uncorrelated among each group of study (p-value=0.002).This might be the limitation of this study because the gender is considered as the confounding factor of HCC incident (Tangkijvanich et al., 1999;Parkin et al., 2003).SNPs on miR-149 is a tumor suppressor in human gastric cancer (Wang et al., 2012) and as a tumor suppressor, this SNPs may be involved in the proliferation and invasion of GM cells via blockade of the AKT1 signaling (Pan et al., 2012).There are studies revealed that the SNPs on miR-149 (rs2292832) with HCC.In Korean population, 159 of HCC and the 201 of healthy controls, revealed CC+TC (AOR=0.536,95% CI=0.335-0.858,p-value=0.009)and TC (AOR=0.542,95% CI=0.332-0.886,p-value=0.015)genotype were significantly tended to reduce the risk of HCC when compare with TT genotype (Kim et al., 2012).Furthermore, in 2014, study has confirmed this association between this SNP (rs2292832) and HCC.The results showed that T allele of miR-149 (rs2292832) associated with increasing risk of Hepatitis B virus associated HCC (Wang et al., 2014).
In conclusion, this study was the first reported considering to study the association of rs2292832 and rs7536540 polymorphisms and the impressibility to HCC in Thai population.Results showed that there were no significant associations between rs2292832 and rs7536540 polymorphisms with the risk of HCC in Thai population.Therefore, rs2292832 and rs7536540 polymorphisms may not be able to use as a genetic markers in Thai population.However, this study had the limitation in unmatched gender among each group.Therefore, further case-control study with gender matched among each group in order to control confounding factor and confirm the association of rs2292832 and rs7536540 polymorphisms with the susceptibility HCC in Thai population.