Potential influence of omentin-1 genetic variants on the clinicopathological features of patients with hepatocellular carcinoma.

Introduction
Hepatocellular carcinoma (HCC) ranks as the fifth supreme prevalent cancer within men globally and the ninth among female, serving as a significant contributor to cancer-related deaths 1. HCC correlates with a low five-year survival rate and rising mortality rates 2, 3. In Taiwan, the second most common reason of cancer-related fatalities is HCC 4, 5. Genetic variation is crucial for susceptibility to HCC and its progression. Most individuals who encounter the recognized infectious or environmental risk mediators (such as alcohol misuse, hepatitis virus infection, or non-alcoholic fatty liver disorder linked to insulin resistance, diabetes or obesity) do not go on to progress HCC. This implies that personal susceptibility plays a role in tumorigenesis 6, 7. Data on the frequency of genotype distribution can help to chart single nucleotide polymorphism (SNP) diversity within a population and investigate the risk and progression of certain disorders 8, 9. Investigations that have recently come to light suggest a link between SNPs in specific genes and the vulnerability to and clinicopathological status of HCC.
Adipokines are bioactive agents secreted by adipose tissue that regulate a range of physiological processes, including inflammation, homeostasis, insulin responsiveness, and immune responses 10, 11. Metabolic and inflammatory pathways are influenced by key adipokines for instance omentin-1, adiponectin, resistin and leptin, which act locally or systemically through endocrine, autocrine, or paracrine processes 12. Adipokines have multifaceted effects in cancer, impacting the tumor microenvironment, cellular proliferation, apoptosis, angiogenesis, and metastasis 13. Omentin-1 have identified adipokine consisting of 313 amino acids, is primarily generated in the small intestine as well as in adipose tissue and omental 14, 15. Omentin-1 exhibits anti-hyperinsulinemic and anti-inflammatory functions 16. It has been documented to play a protective effect in decreasing proinflammatory cytokine generation 17. Previous experimental studies have shown that omentin-1 is positively linked with increased levels of anti-inflammatory mediators 18, 19. In the context of cancer, there is an inverse relationship between serum omentin-1 levels and obesity, indicating that omentin-1 could be a marker for cancer development 20. Moreover, omentin-1 might act as a tumor-suppressor mediator, given that patients with renal cell carcinoma have been observed to have lower serum levels of omentin-1 21. No data are available on the links between carcinogenic lifestyle factors, OMNT1 gene polymorphisms, and HCC. Consequently, this research investigated the impact of carcinogenic lifestyle factors and OMNT1 gene polymorphisms on the likelihood of HCC development in a cohort of Taiwanese. We also investigated the relationships between OMNT1 genotypes and the histopathological prognostic variables of HCC.

Materials and Methods

Study participants
We registered 413 patients with HCC at Chung Shan Medical University Hospital in Taiwan. From the Taiwan Biobank Project, 826 healthy controls (HCs) with no cancer history were randomly chosen and anonymized. Every participant in the study belonged to the Han Chinese ethnicity. Patients with HCC were staged using the 2010 American Joint Committee on Cancer (AJCC) TNM staging system, which takes into account tumor morphology, the number of affected lymph nodes, and metastases 22. Prior to entering the study, each participant gave informed written consent and filled out a structured questionnaire regarding their sociodemographic status, as well as their use of alcohol and cigarettes. A diagnosis of liver cirrhosis was made based on biopsy results, suitable sagittal CT or MRI scans, or biochemical indicators of liver parenchymal damage accompanied by endoscopic esophageal or gastric varices. Before the study began, it received approval from the Institutional Review Board of Chung Shan Medical University Hospital.

Selection and genotyping of SNPs
The OMNT1 SNPs rs2274907, rs35779394, rs4656959, and rs79209815 were selected based on previous reports 23, 24. Every SNP had a minor allele frequency greater than 5%. Genomic DNA was extracted from 3 mL peripheral blood samples using QIAamp DNA Blood Kits (Qiagen, CA, USA). Using previously described assessment techniques 8, 20, 21, allelic discrimination was performed on the SNPs. RT-qPCR experiments and the isolation of RNA were performed following the protocols we published earlier 25, 26.

Analysis of clinical dataset
The GTEx portal (gtexportal.org/home/) serves as a comprehensive public resource for the analysis of gene levels and modulation specific to various tissues. It provides quantitative trait loci (QTLs), histological images, and gene expression data that is open-access 27.

Statistical analysis
To assess the differences between the HCC and control groups, the Fisher's exact test and Mann-Whitney U test were employed, with p-values below 0.05 considered statistically significant. Logistic regression was used to calculate odds ratios (ORs) and their 95% confidence intervals (CIs) for the associations between genotype frequencies and HCC risk. The collected data were analyzed using version 9.1 of the Statistical Analytic System (SAS) software.

Results
The demographic characteristics of the 413 patients with HCC and the 826 cancer-free HCs) did not show significant differences (Table 1). Controls reported alcohol consumption significantly less often than patients (p < 0.001), but there was no difference in cigarette smoking status between the two groups (p = 0.409) (Table 1). The proportions of HCC patients who tested positive for HBsAg (44.8%) and anti- HCV antibodies (36.6%) (Table 1). At the time of enrollment in the study, 310 patients (75.1%) presented with stage I/II HCC, while 103 (24.9%) had stage III/IV disease. Patients with HCC also presented with N1+N2+N3 lymph node status (2.2%), metastasis (5.8%) and vascular invasion (12.3%). Liver cirrhosis was present in most patients (86.2%) (Table 1).
Genotyping results for the OMNT1 SNPs in HCs and HCC patients are shown in Table 2. The homozygous T/T alleles for rs2274907, rs35779394, and rs79209815, as well as the homozygous A/A allele for rs4656959, were the most common (Table 2). After adjusting for age, gender, cigarette smoking, and alcohol consumption (Table 2), none of the genotypes for the four OMNT1 SNPs across different groups exhibited notable associations.
Next, we conducted a comparison of the distributions of clinical aspects and OMNT1 genotypes among HCC patients. No significant influence of SNPs rs2274907, rs35779394 and rs4656959 on clinicopathologic traits in HCC patients (Table 3). However, compared with patients with the T/T genotype, those with at least one polymorphic C allele at the rs79209815 SNP (T/C+C/C genotype) were susceptible to progressing to stage III/IV disease (OR, 1.899; 95% CI, 1.027~3.512; p<0.05) and large tumors (OR, 2.055; 95% CI, 1.109~3.810; p<0.05) (Table 4). The TC or CC genotypes at rs79209815 were related with an increased risk of stage III/IV disease (OR, 2.298; 95% CI, 1.142-4.622; p < 0.05) and larger tumors (OR, 2.298; 95% CI, 1.142-4.622; p < 0.05) in male, but not female, patients compared with the TT genotype (Table 5).
GTEx data indicate that, in lymphocytes and whole blood, individuals carrying the C variant at rs79209815 showed a trend toward augmented omentin-1 expression compared with those with the wild-type TT homozygous genotype (Figure 1).

Discussion
Many cancer reports have confirmed the effectiveness of biomarkers based on genetic aberrations related to tumors in assessing risk, aiding in early diagnosis, and predicting treatment results 28, 29. Approximately 1% of the overall population carries genetic polymorphisms, which are variations in genomic sequences among individuals. Repetitive sequences most frequently exhibit alterations in the form of SNPs 30. An expanding body of investigation has recently underscored the significance of SNPs and other genetic changes in predicting and defining pharmacotherapeutic functions in HCC 31, 32. Moreover, the methodical identification of functional variants linked to cancer risk has demonstrated how SNPs in functional domains affect gene level and tumor susceptibility, highlighting the importance of SNPs in tumor biology 33. These findings are supplemented by thorough reviews that detail the biological and molecular processes through which SNPs affect gene expression, thereby affecting the progression and development of tumor 34. Hypothesis-driven genetic research has informed both case-control and prospective cohort reports that investigated the connection between SNPs and HCC. The studies have underscored the connection between changes impacting multiple biological mechanisms—for instance oxidative stress, DNA repair, and inflammation processes—and the evolution of liver cancer in patients with hepatitis 35. We investigated polymorphisms in the OMNT1 gene and noted their different distributions among HCC patients. Our investigation showed that patients with the T/C+C/C genotypes of rs79209815 exhibited a significantly elevated risk of progressing stage III/IV disease and large tumors.
Adipokines are unique bioactive peptides released by adipose tissues and play a role in various bodily functions 36, 37. To examine the function of adipose tissue in the progression of inflammation and carcinogenesis, many investigators have been investigating this issue for the last two decades 38, 39. Recent studies have documented that omentin-1 plays a vital effect in cell differentiation and the promotion of cancer cell death 40. Many associated studies found that the levels of omentin-1 in circulation among patients with colorectal and renal cell carcinoma differed, suggesting that omentin-1 might play a part in cancer development 41. Recently, omentin-1 SNPs have been investigated in several cancers. For example, in OSCC, the TA and AA genotypes of SNP rs2274907 were related with an augmented risk of progressing to an advanced clinical stage compared with the TT genotype 23. The OMNT1 rs2274907 and rs4656959 variants are protective against perineural invasion, particularly in prostate cancer patients without biochemical recurrence 24. Conversely, research on omentin-1 and HCC is limited. We conducted this study to compare the allelic distributions of OMNT1 gene polymorphisms between HCs and HCC patients. Carriers of at least one C allele (genotypes TC or CC) at the OMNT1 SNP rs79209815 exhibited a heightened risk for developing stage III/IV disease and larger tumors, according to our findings. It is worth mentioning that these correlations were more marked in male patients than in female ones. Furthermore, there was a tendency for the omentin-1 expression to be higher in individuals with the C variant at rs79209815 than in those with the wild-type TT homozygous genotype. Omentin-1 levels are thus crucially linked to the advancement of HCC in patients. It is important to note the limitations of the current study. More research is required, which calls for a larger sample size and a longer follow-up time. Furthermore, an independent cohort of HCC cases from Taiwanese communities and other cohorts found in open-access databases must be used to confirm the current findings.
To sum up, our study is the first to uncover links between OMNT1 gene variants and HCC. The OMNT1 rs79209815 variant (genotypes TC or CC) is linked to a heightened risk of developing stage III/IV disease and larger tumors, especially among male HCC patients, as our findings suggest. Moreover, the wild-type TT homozygous genotype was linked to considerably reduced OMNT1 expression levels in comparison to the TC or CC genotypes of rs79209815, suggesting that this OMNT1 SNP plays a crucial role in HCC development.