DNA Methylation Status of RETN and ADIPOQ Genes in Sporadic Colon Cancer

Background: Colon cancer develops through a complex process that involves epigenetic alterations. Compelling evidence has been achieved that adipocytokines link obesity with colon cancer progression Therefore, understanding the epigenetic modi�cations in adipokine genes might help in clarifying their role in colon cancer pathogenesis. The aim of the present project was to study the DNA methylation status of RETN and ADIPOQ genes in sporadic colon cancer patients. Methods: 70 cancerous colon tissue and adjacent paired non-cancerous tissue was used to determine the DNA methylation status using methylation-specic polymerase chain reaction (MS-PCR) assay. Quantitative real-time PCR (qRT-PCR) was used to determine the expression level of RETN and ADIPOQ genes. Results: In colon cancer tissues, the CpG sites in the three selected promoter regions of ADIPOQ and RETN were hypermethylated in all samples. DNA methylation level at the CpG sites in exon one of the RETN gene exhibited a lower level in the non-cancerous tissue compared to the cancerous tissue and paired blood samples. The RETN mRNA was upregulated. Conclusion: We postulate that DNA methylation status at the CpG sites in exon one of the RETN gene might help uncover cancer signatures in sporadic colon cancer and may be used as a biomarker. The upregulation of the RETN mRNA level might play a role in sporadic colon cancer tumorigenesis.


Introduction
Colon cancer is a hyperplasia of the large intestine and one of the most common causes of cancer-related death in the world 1 .Colon carcinogenesis is a slow and stepwise process that can take years to develop 2,3 .The initiation of colon cancer exhibits additional gene mutations, oncogene activation, loss and gain of chromosomes, microsatellite instability, and CpG island methylator phenotype (CIMP) 3 .As reported previously in the literature, 20% of patients diagnosed with colon cancer have familial or congenital mutations in genes that accelerate carcinogenesis to an early age onset.The remaining 80% tend to develop colon cancer later in life and do not exhibit any obvious genetic causes 1 .Therefore, lifestyle factors that might modify epigenetic patterns, including obesity, have been linked to colon cancer development 3 .A large number of research studies have examined the association between obesity and colon cancer [4][5][6] .The literature suggested that increased body mass index correlates with colon cancer development 7 .Therefore, adipokines such as leptin, adiponectin, resistin, and visfatin were measured to investigate their association with colon cancer [8][9][10] .Resistin, also known as an adipose tissue-speci c secretory factor, is a cysteine-rich peptide secreted by adipocytes, immune cells, and epithelial cells that in humans is encoded by the RETN gene 11,12 .The RETN gene is located on chromosome 19p13.2with four exons and three introns, considering exon one as the untranslated region 13 .Resistin protein is comprised of 90 amino acids and 18 amino acids as a signal sequence.Therefore, resistin pre-peptide has 108 amino acids with 12.5 kDa.Resistin is found in two different biological active forms: an oligomer and a trimer 14 .Although resistin is a small adipokine, it is involved in the activation of cell proliferation, survival, and anti-apoptosis 15 .Furthermore, resistin is also involved in cell in ammation that causes cell adhesion and migration that promotes tumorigenesis 14 .ADIPOQ is the gene of adiponectin located on chromosome 3q27 and comprises three exons and two introns, where exons 2 and 3 are coding regions.Adiponectin protein is 30 kDa with 244 amino acids that is expressed exclusively in adipocytes 16 .It has been acknowledged that a low adiponectin level may in uence colon cancer carcinogenesis [17][18][19] .It has been suggested that adiponectin may have a role in the development and progression of cancer via its pro-apoptotic and/or anti-proliferative effects.Normally, adiponectin attenuating cancer progression through adenosine monophosphate-activated protein kinase (AMPK).
Epigenetic research has shown that human cancer cells harbor epigenetic alterations 20 .Advancements in the rapidly evolving cancer epigenetic regulations have shown that changes in the epigenetic landscape are a hallmark of cancer.Therefore, disruption of epigenetic mechanisms can lead to altered gene activity and malignant cellular transformation.Signi cant progress has been made in the basic understanding of how various epigenetic changes such as DNA methylation, histone modi cation, miRNA expression, and higher-order chromatin structure affect gene activity.DNA methylation is the most broadly investigated epigenetic alteration of genomic DNA and regulating genomic function 21 .Therefore, changes in the methylation state at discrete loci are potentially associated with the control of speci c genes related to cancer pathogenesis 22 .DNA hypermethylation has many roles in tissue-speci c transcription control.Although tissue-speci c DNA hypermethylation occurs more frequently in actively transcribed gene bodies and in intragenic or intergenic enhancers, promoter hypermethylation in uences gene expression most notably at promoter regions that are rich in CpGs 23 .Abnormal increases in methylation at speci c DNA sequences can serve as biomarkers for a variety of diseases 24 .
Previous studies have examined RETN DNA methylation 25,26 .But to date, none has focused on the effect of RETN DNA methylation on sporadic colon cancer.Understanding the epigenetic role in RETN may serve as a prognostic, diagnostic marker, and the reversibility of the changes makes it possible to treat sporadic colon cancer disease.
Therefore, the present study aimed to study the DNA methylation status of RETN and ADIPOQ genes in sporadic colon cancer patients.

Bioinformatics analysis
The presence of promoter CpG islands in the RETN sequence region ranging from 500 bp upstream to 500 bp downstream of the transcription start site was evaluated using the online CpG Island Searcher software (http://dbcat.cgm.ntu.edu.tw/) with default settings.For MS-PCR primers design, bioinformatics link was used as DNA bisul te conversion tool (https://www.zymoresearch.com/pages/bisulte-primer-seeker).Then, the design of MS-PCR primers was followed as described by Herman and his team 27 .For the ADIPOQ gene, two sets of primers were chosen from the literature [27][28][29] .

Subjects
Sporadic colon cancer Saudi females (n = 39, age = 59.46 ± 2.05 years) and males (n = 31, age = 60.10 ± 2.35 years) patients were included in the study.All participants provided written informed consent after receiving information about the purpose of the study.In some cases, rst-degree relatives signed the consent form on behalf of the patients.Tissue samples were obtained from patients assigned for colectomy or endoscopy procedures at Prince Sultan Military Medical City (PSMMC; Riyadh) and King Khalid University Hospital (KKUH; Riyadh).Two samples were collected from each patient, one from cancerous tissue and one from adjacent (> 10cm) non-cancerous tissue.
After surgical removal, the tissue samples from the colectomy procedure were either snap freeze by liquid nitrogen for two minutes or saved in RNA later solution (Invitrogen by Thermo Fisher Scienti c, USA) and stored at -80°C.

DNA extraction and bisul te treatment
Genomic DNA was extracted from peripheral blood samples (2.5 ml) using the Puregene Blood Core Kit C (Qiagen, Germany) and from tissue using DNeasy Blood and Tissue Kit (Qiagen, Germany).DNA integrity and concentration were assessed by NanoDrop 2000c spectrophotometer (Thermo-Scienti c).DNA (up to 1µg DNA) was then treated with sodium bisul te using the EpiTect Fast DNA Bisul te Kit (Qiagen, Germany) according to manufacturer protocol.
CpG Methylated Human Genomic DNA Control (Thermo scienti c, USA) was used to ensure conversion e ciency.

Methylation Speci c-PCR (MS-PCR)
To determine the methylation status of the two selected genes (RETN and ADIPOQ), MS-PCR was used for tissues and blood samples.To validate primer design, fully methylated and fully unmethylated DNA controls (EpiTect Control DNA and Control DNA Set (Qiagen) were used.Also, MS-PCR primers were tested against un-treated DNA with bisul te to test primer's speci city.It was considered only the speci c primers.The experiment was duplicated for methylated results to con rm methylation status.Bisul te-treated DNA templates were PCR ampli ed using HotStarTaq (5units/µl) from Qiagen (Germany), dNTPs (20mM) from Thermo (USA), 10X buffer from Qiagen (Germany), 10µM each primer, and nuclease-free water up to 25µl.Ampli cation conditions for region and exon one at the RETN gene were: 95°C for 15 minutes; 95°C for 30 seconds, 59°C for 30 seconds, 72°C for 1minute, for 40 cycles, and 72°C for 10 minutes.Ampli cation conditions for promoter region two and three at the ADIPOQ gene were: 95°C for 15 minutes; 95°C for 10 seconds, (variable for each primer, Table 1) for 10 seconds, 72°C for 30 seconds, for 40 cycles, and 72°C for 10 minutes.The primers for the MS-PCR are shown in Table 1

RNA extraction and real-time qPCR
Total RNA was isolated from blood using PAXGene Blood RNA Kit (Qiagen, Germany) and from tissues using RNeasy Plus Mini Kit (Qiagen, Germany) as the manufacturer described.Total RNA concentration and purity were determined using NanoDrop 2000c spectrophotometer (Thermo-Scienti c).
RETN mRNA and ADIPOQ mRNA were quanti ed by real-time PCR.400ng/µl of RNA was used to synthesize complementary DNA (cDNA) by the Reverse Transcription System (Promega, USA) using oligo-dT primers according to the manufacturer's instructions.Then, Real-time PCR ampli cation reactions from 1µl of the synthesized cDNA were prepared using 10µl of 2X Taqman® Universal Master Mix (Applied Biosystems, USA), 1µl of 20X Taqman® Gene Expression Assays (Applied Biosystems, USA), and nuclease-free water to a nal volume of 20 µl.Q-PCR ampli cation was performed in a StepOne Plus (Applied Biosystems, USA) using the default setting for the ΔΔCt method.Relative gene expression levels were calculated using the comparative Ct (ΔΔCt) method and GAPDH (Hs99999905_m1) as the internal control.The expression assays (primers), ADIPOQ (Hs00605917_m1), and RETN (Hs00220767_m1) were purchased from Applied Biosystems.All reactions were carried out in triplicate.

Statistical analysis
Data are expressed as means ± standard error of the mean.The 2×2 contingency tables were used to determine the methylation frequency of MS-PCR results using Fisher's exact test.Comparisons between cancerous tissues and non-cancerous tissues data were made using Wilcoxon matched-pairs signed-rank test as data non-parametric.To evaluate the correlation between blood paired with cancerous tissue, Pearson's correlation or Spearman's rank correlation were performed for parametric and non-parametric data, respectively.Statistical analyses were performed using the computer software GraphPad Prism version 9 (San Diego, USA).All statistical tests considered two-tailed P-value < 0.05 was statistically signi cant.

Frequency of methylation
All the tissues and the paired blood samples at the CpG sites in the two promoter regions of the ADIPOQ gene showed a high frequency (92-100%) of DNA methylation in all representative samples.There was also a high level of DNA methylation at the CpG sites in the promoter region of the RETN gene.Hypermethylation was found in 85.7% cancerous tissue, 91.1% non-cancerous tissue, and 83.3% paired blood samples.The region that is downstream of the transcriptional start site (TSS) in exon one of the RETN gene showed 67.1%, 51.1%, and 62.5% methylation in colon cancerous tissue, non-cancerous tissue, and paired blood samples, respectively (Fig. 1).The Fisher test did not show any signi cant differences in the frequency of methylation between cancerous tissue paired with noncancerous tissue at the CpG sites in the region of RETN promoter (P = 0.14) and in exon one (P = 0.83).

Correlation of hypermethylation between cancerous tissue and paired blood samples
A positive correlation was found between cancerous tissue paired blood for both targeted genes (Table 2).The result suggests that blood is a useful non-invasive sample to study both genes for the level of DNA methylation.

Gene expression for ADIPOQ and RETN genes
Gene expression was quantitatively for ADIPOQ and RETN using TaqMan gene expression assay.Sixtyeight tissue samples (cancerous paired non-cancerous tissue) and 21 paired blood samples were assayed.The TaqMan assay could not detect the ADIPOQ gene transcript, either in tissue or blood.The single most striking observation to emerge from the data comparison using paired t-test between cancerous and adjacent non-cancerous tissue revealed that RETN expression is downregulated (P = 0.154).This result may implicate DNA methylation in exon one may have an effect on gene activity.Interestingly, there was a signi cant positive correlation between cancerous tissue and paired blood (P = 0.0006).This correlation suggests that blood is a good representative sample to study the RETN gene expression in colon cancer.

Discussion
Genetic cancer cannot explain sporadic cancer or cancer development in individuals with no family history of cancer.
Nonetheless, epigenetic mechanisms can describe the increased risk for the development of sporadic cancer.Thus, epigenetic markers that are consistently dysregulated among cancers present an opportunity to use them as cancer biomarkers for diagnosis, risk assessment, and prediction of therapeutic response.Our study shed light on the DNA methylation of RETN and ADIPOQ genes and their involvement in the progression of sporadic colon cancer to serve as a base for future studies.It is well known that obesity causes dysregulation of adipokines, and that is associated not only with obesity but also with malignancy.Studying aberrant DNA methylation on promoter regions on adipokine genes might help decipher the molecular mechanisms underlying the effect of obesity in sporadic colon cancer development.To the best of our knowledge, this is the rst study to investigate DNA methylation status at CpG sites of RETN and ADIPOQ genes in sporadic colon cancer tissue.
The number of genes known to undergo promoter hypermethylation in cancer has grown substantially.Our exploring study using cancerous tissue, matched normal tissue, and paired blood samples from colon cancer patients demonstrated hypermethylation at the CpG sites in the two promoter regions of the ADIPOQ gene.Additionally, the expression level of adiponectin was undetectable.Our result agrees with previous studies.Haghiac and his team showed that obesity in pregnancy is associated with an increase in ADIPOQ DNA methylation and lower mRNA concentration 28 .A year later, another group have observed hypermethylation in the same promoter region that we explored (region one) and con rmed down-regulation for adiponectin level in prostate cancer tissue via promoter hypermethylation 29 .Given that hypermethylation is an important factor in regulating gene expression, we hypothesize that the undetected ADIPOQ gene transcript in our study is due to the frequency or location of the DNA methylation on the ADIPOQ promoter.Another possible explanation is the developmental stages of cancer.It is well known that the role of adiponectin in colorectal neoplasm is a direct anti-tumorigenic effect 29 .So, de ciency of adiponectin seems to be associated with the development of an early neoplasm rather than advanced colon cancer.
Therefore, we suggest that the promoter hypermethylation that we observed in the present study is not sporadic colon cancer-associated methylation but might be associated with the repression of the ADIPOQ mRNA transcription.
We also observed hypermethylation at the CpG sites in the promoter region of RETN gene in cancerous tissue, adjacent normal tissue, and paired blood samples.Whereas DNA methylation level at the CpG sites in exon one of the RETN gene a lower level in the non-cancerous tissue compared to the cancerous tissue, and the blood samples.The region that is downstream of the transcriptional start site (TSS) in exon one of the RETN gene showed a cancer-speci c methylation pattern.This region was more informative because less methylation in the adjacent non-cancerous tissue than the cancerous tissue was detected.This nding raises the hypothesis that methylation at the CpG sites in exon one might contribute to the disease and can be suggested as a biomarker to sporadic colon cancer.Also, the outcome also suggests that the methylation pattern in exon one of the RETN may be important for understanding the molecular mechanisms linking to the development of the disease.Here we believe we are identifying novel colon cancer DNA methylation changes associated with sporadic colon cancer.We postulate that DNA methylation at the CpG sites in exon one of the RETN gene might help uncover cancer signatures in colon cancer and may be used as a prognostic biomarker.
Moreover, our data showed that the RETN mRNA was upregulated in the tissues and blood samples.Previous studies con rmed a higher level of resistin in patients with colon cancer than healthy controls, indicating that resistin levels may be positively correlated with the risk of colon cancer [30][31][32] .In 2002, Sadashiv and his group measured resistin mRNA in subcutaneous adipose tissue (SAT) in postmenopausal obese and non-obese women and showed that the expression of resistin in serum and the level of RETN mRNA were upregulated in postmenopausal obese women 33 .In contrast, when resistin was measured in visceral adipose tissue (VAT), it was found that the expression of resistin was downregulated at the transcriptional level and upregulated in the serum of the postmenopausal obese group 33 .They postulated that the mRNA resistin level variation in both tissues could pinpoint a posttranslational mechanism occurrence.Therefore, they suggested that additional research is needed to explore the regulation and biological function of resistin in humans.
Although the role of resistin in colon cancer is far from being elucidated, several mechanisms may be involved in explaining these outcomes.In fact, a few years later it was reported that the plasma resistin was inversely associated with the extent of methylation at SNP − 420 in the promoter of the RETN gene 34 .They suggested that the association could have genetic and epigenetic effects on the expression of the RETN gene and the level of plasma resistin.The result was supported by another group who reported the effect of hypomethylation on the RETN gene on PCOS causing upregulation of resistin mRNA 34 .Given that hypermethylation is an important factor in regulating gene expression, we suggest that the hypermethylation at the CpG sites in the promoter region of the RETN gene is not colon cancer-associated methylation.It might have an effect on RETN gene expression, and hence, resistin action.
While DNA methylation as cancer biomarkers is still a relatively new area, the advantages of using DNA methylation as a cancer marker are evident.Furthermore, the upregulation of RETN mRNA in the tissues and blood samples may regard as a fundamental role of DNA methylation in the regulation of gene activity.It is known that the development of CpG island hypermethylation pro les for every form of human tumors will yield valuable pilot clinical data in monitoring and treating cancer patients.Therefore, more analysis is needed to determine if DNA methylation in exon

Supplementary Files
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Figure 1 MS
Figure 1 The Institutional Review Board approved the study at Prince Sultan Military Medical City (PSMMC), Oncology Department (project no.995 ref no.#HAP-01-R-015; approval date: 10 October 2017; Riyadh; Saudi Arabia and King Khalid University Hospital (KKUH), College of Medicine (project no.E-17-2732 ref no.18/0068/IRB; approval date: 28 December 2017).All experiments were performed in accordance with relevant guidelines and regulations.
The volunteer's exclusion criteria required individuals to have hereditary colon cancer, treated with either chemo-or radiotherapy.

Table 2
Correlation of hypermethylation between cancerous tissue and paired blood samples analysis