Diagnostic role of NPY methylation in patients with colorectal cancer

: Objectives : A growing number of studies have shown that methylation biomarkers play an important role in on-cogenesis. This study aimed to explore the diagnostic role of neuropeptide Y (NPY) methylation in colorectal cancer (CRC). Methods : mRNA and protein expression, methylation, survival benefits, and immune cell infiltration were analyzed using bioinformatics tools across all tumors from The Cancer Genome Atlas. NPY methylation in CRC was further validated in CRC tissues, fecal samples, and cell lines. Analyses of NPY methylation were performed using Sequenome EpiTYPER and quantitative PCR. Retrieval of NPY expression in cell lines was tested using real-time PCR and western blotting. Results : Bioinformatic analysis showed that the methylation level of NPY increased in most carcinomas ( P <0.05). Moreover, statistical correlations were observed between NPY transcriptional expression and CD4 + T cells, macrophages, and dendritic cells in colon cancer ( P <0.05). Similar results were obtained for CD4 + T cells, neutrophils, and NPY in rectal cancer ( P <0.05). Our results showed that NPY was hypermethylated in CRC tissues and fecal exfoliated cells ( P <0.05). Fecal NPY methylation was observed in 82.5% sensitive for primary tumors, 46.3% for intestinal polyps (including adenomatous, serrated, and inflammatory polyps), and 23.4% of healthy controls. Overall, fecal NPY methylation was 76.6% specific. For cell lines, in vivo experiments demonstrated that 5-aza-2′-deoxycytidine downregulated the methylation of NPY and restored its mRNA level ( P <0.05). Conclusions : This study indicates that NPY is hypermethylated in CRC, and that NPY methylation in fecal DNA is a potential noninvasive diagnostic biomarker for Chinese patients with CRC.


Introduction
The incidence and mortality of colorectal cancer (CRC) in China have increased annually, and most patients have been diagnosed at a median or advanced stage [1] .CRC develops in a multi-step process, from precancerous lesions to CRC. Early screening of the general at-risk population can significantly reduce the incidence of CRC by early prevention and intervention. Endoscopy, blood tests, and stool detection are common screening methods for CRC [2] . Methylated cell-free DNA has been developed as a biomarker in recent years, owing to its noninvasive detection and cost effectiveness [3] .
Epigenetics is a promising mechanism that initiates and regulates genetic expression without affecting the genome sequence. DNA methylation, m6A modification, and histone acetylation are the classic means of epigenetic regulation [4] . Collectively, these findings illustrate the potential therapeutic benefits of epigenetic methylation regulation for cancer treatment. The methylation of cytosine at CpG dinucleotides is associated with transcriptional activation or repression [5] . Therefore, it is important to explore epigenetic functions to improve the diagnosis and treatment of cancer based on their biological significance.
Neuropeptide Y (NPY) is a sympathetic neurotransmitter composed of 36 amino acids and is mainly distributed in brain tissues (medullary brainstem and cerebral cortex). It is released by the peripheral sympathetic neurons under chronic stress and hypoxia. This peptide exhibits pleiotropic effects via Y1-Y5 receptors [6] . The physiological effects of NPY include ileal immobilization, decreased gastric and pancreatic secretion, and increased absorption of intestinal water and electrolytes [7] . An increased density of NPY neurons has been reported in the enteric nerve plexus of patients undergoing ileal surgery for Crohn's disease [8] . In the cardiovascular system, NPY is associated with the physiological processes of vasoconstriction, heart remodeling, and angiogenesis, leading to pathological processes such as hypertension, atherosclerosis, myocardial infarction, arrhythmia, and heart failure [9] . NPY has been used as an antidepressant and anxiolytic therapy [10] . In addition, increasing evidence has demonstrated that NPY is involved in breast cancer [7] , liver cancer [11] , prostate cancer [12] , pancreatic cancer [13] , bladder cancer [14] , endometrial cancer [15] , esophageal squamous cell carcinoma [16] , head and neck tumors [17] , venturia sarcoma, and neuroblastoma [18] . The NPY gene was found to be methylated in CRC [19−20] with potential predictive value [21,22] . However, there have been no reports on the use of fecal NPY detection for the early diagnosis of CRC in a Chinese cohort. In summary, this study aimed to explore the diagnostic role of NPY methylation in Chinese patients with CRC.

Gene expression and survival analysis of NPY
NPY expression in tumor cell lines was analyzed using the Cancer Cell Line Encyclopedia database ① . GEPIA2 ② was used to compare the differential expression and survival benefits in terms of overall survival (OS) and disease-free survival (DFS) between tumor and adjacent normal tissues for NPY across all tumors. The protein and methylation expression of NPY were analyzed using the clinical proteomic tumor analysis consortium and methylation part of UALCAN ③ , respectively.

Immune infiltration analysis of NPY
We used TIMER ④ to conduct a comprehensive systematic analysis of immune infiltrates across all cancer types. The relationship between NPY and immune cell infiltration was estimated by deconvolution. The results are plotted as scatter plots. Pearson's correlation analysis was used to evaluate the relationship between NPY expression and immune cells.

Clinical colorectal cancer samples and cell lines
From January 2013 to December 2017, 42 tumors and 40 corresponding normal paraffin-embedded tissue samples were collected from the Sun Yat-sen University Cancer Center, Guangzhou, China. A total of 208 fecal exfoliated cell samples from newly diagnosed or healthy patients were obtained from the Department of Colorectal Surgery, Departments of Medical Oncology and Screening Center for Cancer Prevention of Sun Yat-sen University Cancer Center between September 2016 and December 2018. Patients with no detailed follow-up data were excluded from the study. Human cancer cell lines (HCT116 and SW480) were purchased from the cell bank of the Chinese Academy of Sciences in Shanghai. Incomplete Leibovitz L-15 was supplied by SW480 (KeyGen, Nanjing, China). HCT116 cells were cultured in McCoy's 5A medium (HyClone, Logan, UT, USA). All cells were incubated with 10% serum (Gibco, Waltham, MA, USA) at 37 °C and 5% CO 2 .

Sequenom EpiTYPER analysis
The target genomic DNA was extracted and bisulfited using a Qiagen Nucleic Acid Isolation Kit (Hilden, Germany) and a Zymo Research Conversion Kit (Irvine, CA, USA). NPY methylation was evaluated quantitatively using the Agena Sequenom platform (San Diego, CA, USA). The forward primer for NPY was 5′-aggaagagagAAGTTTTGTCGCGATTCGTT-3 ′, and the reverse primer was 5 ′-cagtaatacgactcactatagg-gagaaggctCTCCCACCCCTAAACAAAC-3 ′. A total of 15 CpG sites were examined. The methylation results were analyzed and extracted using EpiTYPER (Agena).

Methylation analysis of NPY from fecal exfoliated cells
Serum was collected and centrifuged at 20,000 g within 5 h of sampling. The samples were stored at −80 °C until analysis. DNA was extracted using a stool nucleic acid kit (Qiagen). The entire DNA was bisulfite-converted using the EZ DNA Methylation Lightning Kit (Zymo) following the manufacturer's instructions. In total, fecal DNA was mixed with a primer/probe mix (BioRad, Hercules, CA, USA) in a volume of 20 μL. The reaction conditions were as follows: 95 °C for 3 min, followed by 35 cycles of 95 °C for 15 s, 62 °C for 30 s, and finally 98 °C for 10 s. The results were analyzed by fluorescence quantitative PCR (ABI).

Western blotting
The cells were collected and lysed in a mixture of RIPA buf- . The protein concentration was determined using a BCA protein assay kit (Beyotime). The target protein was then separated and electroblotted onto a polyvinylidene fluoride ultrafiltration membrane (Merck, Burlington, MA, USA). The incubation concentrations of NPY and β-actin antibodies were 1∶200 (Abcam, Cambridge, UK) and 1∶1000 (Santa Cruz Biotechnology, Dallas, TX, USA), respectively. The results were compared with those of the naked eye using a Bio-Rad imaging system (Bio-Rad, USA).

Statistical analysis
SPSS software (version 26.0) was used for the statistical analysis. The Chi-square test was used to calculate the relationship between methylation biomarkers and clinicopathological characteristics. The t-test or one-way ANOVA were used to compare the differences in the overall average number of methylations between the groups. The diagnostic efficacy of the methylation biomarkers was evaluated using ROC curves, and the Kaplan-Meier method was used to analyze the relationship between NPY and patient survival prognosis. Statistical significance was set at P<0.05.

Gene expression and survival data of NPY in pancancer
As depicted in Fig. 1a and 1b  and pathological stage revealed a significant correlation with colon cancer, testicular germ cell tumor, and uterine corpus endometrial carcinoma (Fig. 1d, P<0.05). Fig. 1e demonstrates that low NPY was linked to poor OS in patients with pancreatic adenocarcinoma (P<0.05). However, no significant correlation was found between NPY and DFS across all listed tumors (Fig. 1e, P>0.05). Statistically positive correlations between CD4 + cells, macrophages, dendritic cells, and NPY transcription were observed in COAD (Fig. 3, P<0.05). Similar results were found for CD4 + cells, neutrophils, and NPY in the READ group (Fig. 3, P<0.05).

Methylation role of NPY in colorectal cancer tissues
We investigated 42 CRC tissues to validate the epigenetic function of NPY methylation in specific tumors. Table 1 summarizes the clinicopathological characteristics of the patients. Compared to adjacent normal tissues, NPY was significantly hypermethylated in tumors (Fig. 4a, P<0.05). Moreover, all target CpG sites showed the same statistical difference (Fig.  4b, P<0.05). In addition, no obvious correlation was found between NPY methylation and sex, fecal immunochemical test results, tumor location, tumor differentiation, CEA, TNM stage, etc. (Table 1, all P>0.05). To assess the prognostic value of NPY methylation, patients were divided into two cohorts. The diagnostic sensitivity and specificity of NPY methylation for CRC were 78.5% and 87.5%, respectively (Fig. 4c). The area under the curve (AUC) was 0.83. The median follow-up was 878 d. According to the Kaplan-Meier analysis, the NPY methylation level was not an independent predictor of OS, but showed a correlative trend in patients with CRC (P=0.32, Fig. 4d).

ROC curve analysis of NPY methylation in fecal samples
Clinicopathological characteristics of the fecal samples are presented in Table 2. As depicted in Fig. 5 and Table 3, the sensitivity, specificity, and AUC values of the fecal NPY methylation test (Fig. 5a)

Regulation of NPY methylation in colorectal cancer cell lines
The function of NPY methylation was further explored in CRC cell lines (HCT116 and SW480). All cells were treated with various concentrations of 5-AZC for 72 h (0 µmol/L vs. 5 µmol/L vs. 10 µmol/L). NPY methylation gradually reduced after 3 d (Fig. 6a, P<0.05). Meanwhile, mRNA levels were increased (Fig. 6b, P<0.05). However, the protein expression was not consistently enhanced (Fig. 6d, P>0.05). Existing data suggest that NPY methylation may be due to the dysregulation of NPY transcriptional expression in CRC.

Discussion
Recently, methylation biomarkers have shown outstanding prospects in cancer diagnosis [3] . The present study found:  Previous studies have demonstrated NPY polypeptides and their receptors have some potential for tumor-targeted ther-apy; however, the molecular mechanism for their effects remain obscure. Increased NPY expression is associated with   tumor cell growth, metastasis, angiogenesis, hypoxic tumor microenvironment, drug tolerance, and survival prognosis in neuroblastoma and Ewing's sarcomas [23] . The continuous activation of NPY/Y2R in neuroblastoma is associated with tumor cell growth through the p44/42-MAPK pathway, and apoptosis or drug resistance via the BCL-2 pathway [24,25] . Plasma NPY expression was elevated in 232 patients with Ewing's sarcoma, but there was no significant correlation with patient survival [26] . Spontaneous activation of NPY1R/5R receptors can lead to the death of Ewing's cells. The hypoxic microenvironment upregulates NPY and Y5R expression via Y2R [26] . Combined with IRF5, TNFRSF10C, and HOXA9, NPY is a liver cancer-specific hypermethylated tumor suppressor gene with diminished gene expression [11] . NPY antagonists reduce the proliferation and progression of prostate cancer cells by blocking the continuous phosphorylation of ERK1/2 and cAMP accumulation [27] . NPY is highly expressed in prostate intraepithelial neoplasia and prostate cancer, and is an independent risk biomarker for postoperative recurrence survival and tumor progression time [12] . In addition, scholars have reported that increasing NPY gradient concentrations promote breast cancer cell proliferation by regulating ERK1/2 phosphorylation, which could be reversed by NPY receptor antagonists [28] . Others have suggested that NPY is a differentially methylated gene in Chinese patients with breast cancer [7] . A case-control study reported that proline at NPY rs16139 presented a lower risk of pancreatic cancer than the leucine genotype [13] . Gene Expression Omnibus database analysis revealed that NPY was hypermethylated in bladder cancer [14] , endometrial cancer [15] and oral squamous cell carcinoma [29] . In contrast, NPY can alleviate cancer pain caused by the spinal pain signaling pathway [30] . The expression of NPY affects the imbalance in food intake, not only leading to obesity but also to cancer-related cachexia. Being overweight or obese increases the risk of CRC, breast, and pancreatic cancer [31] . Studies have reported that cachexia is present in more than 60% of end-stage patients. Animal experiments have confirmed that tumor resection can upregulate NPY expression in mice [32] . It affects tumor growth and apoptosis by dysregulating the balance between energy metabolism and immune function [33] . NPY methylation showed a good ability to identify patients with a favorable responses to radiotherapy and chemotherapy (AUC=0.93) or survival benefits in esopha-geal squamous cell carcinoma. In addition, it was hypermethylated in patients with kidney cancer [34] and head and neck tumors [17] , and was not significantly related to patient OS. Interestingly, NPY is related to intestinal inflammation by binding to Y1 and Y2 receptors on immune cells (T lymphocytes, macrophages, monocytes, and dendritic cells) [35] . Consistent with our findings, these findings illustrate the potential therapeutic benefits of combining NPY with immune cell analysis. For CRC, some researchers have conducted a methylation test for 14,000 genes (27,578 CpG sites) and confirmed that NPY was hypermethylated in CRC [20] . The sensitivity and specificity of NPY combined with WIF-1 and PENK methylation detection in CRC were 87% and 80%, respectively [21] . Others found that NPY dysregulation predicted the effects of immunotherapy or chemotherapy in 82 patients with metastatic CRC [22] . Using digital PCR, methylated free DNA was found in 80% of patients with metastatic CRC and in 45% of patients with limited-stage CRC [36] . Plasma digital PCR detection of NPY methylation is useful for tumor treatment and follow-up monitoring [37] . In line with previous studies, we confirmed that NPY was hypermethylated in Chinese patients with CRC. Fecal NPY methylation was 82.5% sensitive for primary tumors, 46.3% for intestinal polyps (adenomatous, serrated, and inflammatory polyps), and 23.4% for healthy controls. Meanwhile, fecal NPY methylation was 76.6% specific. Previous studies have reported that NPY hy- permethylation is associated with the invasion of CRC cells [38] . Furthermore, we found that NPY hypermethylation is related to the downregulation of gene transcription. However, we found that NPY demethylation drugs failed to significantly restore protein expression, which may be attributed to the following factors. First, the concentration of demethylation drugs needed to significantly upregulate protein expression was >10 μmol/L. Second, the expression of NPY protein may be affected by the demethylation of other genes. Third, NPY expression in CRC cell lines was extremely low. Finally, western blotting lacks necessary sensitivity. In contrast, no obvious correlation was found between NPY methylation and clinical parameters or OS benefits in our study, which was different from other heterogeneous tumors. This study had some limitations. First, as a diagnostic marker, the sample size was insufficient, with only 208 stool samples used in this study. Second, this study did not explore the detailed mechanism of CRC, which needs further investigation. Third, fecal NPY methylation showed poor diagnostic specificity for CRC. The combined detection of multiple genes can improve this method to a certain extent. Therefore, it is necessary to explore new programs using other genes to improve their detection sensitivity and specificity. Finally, the failure rate of fecal NPY detection was high with an unsatisfactory burden. Methods of fecal DNA extraction to purify human-derived genes should be standardized, including nucleic acid extraction, PCR detection, and strict quality control.

Conclusions
This study indicated that NPY is hypermethylated in CRC and that NPY methylation in fecal DNA may be a potential noninvasive diagnostic biomarker for Chinese patients with CRC.