Colorectal cancer (CRC) is one of the most common malignancies worldwide, and the second cause of cancer related deaths in developed countries (1,2). CRC is the 3rd and the 4th commonly diagnosed cancer in Iranian men and women, respectively (3). The lack of clinical manifestations in CRC patients until the late stages of cancer is a common disease characteristic, which results in poor prognosis and high mortality. The process of carcinogenesis in primary adenomas, which are precursor lesions of colon cancer that eventually develop into colorectal carcinomas, is slow, and is a cause of the late diagnosis (4). Eighty percent of early-diagnosed patients are referred for tissue resection and are eventually cured (5). In order to reduce the morbidity and mortality of the disease, early diagnosis and treatment of CRC appears to be of critical importance, since there is a large preclinical asymptomatic stage in CRC patients (6).

There are several CRC screening methods, such as fecal occult blood testing (FOBT), barium enema, flexible sigmoidoscopy and colonoscopy. FOBT and colonoscopy are commonly used clinically; however, they have some technical restrictions and disadvantages (7). FOBT, while a simple method in practice, does not have high sensitivity and specificity. Considering that colonoscopy is the ‘gold standard’ method for CRC screening, its invasiveness nature and complicated required preparation procedure make patients reluctant to choose it as an acceptable screening method (7). Therefore, developing new and useful screening methods is a high priority (8).

Sporadic CRC occurrence is due to accumulation of genetic and epigenetic changes, which cause normal epithelial cells to transform into the adenocarcinoma cells (9). There is increasing evidence that widespread epigenetic alterations are the key features of most of cancer types (10,11), and these changes may be important in the pathogenesis of CRC. Aberrant DNA methylation is one of the best known and well-defined epigenetic changes in tumors, and is a frequent mechanism for inappropriate gene silencing among tumor suppressor genes (12–14). DNA methylation is of particular interest, as it occurs in the primary stages of carcinogenesis, and hence, can be used as a marker for early detection of CRC (15).

Septin 9 (SEPT9) is a member of the septin gene family, which are highly conserved and encode GTP-binding proteins. Septins are multidomain proteins which together form filamentous structures that form part of cytoskeleton (16,17). Furthermore, septins belong to P-loop GTPases superclass and were first identified in yeast as key genes in cell division (18). These proteins have prominent roles in multiple cellular processes, including cell membrane rigidity, establishment of separate cellular domains by creating membrane diffusion barriers, providing scaffold for localization of proteins to certain subcellular regions and determination of cell polarity (16,18,19). The precise mechanism of SEPT9 molecular function in colon cancer pathogenesis has not yet been clearly described (20), however, several studies suggest possible roles in different malignancies, including leukemia (21), breast and ovarian cancer (22–24), brain tumors (25) and CRC (19,26–28).

The methylation status of the SEPT9 gene has been examined previously in CRC patients, as well as cases with precancerous lesions including adenomas in several studies (29,30). In a recent study conducted by Ahmed et al (31), a methylation panel including the SEPT9 gene was analyzed in CRC patients, and concluded that SEPT9 promoter methylation is a promising biomarker with the ability to discriminate CRC tissues as well as adenomas, from normal mucosa.

The NTRK neurotrophin receptor family includes NTRK1 (TrkA), NTRK2 (TrkB) and NTRK3 (TrkC), which, in conjunction with their ligands (NGF, BDNF and NT4/5, NT-3, respectively) are important in development of the nervous system (32). It has been demonstrated previously that NTRKs have oncogenic effects in some cancer types, such as breast cancer and liver cancer (33). Recent studies have demonstrated that NTRK1 and NTRK3 may be dependent receptors, which depend on availability of their ligands to select their specific cell signals; these receptors are defined as having the ability to induce opposite effects in the presence or absence of their ligands (34,35). The availability of the ligand leads to the transduction of a positive cellular survival or differentiation signal, whereas the induction of apoptosis is a result of the absence of ligand (36). Observations which have demonstrated NTRK3 is a beneficial prognostic factor in certain cancers, including melanoma and medulloblastomas, indirectly support that NTRK3 has a dependence receptor role and is a conditional tumor suppressor gene (37,38). Therefore, these findings suggest that NTRK3 may act as a conditional tumor suppressor gene in CRC.

Specific somatic missense mutations in NTRK3, which probably inhibit its function, have been identified in colorectal cancer, as well as breast, lung, and pancreatic cancers (39,40). Considering the possibility of NTRK3 as a CRC tumor suppressor gene, based on the discovery of its mutant and methylated forms in CRC, Luo et al (41) conducted a study in order to define the effect of aberrant methylation on NTRK3 expression, and also to define whether NTRK3 has oncogenic or tumor suppressor functions in CRC cell lines. The authors concluded that aberrant methylation of NTRK3 is prevalent in CRC and adenomas that consequently silences its expression, which suggests its tumor suppressor role. The results exhibited NTRK3's function as a dependent receptor which means that binding its ligand, NT-3, it can induce proliferation while the absence of NT-3 leads to NTRK3-mediated apoptosis. Overall, those findings suggested NTRK3 as a novel conditional tumor suppressor gene in CRC.

The present study aimed to analyze the methylation status of SEPT9 and NTRK3 gene promoters in order to examine their ability to differentiate CRC tissues from normal mucosa, and to assess the validity of NTRK3 as a methylation marker in clinical CRC samples.

The aberrant methylation in the promoter regions of genes is a prevalent event in a number of cancers including CRC (42). There are several methods to identify the methylation status of genomic DNA in specific sites; of those, MS-HRM is a highly sensitive semi-quantitative method, which can detect methylation level of specific region of bis-DNA precisely.

The MS-HRM assay was performed in order to identify the methylation status of SEPT9 and NTRK3 gene promoters. The methylation levels of SEPT9 and NTRK3 genes in CRC were high compared with matched normal tissue (P<0.001). There was not any significant association between clinical features and pathological findings of patients and hyper-methylation of analyzed genes in this study. The results of the present study demonstrated that the first and second studied locations of SEPT9 promoter region were not associated with age, gender, TNM stage, and grade of tumor, while the first location was just associated with tumor location. Similarly, NTRK3 methylation was not associated with such clinical and pathological parameters. Previous studies have reported that the proportion of methylated SEPT9 genes augmented with the progression of CRC (30,42), whereas our results demonstrate that this proportion of SEPT9 was similar between different stages and tumor grades of CRC cases. These results are in agreement with the results of other similar studies such as Su et al (43). However, these findings may be biased due to the small number of cases included in the study. Statistical analysis indicated that there is no association between methylation level of first and second location of SEPT9 gene, which suggests that their methylation process is independent of each other, however, the overall methylation in either tumor or normal matched adjacent tissue was higher in the second location in comparison to the first location, which may be due to the importance of this location in methylation-mediated silencing of SEPT9. In addition, the sensitivity and specificity of the second analyzed location for detection of CRC tissues form normal ones was higher than the first location. This finding suggests that this location may be more significant in SEPT9 hypermethylation and the development of CRC, and can detect tumor tissues more precisely then first location. In an study established by Wassekort et al (44) it has been shown that hypermethylation in a specific CpG island of SEPT9 promoter (including our studied locations) is probably an early event in adenocarcinoma progression. Furthermore, Wassekort et al (44) have proved that there is a direct link between this region and the region cross-examined by EpiproColon test that detects methylation of SEPT9 in cell free DNA. Our results, in concordance with the published data, supports that SEPT9 methylation in this CpG island can be a useful biomarker in CRC diagnosis. Tóth et al (20) analyzed SEPT9 methylation in both tissue and plasma of healthy, adenoma and CRC cases quantitatively, and detected methylated SEPT9 in all tissue samples at different levels regardless of the group. Methylated SEPT9 levels in CRC and adenoma tissue samples were not significantly different; however, its levels in healthy tissue samples were much lower and considerably distinct from either adenoma or CRC (20). Overall, our findings supported previous studies for SEPT9 gene being a promising marker for detection of CRC.

The NTRK3 gene has been recently demonstrated to become hypermethylated and silenced in CRC cell lines, which suggests its role as a tumor suppressor gene in colorectal cancer (41). The present study analyzed NTRK3 promoter methylation in order to determine whether it can act as a biomarker in diagnosis of CRC or not. Our results showed that the overall mean of methylation level in either normal tumor free tissue or CRC tumor tissue samples was high in which there were just two samples with 0% methylation. Although our results are similar to those of Luo et al (41) and obviously NTRK3 promoter methylation is able to discriminate the tumoral samples from normal tumor free samples with acceptable sensitivity and specificity, however, its high level methylation in tumor adjacent tissue suggests that it may start methylation process far before the appearance of any pathological features in cancerous cells. However, this hypothesis should indeed be further analyzed with samples in very initial stages of carcinogenesis.

In conclusion, based on our findings, SEPT9 methylation can be used as a diagnostic marker independent of different clinicopathological features of CRC patients. In particular, the second location is a promising candidate, considering its high sensitivity and specificity and also the accuracy of the test. For NTRK3 gene, we suggest further analysis with large sample size and specially samples in very initial stages of colorectal carcinogenesis in order to define its potential as a diagnostic biomarker in CRC.