Background

Colorectal cancer (CRC) ranks as the third most prevalent form of cancer globally, with a reported incidence of over 1.84 million new cases in 2018. This places CRC behind only lung and breast cancers in terms of frequency [1, 2]. Colorectal cancer (CRC) has a higher prevalence in males compared to women, with an approximate difference of 30%. The incidence of CRC is generally low in adults below the age of 50, regardless of gender, but significantly rises with advancing age [3, 4]. Approximately 70% of people develop sporadic colorectal cancer (CRC) in the normal epithelial lining of the colon and rectum, without any hereditary predisposition [5]. Less than 10% of individuals diagnosed with colorectal cancer (CRC) exhibit a hereditary susceptibility, including conditions such as Familial Adenomatous Polyposis (FAP) and Hereditary Non-Polyposis Colorectal Cancer (HNPCC) [6, 7].

The completion of the human genomic project has recently drawn the attention of biomedical researchers to single nucleotide polymorphisms (SNPs) as a potential factor in the development of colorectal cancer (CRC) [8, 9]. Single nucleotide polymorphisms (SNPs) are inherent variations in DNA sequences that distinguish them from gene mutations. These variations are present in the general healthy population at a minimum frequency of 1% [6]. Single nucleotide polymorphisms (SNPs) are present in nearly all genes at varying rates and have the potential to impact the protein products of genes, potentially leading to an increased vulnerability to cancer. Among the genes that are currently recognized to possess single nucleotide polymorphisms (SNPs), a subset of particular interest is constituted by those that encode for microRNAs (miRNAs). MicroRNAs (miRNAs) are a group of small, single-stranded noncoding RNAs that have significant involvement in the control of gene expression by translational repression or degradation of target mRNA. These miRNAs have a profound impact on essential activities in a wide range of physiological processes [10,11,12]. Dysregulation of microRNAs (miRNAs) has been implicated in a diverse array of human disorders and can exert influence on the initiation and advancement of tumors through modulation of proto-oncogenes and tumor suppressor genes [13, 32]. In the process of PCR design, the focus was on four specific high-frequency single nucleotide polymorphisms (SNPs), namely Mir146a, Mir423, Mir370, and Mir196a2. These SNPs were targeted inside DNA fragments of varying lengths, specifically 257 bp, 215 bp, 306 bp, and 319 bp, respectively. The oligonucleotide sequences for PCR are presented in Table 1. The PCR experiments were carried out using a lyophilized PCR AccuPower PreMix (Promega Co., USA), with a final volume of 20 mL for each amplified fragment. Following the execution of polymerase chain reaction (PCR) tests, it was verified that the resulting PCR products exhibited sizes consistent with the anticipated values using electrophoresis conducted on agarose gels.

Table 1 The specific PCR primers designed for the amplification of the microRNA SNP
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SSCP technique

The genoty** studies were performed with the PCR-single-strand conformation polymorphism (SSCP) technique. Subsequently, the PCR products were put into polyacrylamide gels with an 8% concentration, and the samples were allowed to migrate until they reached the bottom of the gels. The gels were subjected to staining with Ethidium bromide in accordance with the prescribed methodology [33]. The SSCP banding patterns were confirmed using Sanger dideoxy-sequencing, following the recommended protocols provided by Macrogen Inc., a company based in South Korea. The DNA chromatogram of each genotype was viewed utilizing Snap Gene Viewer version 4.0.4, a software produced by Insightful Science in Canada. The alignment of the reported variation with the matching DNA sequences was conducted using Bio Edit software, specifically version 7.1.

Statistical and functional analysis

The chi-square test was employed to assess the disparities in genotype distribution between the subjects and the control group. The odds ratio (OR) and 95% confidence interval (95% CI) were computed to assess the relative risk of colorectal cancer (CRC) and to examine the associations between the single nucleotide polymorphism (SNP) and clinicopathological features of individuals diagnosed with colorectal cancer. The predetermined level of significance was established at a value less than 0.05. The statistical analyses were conducted using SPSS version 28.

Results

Demographic characteristics of study subjects

The research encompassed a comprehensive sample size of 100 cases of colorectal cancer (CRC) and 100 control subjects. The clinical and pathological attributes of these individuals are detailed in Table 2. Significant statistical differences were seen between the patients and controls in relation to smoking status, gender, age, and alcohol intake. There were no statistically significant differences found between the two groups in terms of inclination and education level. The postoperative pathological stages were categorized as follows: stage I, with a total of 50 cases accounting for 50% of the sample; stage II, with 25 cases representing 25% of the sample; stage III, with 20 cases accounting for 20% of the sample; and stage IV, with 5 cases representing 5% of the sample.

Table 2 Clinicopathological characteristics of CRC cases and controls
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Genoty** analysis

Four single nucleotide polymorphisms (SNPs) with the highest frequency were individually chosen. This study selected four single nucleotide polymorphisms (SNPs) as microRNAs (miRNAs): Mir146a, Mir423, Mir370, and Mir196a2. The analysis revealed four distinct banding patterns corresponding to the selected SNPs, indicating that the Mir146a, Mir423, Mir370, and Mir196a2 SNPs exhibit three genotypes each. The sequencing studies that were done successfully confirmed the presence of the three anticipated genotypes for all high-frequency single nucleotide polymorphisms (SNPs) that were examined. The electropherograms of the four examined single nucleotide polymorphisms (SNPs) exhibited the following genotypes: Mir146a (GG: GC: CC), Mir 423 (AA: AC: CC), Mir370 (GG: GA: AA), and Mir196a2 (CC: CT: TT) (Fig. 1).

Fig. 1
figure 1

The present work presents a schematic diagram illustrating the process of microRNA genoty** through the utilization of the PCR-SSCP-sequencing approach. In this study, we design four unique primers, denoted as A1, B1, C1, and D1. These primers were designed to amplify DNA fragments of certain lengths, 257 bp, 215 bp, 306 bp, and 319 bp. The purpose of these amplifications was to flank the regions associated with Mir146a, Mir423, Mir196a2, and Mir370, respectively. The genoty** technique employed in this study was PCR-SSCP as shown in figures A2, B2, C2, D2, specifically targeting SNPs. It was observed that all the targeted SNPs exhibited three distinct patterns of nucleic acid changes. The sequencing reactions of the targeted loci, as positioned in the amplified PCR fragments, are denoted as A3-A4, B3-B4, C3-C4, and D3-D4

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In order to assess the Hardy–Weinberg equilibrium (HWE) of the research population, an analysis of the genetic diversity of the discovered polymorphic single nucleotide polymorphisms (SNPs) was conducted. Based on the chi-square values, the compatibility of the polymorphisms of all four discovered polymorphic single nucleotide polymorphisms (SNPs) with the Hardy–Weinberg equilibrium (HWE) was confirmed in both the control and colorectal cancer (CRC) groups at a significance level of 0.05 (see Table 3).

Table 3 Hardy–Weinberg equilibrium (HWE) for Mir146a, Mir 423 A\C, Mir196a2 C\T, and Mir370 A\G SNPs in patients and control groups
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The distribution of Mir146a genotype in patients with colorectal cancer (CRC) was seen as follows: 45 individuals (45%) exhibited the homozygous GG genotype, 40 individuals (40%) exhibited the homozygous CC genotype, and 15 individuals (15%) exhibited the heterozygous GC genotype. The distribution of the Mir146a polymorphism genotypes in the control group was as follows: 65 participants (65%) had the homozygous GG genotype, 28 subjects (28%) had the homozygous CC genotype, and 7 subjects (7%) had the heterozygous GC genotype. The frequencies of G and C alleles in the subjected group were 52% and 57.5%, respectively, whereas in the control group, they were 68.5% and 31.5%, respectively. The distribution of genotypes for the miR146a gene in both patient and control groups is presented in Table 3.

In the patient group with colorectal cancer (CRC), the frequencies of the homozygous alleles AA and CC for the Mir423 polymorphism were observed to be 40% each. Additionally, the frequency of the heterozygous allele AC for this polymorphism was found to be 20% in the same patient group. In contrast, the healthy control group exhibited frequencies of 60%, 10%, and 30% for the polymorphic alleles AA, CC, and AC, respectively. The frequencies of the A and C alleles in the patient group were 50% and 50%, respectively, whereas in the control group, the rates were 65% and 35%, respectively.

In the study, the frequency of Mir370 polymorphism genotypes (AA, AG, and GG) was detected in both the patient and control groups. In the patient group, the observed frequencies were 40% for AA, 25% for AG, and 35% for GG genotypes. Conversely, in the control group, the observed frequencies were 70% for AA, 15% for AG, and 15% for GG genotypes. The frequencies of the A and G alleles in the sick group were 52.5% and 47.5%, respectively. In contrast, the control group had frequencies of 77.5% and 22.5% for the A and G alleles, respectively.

The frequencies of the Mir196a2 polymorphism alleles CC and TT in the patient group with colorectal cancer (CRC) were 50% and 32%, respectively, while the frequency of the heterozygous allele (CT) was 18%. In contrast, the healthy control group had frequencies of 70%, 15%, and 15% for the polymorphic alleles CC, TT, and CT, respectively, as indicated in Table 3 (Tables 4, 5).

Table 4 Genotype frequencies of Mir146a, Mir423, Mir196a2, and Mir370 in both the control (n = 100) and patient subjects (n = 100) and their association with CRC risk
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Table 5 Genotype frequencies of mir-146a, mir-423 and mir-196a2, mir370 in patient subjects (n = 100) and their association with tumors stage
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Discussion

The high death rates observed in diverse populations for colorectal cancer (CRC) can be attributed to the significant number of cases being diagnosed at later stages. It is worth noting that CRC is the fourth most prevalent kind of cancer globally [34]. The issue at hand is a significant public health concern across several populations [35]. According to the World Health Organization (WHO), it has been projected that the incidence of new cases of colorectal cancer (CRC) and the associated mortality rates will see a significant rise of 77% and 80%, respectively, by the year 2030 [36, 37]. MicroRNA (miRNA) has been extensively studied and shown to have a strong correlation with the initiation and advancement of many cancer types. A number of microRNA (miRNA) biomarkers have been discovered as possible risk factors for colorectal cancer (CRC) [38, 39]. The increasing prevalence of colorectal cancer (CRC) among various populations prompts inquiries into the potential risk linked to certain alleles of MicroRNA (miRNA) in affected individuals. The objective of this study was to investigate the correlation between microRNA (miRNA) polymorphism and colorectal cancer (CRC) in the Iraqi population by employing genoty** techniques to analyze four frequently seen single nucleotide polymorphisms (SNPs) in microRNA (miRNA) sequences.

MicroRNAs (miRNAs) have a crucial role in regulating a wide range of biological and physiological processes. Consequently, their dysregulation, which can be caused by mutations, transcriptional changes, and epigenetic modifications, appears to be associated with this phenomena [44]. The selection of the PCR-SSCP method in the present genoty** strategy was based on its cost-effectiveness and high sensitivity in identifying genetic difference within the 200–350 bp range [45].

This study revealed a significant association between Mir146a and colorectal cancer (CRC) within the sample group. The potential correlation between these single nucleotide polymorphisms (SNPs) and the initiation and progression of colorectal cancer (CRC) can be clarified by further examination. The current investigation revealed that individuals with the GG genotype of the Mir146a gene exhibited an increased vulnerability to colorectal cancer (CRC). The observed result is not unexpected, as previous studies have demonstrated that the activity of Mir146a, Additionally, it has been found that individuals with the GG genotype have higher expression levels of Mir146a, leading to an increase in its activity [46, 47]. Several investigations have been undertaken on colon, lung, breast, and hepatic cancer, which support our discovery of an elevated cancer risk associated with the Mir146a CG genotype [48,49,50]. The study on colon cancer did not acknowledge this particular correlation [51]. A subsequent investigation was conducted on a community in Italy, wherein it is widely acknowledged that cancer arises as a result of the interplay between genetic factors and environmental influences. On the contrary, a meta-analysis conducted on single nucleotide polymorphism (SNP) studies of colorectal cancer (CRC) microRNAs (miRNAs) revealed that individuals of Asian descent exhibit a tendency toward a correlation between the mir-146a SNP and an elevated risk of CRC [52].

Several studies have demonstrated that Mir146a, along with its prevalent polymorphism, rs2910164, which involves a G to C substitution, is found inside the sequence of the Mir146a precursor. In the present work, we want to investigate this miRNA hotspot in colorectal cancer (CRC) for the first time. The single nucleotide polymorphism (SNP) in question converts a G:U base pair to a C:U mismatch. As a consequence, there is a decrease in the levels of both pre- and mature Mir146a [57, 58]. Another study asserts that the Mir423 single nucleotide polymorphism (SNP) plays a protective effect in relation to the risk of breast cancer [22]. Our study conducted an observation of an elevated susceptibility to colorectal cancer (CRC) in individuals with the Mir423 single nucleotide polymorphism (SNP) CC genotype. A study conducted by [58] demonstrated an elevated susceptibility to colorectal cancer (CRC) when Mir 423 is accompanied by other mutations. These findings demonstrate a multifaceted interplay between single nucleotide polymorphisms (SNPs), other genetic variations, and the combined effects of environmental and ethnic factors.

The current study aimed to examine the genetic polymorphism of Mir 370 in colorectal cancer (CRC). Specifically, we explored the mir-370 single nucleotide polymorphism (SNP) rs2279398G > A in case–control studies to assess its association with CRC risk. Our findings revealed that this association exhibited diverse directions [59, 60]. Numerous studies have demonstrated that the rs2279398 polymorphism, which is situated in the 3’-UTR region of the DOK3 gene, has the ability to impact the efficacy of miRNA binding. Consequently, this polymorphism may have a role in the modulation of tumor suppression, while previous studies have suggested that the findings presented here could offer valuable SNP information for elucidating the specific biological implications of cancer progression and treatment resistance in individuals with colorectal cancer (CRC). In a similar vein, Zhang and colleagues have documented that the presence of the KRAS 3'-UTR polymorphism has the potential to serve as a predictive factor for the response of patients with wild-type KRAS colorectal cancer who are undergoing cetuximab monotherapy [61, 62]. However, despite the growing body of evidence indicating the significance of DOK3 in several facets of carcinogenesis, there remains a lack of clarity on the potential impact of the DOK3 (rs2279398) polymorphism on protein expression.

While rs11614913 SNP may not be as widely recognized as rs2279398 SNP, it has also been associated with the progression of colorectal cancer. Additionally, it has been found to be associated with elevated susceptibility to various other types of cancer [44, 72].

The main limitation of this study is the small sample size, which may affect the reported results by not fully reflecting the actual effects on the development and advancement of colorectal cancer (CRC). The utilization of a small sample size in this study may potentially affect the statistical significance of the findings. Therefore, it is advisable to reproduce the aforementioned technique using a larger sample size in order to acquire more reliable conclusions generated from a thorough investigation.

In summary, the genoty** investigations conducted on four variants within microRNAs demonstrated a significant association between the polymorphisms of Mir146a, Mir196a2, Mir370, and Mir423 single nucleotide polymorphisms (SNPs) and colorectal cancer (CRC) in the Iraqi people. Individuals harboring the Mir423: AC and Mir370: AG genotypes exhibited a heightened vulnerability to colorectal cancer (CRC). Both single nucleotide polymorphisms (SNPs) possess the potential to serve as biomarkers for the evaluation of colorectal cancer (CRC) in the Iraqi population being studied. However, it is imperative to do additional research in order to substantiate the molecular diagnostic capabilities that have been alluded to on a more extensive level.

Conclusions

The study of our aggregated data reveals that single nucleotide polymorphisms (SNPs) in microRNA have a notable and distinct correlation with the heightened susceptibility to colorectal cancer (CRC). Nevertheless, this study proposes the utilization of a more comprehensive range of examined samples in order to obtain additional insights into the association between both single nucleotide polymorphisms (SNPs) and the susceptibility to colorectal cancer (CRC).