Association between colorectal cancer, the frequency of Bacteroides fragilis, and the level of mismatch repair genes expression in the biopsy samples of Iranian patients

Background Deficient DNA mismatch repair (MMR) can cause microsatellite instability (MSI) and is more common in colorectal cancer (CRC) patients. Understanding the carcinogenic mechanism of bacteria and their impact on cancer cells is crucial. Bacteroides fragilis (B. fragilis) has been identified as a potential promoter of tumorigenesis through the alteration of signaling pathways. This study aims to assess the expression levels of msh2, msh6, mlh1, and the relative frequency of B. fragilis in biopsy samples from CRC patients. Materials and methods Based on the sequence of mlh1, msh2, and msh6 genes, B. fragilis specific 16srRNA and bacterial universal 16srRNA specific primers were selected, and the expression levels of the target genes were analyzed using the Real-Time PCR method. Results Significant increases in the expression levels of mlh1, msh2, and msh6 genes were observed in the cancer group. Additionally, the expression of these MMR genes showed a significant elevation in samples positive for B. fragilis presence. The relative frequency of B. fragilis in the cancer group demonstrated a significant rise compared to the control group. Conclusion The findings suggest a potential correlation between the abundance of B. fragilis and alterations in the expression of MMR genes. Since these genes can play a role in modifying colon cancer, investigating microbial characteristics and gene expression changes in CRC could offer a viable solution for CRC diagnosis.


Introduction
Colorectal cancer (CRC) is one of the most prevalent malignancies afflicting both men and women [1].Globally, CRC ranks as the second cause of cancer-related deaths, claiming the lives of many, and is the third most common cancer worldwide.Fortunately, the incidence of CRC in Iranian individuals is comparatively lower than in Western countries [2].As of 2020, reports indicate that 1.9 million people are diagnosed with CRC annually [3,4].Given its considerable lethality, swift and early diagnosis and intervention become imperative [5].Individuals with CRC exhibit noticeable alterations in gut microbiota compared to healthy individuals.Notably, there is an increase in the presence of B. fragilis, Fusobacterium, Enterobacteriaceae, Campylobacter, Erysipelotrichaceae, Collinsella, and Peptostreptococcus in the faeces of CRC patients.Numerous studies underscore the significance of B. fragilis as an enterotoxin-producing bacterium, playing a pivotal role in the initiation and progression of CRC.This involvement occurs through modulation of the mucosal immune response, epithelial cell modification, and the induction of adenoma in the primary stages of CRC [6][7][8][9].
Recent investigations confirm the increase of toxigenic B. fragilis species in CRC patients.Furthermore, the presence of toxigenic B. fragilis markers has been validated in the colon and terminal ileum of patients with ulcerative colitis, a population prone to developing colon cancer [10,11].It is demonstrated that B. fragilis is a predominant and consistent pathogen in stool mucosa and colon tissue samples of CRC patients [12].
While CRC primary diagnosis conventionally relies on colonoscopy, molecular markers such as carcinoembryonic antigen in serum are employed in clinical settings for CRC diagnosis [13].However, identifying markers indicative of the transformation from adenomatous polyp to adenocarcinoma in the disease's early stages remains elusive [14].Addressing this gap, the identification of diagnostic markers could expedite CRC diagnosis and impede its progression.
The Mismatch Repair (MMR) system, integral to DNA homeostasis, is among the enzyme systems crucial for maintaining genomic stability.MMR loss leads to the rapid accumulation of potential mutations, predisposing individuals to specific cancer types [15].
Mutations in MMR proteins result in Microsatellite Instability (MSI), a genomic instability syndrome implicated in Lynch syndrome and gastrointestinal cancers.Lynch syndrome primarily arises from germ cell mutations, predominantly in mlh1 or msh2, and to a lesser extent in msh6 and rarely pms2 [13].MMR genes encode proteins that recognize and repair errors that occur during cell replication.In individuals with mutations in MMR genes, such as msh2, msh6, and mlh1, the risk of CRC is significantly increased [14].
Scientific evidence indicates a substantial increase or decrease in the relative expression of MMR system genes in various human cancers [16].Recent studies underscore a significant elevation in the relative expression of MMR system genes in CRC patients.Consequently, evaluating the extent to which their expression fluctuates in different diseases can serve as a biomarker for cancer diagnosis and treatment, particularly in the context of colorectal cancer.In this study, the expression of mlh1, msh2, and msh6 genes and the relative presence of B. fragilis in biopsy specimens of patients with CRC and healthy individuals were investigated by Real-Time PCR to determine whether the presence of this bacterium affects the expression of selected genes that are involved in CRC development.

Sampling
In this study, two separate groups of individuals were evaluated.The first group consisted of twenty healthy individuals suspected of CRC who underwent a colonoscopy, while the second group included a total of 40 patients with CRC.Colonoscopy biopsies were got from the right (from the cecum to transverse colon) and left (from descending colon to the rectum) colons of patients.Tissue biopsies were collected in Transystem tubes containing normal saline and RNA-later, and were kept at − 20 °C until analysis.

DNA and RNA extraction and cDNA synthesis
Biopsy samples have been extracted using special DNA and RNA extraction kits (ROJE Company-Iran) to analyze tissue samples.In the following step, a spectrophotometer (Nano Drop, 2000) was used to measure the concentration and purity of the extracted DNA.As well, cDNAs were synthesized using a cDNA synthesis kit (RT-Roset, ROJE Company-Iran).

Real-time PCR
In order to run Real-Time PCR, specific primers mentioned in Table 1 were utilized to assess the selected genes expression modification and the relative abundance of B. fragilis.Quantitative PCR reactions were performed on Real-Time PCR Applied Biosystems 7900 using SYBR® select Master Mix in 20 µl reactions.Cycle conditions for the mlh1, msh2, and msh6 genes were as follows: 95 ° C for 10 min and 40 cycles at 95 ° C for 20 s, 55 ° C for 30 s and 72 ° C for 30 s. Cycle conditions for the detection of B. fragilis were as follows: 95 ° C for 10 min, and 40 cycles at 95 ° C for 20 s, 56 ° C for 30 s, and 72 ° C for 30 s.

Reference gene for qPCR
The gapdh cellular gene was applied to normalize the target genes expression in biopsy samples.Also, the bacterial universal 16srRNA gene was used as a reference gene to investigate the relative abundance of B. fragilis (Table 1).In order to ensure the accuracy of the results, all qPCR reactions were conducted in duplicate for controls and tests.

Statistical analysis
Biopsy samples from the control group (n = 20) and cancer group (n = 40) in terms of presence, relative frequency of B. fragilis, and relative expression of mlh1, msh2, and msh6 genes were analyzed.The formula 2 −ΔΔCt was used to determine the relative expression of each mentioned MMR genes to gapdh RNA.ΔΔCt = ΔCt (Target)-ΔCt (Reference).
The following formula was used to calculate the fold change in the expression of target genes.
2 −(ct target − ct reference) Tumor / 2 −(ct target − ct reference) normal Data analysis was performed using SPSS version 21 and PRISM software version 8. Quantitative data were summarized as mean and reaction progression deviation.Quantitative data were checked for normal distribution, and if normality test was passed, analysis of variance (Non-parametric ANOVA) with a significant level (P value < 0.05) was used.

Samples
According to demographic information, 52% of patients in cancer group were women and 48% of them were men.The age range of the women was between 50 and 60, while men ranged from 50 to 80 years.The individuals in the control group included 45% women and 55% men, with the highest age range between 30 and 40 and 30-50 years for women and men, respectively.The most common symptoms that led to colonoscopy in patients were anemia (34%), abdominal pain (31%), blood in the stool (19%), and rectal bleeding (16%).Figure 1 demonstrates the involvement of different parts of the colon in patients with CRC, obtained after gastroenterology examinations and pathology results.Based on morphological diversity, tissue samples included adenocarcinoma (87%) and adenoma (13%).The tissue samples obtained from the patients are related to the proximal and distal regions of the intestine.Also, Table 2 provides complete descriptions of cancer samples.

The expression level of target genes
The gapdh gene was utilized as a control to investigate the expression of mlh1, msh2, and msh6.Real-Time PCR was used in order to estimate gapdh gene expression levels in control and cancer groups.Based on obtained results, a comparison of mlh1 gene expression in control and cancerous groups indicates that the mlh1 gene in the cancer group significantly increased compared to the control group (P value = 0.0139) (Fig. 2).Moreover, msh2 gene significantly increased in cancer group compared to the control group (P value = 0.0128) (Fig. 2).Comparison of msh6 expression in healthy individuals and cancer patients presented a significant elevation in msh6 expression in the cancer group compared to the control one (P value = 0.0001) (Fig. 2). Figure 3 contains information about the expression of genes in the control and cancer groups.Based on fold change analysis of MMR genes, the level of mlh1 gene expression was 5 times higher in the cancer group in comparison with the control group (P value = 0.0139).Additionally, msh2 and msh6 genes expression increased by 6 and 7 times in cancer group compared to the control group, respectively (P value = 0.0128 and P value = 0.0001) (Fig. 4).The relative abundance of the bacterium was estimated using the 16srRNA gene primers specific for B. fragilis.The results showed that the frequency of B. fragilis in the cancer group was significantly higher compared to the control group (P value = 0.0378) (Fig. 5).

B. fragilis abundance in cancer and control groups
Real-time PCR was performed using 16s rRNA specific for B. fragilis for all samples.The relative frequency of B. fragilis was significantly higher (80%) in the cancer group compared to the control group (50%).The prevalence of B. fragilis in men with cancer was higher than in women, so that the presence of this bacterium was reported in 55% in men and 44% in women.The prevalence of B. fragilis varied among cancer patients of different genders and age groups, with a higher prevalence observed among men and women aged 50-60.

Relative abundance of B. Fragilis in different types of CRC samples
In another part of the study, the relationship between the location of the tumor and the frequency of B. fragilis was investigated, which showed that B. fragilis was observed in 68.8% of tumors located in the distal part of the colon and in 31.2% of tumors located in the proximal part of the colon and rectum.Consequently, tumors located in the distal part of the colon were more associated with B. fragilis.Results indicated B. fragilis was more prevalent in cancer specimens with adenocarcinoma morphology than in other morphologic types.B. fragilis was detected in 67.5% of cancer specimens with adenocarcinoma morphology and 4% with adenoma morphology.Results also showed different relative distribution of B. fragilis in various parts of the large intestine.Accordingly, the most abundance was observed in the rectum, sigmoid colon, cecum, ascending colon, descending colon, hepatic flexion, and transverse colon, respectively.

Changes in the relative expression levels of the selected genes in the presence and absence of B. fragilis
Specifically, the level of expression of mlh1, msh2, and msh6 was compared in the cancer group with and without the presence of B. fragilis.This indicated an increase in the expression of the mentioned genes in cancerous samples in the presence of B. fragilis compared to conditions where B. fragilis is not present.In order to evaluate the expression level of mlh1, msh2, and msh6 genes, a fold change analysis was done.Based on our results, msh2 and msh6 expression levels were raised approximately 6.5 times in the cancer group compared to the control group, and mlh1 expression levels by about 5 times (Fig. 6).

Discussion
CRC is one of the most common types of cancer diagnosed worldwide.The occurrence of CRC has been attributed to various factors, with the age of the affected individual being recognized as one of the most significant known risk factors [16].Several reports indicate that the risk of CRC increases significantly during the fifth decade of life.Despite this, CRCs are rare in individuals under the age of 50 [17].The age range of patients in this study was 50 to 80 years.A total of 48% of this population was male and 52% was female.In a study conducted by Mirzapoor Abbasabadi et al. in Iran, the age range of the patients was 59.5 years, and 59.6% of the subjects were men, which was different from our study [18].The results of the current study also demonstrated a higher commonness of CRC in the left part of the colon than in the right part, which was similar to Raza et al., study [19].In contrast to our study, Komiya et al. found a higher incidence of CRC in the ascending colon.Their results suggest that CRC occurs more frequently in the rectum [20].
Considering the importance of CRC, studying its causes is crucial.It is possible to provide effective prevention and treatment by understanding the causes of these diseases.There are several molecular methods for the early diagnosis of CRC, which can be mentioned as Germ-line APC mutations, mutant alleles of K-ras genes, and alteration in MMR genes [21,22].The findings of this study show a significant increase in the relative expression of msh2, msh6, and mlh1 genes in cancer samples compared to the control group.Many studies have investigated the mlh1 gene and its role in cancers, especially CRC [23][24][25].Defects in MMR genes (mlh1, msh2, msh6) lead to MSI, which is characteristic of hereditary non-polyposis CRC.MSI is a state of genetic variability (prone to mutation) that results from impaired DNA mismatch repair.However, high-frequency MSI occurs in approximately 15% of CRC and other tumors, where MMR defects are caused by epigenetic inactivation of the mlh1 gene by DNA methylation [26].A study conducted by Engel et al. in 2019 on tumor tissue stated that the risk of adenoma due to mutation of msh2 and msh6 genes is significantly higher compared to mlh1 [27].According to the results of the present study, the expression level of the mlh1 gene in cancer samples increased about 5 times compared to the control group, but this increase was less compared to the other two genes.According to another study conducted by Wang et al. in 2019, the expression level of msh2 and mlh1 was examined in the tumor tissue of patients after surgery.In 91% of colorectal carcinomas, the mlh1 gene was not expressed [28].Mutations in the mlh1 and msh2 genes are primarily responsible for the decrease in expression of these genes.Due to the dominance of these two genes in the MMR system, their detection is imperative to understanding the pathogenesis of sporadic CRC [29,30].An additional gene of the MMR system examined in this study is msh2, which encodes a protein vital to DNA repair.In a study conducted by Liccardo et al. in Italy in 2020, it was observed that msh2 gene expression in cancer samples was increased compared to the control group.It has also been mentioned that the overexpression of mlh1 or msh2 genes causes apoptosis or mutated and genetically unstable phenotype [31].According to several studies, it was proven that the overexpression of mlh1 and msh2 genes potentially leads to adverse consequences.When these two genes were upregulated in vitro under the control of the cytomegalovirus promoter, apoptosis was induced in a human cell line [31].
In the results of the present study, it was observed that the expression level of the msh2 gene in cancer samples is almost 6 times higher than in the control group, which is in line with recent studies.In Ekundina et al., study, the mean percentage reactivity of msh2 in normal, colonic polyps, and colorectal carcinoma was 43.2%, 56.6%, and 90.1% respectively, while the mean percentage reactivity for msh6 was 40.5%, 56.2% and 92% respectively [32].The level of msh2 and pms2 protein expression has a positive relationship with tumor size, the degree of tumor invasion to the depth of the tissue, and metastasis to the lymph nodes [33].According to the studies, mutations related to the expression of the msh6 gene are associated with a lower risk of cancer compared to the mutations of the mlh1 or msh2 genes, and those who carry mutations in the expression of the msh6 gene at an older age are also more likely to develop CRC.Several studies have emphasized the significance of the mlh1 and msh2 genes in the MMR system.Mutations in either of these genes lead to a loss of function and contribute to tumor formation, particularly in the proximal colon.Hyper methylation, a common occurrence in sporadic tumors, is notably more prevalent than in MSIpositive hereditary tumors.Additionally, multiple studies have indicated that the overexpression of the mlh1 gene and/or the msh2 gene is linked to tumor metastasis in various organs.
In addition to changes in MMR gene expression, alterations in the abundance of gut microbiota can also be seen in CRC patients, and identifying these two factors as biomarkers for diagnosis is crucial.The results of examining the expression of msh2, msh6, and mlh1 genes in comparison with the presence and absence of B. fragilis in cancer samples showed that in cancer samples with B. fragilis, there is a greater increase in expression than in cancer samples without this bacterium.B. fragilis is regarded as one of the most influential pathogens in the occurrence and spread of colon cancer [34].According to the results of the present study, the relative frequency of B. fragilis in cancer samples has increased about 5 times compared to control samples.Dadgar-Zankbar et al. conducted a study in Iran and found B. fragilis was significantly higher in tumor tissues than in adjacent healthy samples (100% vs. 86% respectively) [35].Several studies have stated that B. fragilis toxin is associated with various diseases, including CRC, which can be referred to the study conducted by Boleij et al. in 2015 on intestinal mucosa samples from patients with intestinal neoplasia [36].These results indicated that CRC is associated with the B. fragilis toxin gene in the late stages.They also stated that exposure to B fragilis toxin is common, which may be a risk factor for developing CRC.Based on all of these findings, it is pertinent to investigate the abundance of this bacterium in CRC samples.This will enable us to predict cancer progression more quickly and prevent its development.Furthermore, because MMR genes play an important role in cancer development and progression, further studies may be able to establish their importance as factors in the proper diagnosis of cancer or its advanced stage.The limitations of the current study included low sample size, lack of access to samples of different stages of CRC to evaluate biomarkers' expression in each stage, lack of investigation of fecal microbiota samples for further confirmation and in-depth verification of mechanisms by which B. fragilis lead to the damaging effect on the gastrointestinal tract.

Conclusion
This study reveals heightened expression of specific MMR genes in cancer samples compared to controls.Moreover, CRC biopsy samples exhibit increased bacterial frequency compared to healthy counterparts.Significantly elevated expression of the examined MMR genes is observed in B. fragilis-positive cancer samples versus those without this bacterium.Investigating B. fragilis presence in confirmed or suspected CRC samples is crucial for expedited cancer diagnosis and prevention.Positive molecular diagnostic tests or elevated risk marker expression potentially identify CRC patients eligible for surveillance or intervention.

Fig. 1
Fig. 1 Types of cancer samples examined in this study

Table 2
Pathological information of patients with CRC F: female, M: male