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This study examines the association between MLH1 gene expression in peripheral blood and colon cancer, utilizing a case-control approach to compare expression levels in patients and matched healthy controls.
MutL homolog 1 (MLH1) is a component of the heterodimeric complex MutLα that detects and fixes base-base mismatches and insertion/deletion loops caused by nucleotide misincorporation. In the absence of MLH1 protein, the frequency of non-repaired mismatches increases, resulting in organ cancer. The current study sought to quantify MLH1 gene expression and its relationship with tumor invasion (T) and lymph node invasion (N) in blood samples from patients with colorectal cancer (CRC). Blood samples were obtained from 36 CRC patients. RNA was extracted, and cDNA was synthesized using a kit. The primers were built using the exon-exon junction approach, and MLH1 and β-actin genes were tested 3x using real-time polymerase chain reaction (Real-Time PCR). Gene expression analysis software was used to analyze the data, and a t-test was used to examine the expression of MLH1 and its connection with T and N variables. In this study, 36 patients with colorectal cancer, including 15 (41.6%) women and 21 (58.4%) men, with a mean age of 57.35 ± 4.22 years and in the age range of 26-87 years, were included. The results showed that the ratio of MLH1 gene expression in patients decreased compared to that in healthy individuals, and the decrease in gene expression at different stages of the disease was significant. The results of this study showed that the reduction of MLH1 gene expression has an effective role in the development of CRC.
Colon cancer (CRC) is one of the most common types of cancer. It is the fourth leading cause of cancer-related death worldwide1. CRC is more frequent in males than in females, and it is three to four times more common in industrialized countries than in developing countries. The age-standardized (global) incidence rate per 1 x 105 of CRC incidences is 19.7 in both sexes, 23.6 in men, and 16.3 in females2. Epidemiological studies have shown strong environmental and lifestyle associations with CRC. Obesity, red/processed meat, tobacco, alcohol, androgen deprivation therapy, and cholecystectomy are all associated with modestly increased CRC risks2,3.
Chromosomal instability, microsatellite instability, and CpG island methylator phenotype (CIMP) play an important role in the tumorigenesis of CRC4. According to previous studies, approximately 250 different mutations have been identified in patients with CRC, which is equivalent to approximately 55% of known mutations related to DNA mismatch repair (MMR) genes. Defects in mismatch repair proteins can be caused by germline mutations in the MSH6, MLH1, PMS2, and MSH2 genes, and most of these mutations are found in MLH1 and MSH2 genes4,5. The most important protein in the MMR system, which is usually involved in CRC, is MLH1. Recent studies have shown that any change in MLH1 expression may increase the risk of CRC. Germline mutations in MLH1 are responsible for Lynch Syndrome, an inherited type of CRC. In addition, 13%-15% of diffuse colon cancer cases are caused by MLH1 deficiency based on somatic promoter hypermethylation6,7,8.
MLH1 gene is located on the short arm of chromosome 3 at position 22.2 and contains 21 exons9. The protein encoded by the MLH1 gene can cooperate with an endonuclease involved in mismatch repair, PMS2, to generate MutLα, which is part of the MMR system. MutLα is mainly involved in the repair of base-base mismatches and deletion and addition loops as a result of incomplete DNA replication. In addition, the encoded protein is involved in DNA damage signaling and can be converted to the ɣMutL form with the MLH3 protein, which is involved in DNA mismatch repair observed in meiosis10,11,12. Studies have shown that MLH1 is involved in other major cellular activities, including regulation of cell cycle checkpoints, apoptosis, crossover recombination, and mitotic incompatibility13.
The MLH1 gene plays a key role in the DNA mismatch repair (MMR) system. A defect in the function of this gene can lead to the accumulation of genetic mutations and, as a result, the development of colorectal cancer14. Previous studies have shown that about 55% of mutations associated with MMR genes in patients with CRC are related to MLH1 gene mutations. In addition, decreased MLH1 gene expression can lead to Lynch syndrome, which is an inherited form of colorectal cancer15,16. Also, MLH1 gene defect based on somatic promoter hypermethylation has been observed in 13%-15% of sporadic colorectal cancer cases17. These scientific evidence show that the MLH1 gene acts as an important biomarker in colorectal cancer, and its expression analysis can provide valuable information about the function of the MMR pathway and the genetic risk of CRC18. Measuring MLH1 expression levels in the peripheral blood of patients with colon cancer can provide valuable information about the functionality of the MMR pathway, which is often disrupted in colon cancer. This method can be used for prognostic purposes and to understand genetic susceptibility to colon cancer19,20. A study on the relationship between MLH1 415 locus G to C mutation and sporadic colorectal cancer in Chinese patients found that the frequency of the MLH1 C/C genotype was significantly higher in sporadic CRC patients than in controls, suggesting a genetic susceptibility to sporadic CRC in Chinese patients21. Another study compared the gene expression of CRC genetic biomarkers in peripheral blood and biopsy samples of inflammatory bowel disease (IBD) patients, highlighting the potential of peripheral blood gene expression analysis for understanding colon cancer-related biomarkers22.
Considering the important role of the MLH1 gene and studies conducted in recent decades with molecular analysis by profiling mRNA expression, cancers have been classified with higher accuracy. The purpose of this study was to quantitatively investigate the expression of MLH1 in peripheral blood samples of patients with CRC using real-time PCR, and to investigate its relationship with pathological factors, stages of tumor progression to the layers of the intestinal wall (T), and stages of invasion to lymph nodes (N). This study was carried out in 36 CRC patients to potentially establish quantitative changes in gene expression as biomarkers for CRC screening, prognosis, and diagnosis using peripheral blood samples.
A case-control research was conducted at Affiliated Hospital 2 of Nantong University between April 2021 and May 2023. Engage with the hospital's administrative department to establish the study framework. Ethical approval was obtained by submitting the study proposal to the Nantong University Ethics Committee. Ethical guidelines were followed to ensure confidentiality and informed consent.
1. Patient recruitment and study design
2. Extraction and purification of RNA
3. Primer design for real-time PCR
4. Real-time PCR
5. Immunohistochemistry and genetic analyses
6. Statistical analysis
In this study, 36 patients with colon cancer were examined for MLH1 gene expression in the peripheral blood and its relationship with colon cancer. Analysis of demographic variables showed that 15 patients (41.6%) were women, and 21 patients (58.4%) were men. The mean age of the patients was 57.35 ± 4.22 years, and the age range was 26-87 years. The body mass index (BMI) status of the patients showed that 14 patients (38.8%) had a normal BMI (18.5 and 24.9 kg/m2), and 22 patients (61.2%) did not ...
This study was conducted with the aim of investigating the expression of the MLH1 gene. In this study, it was shown that the level of MLH1 gene expression was decreased in sick people compared to healthy people. Based on fold change studies, it has been shown that the expression of the MLH1 gene in Stage II, Stage III, and Stage IV in sick people compared to healthy people had a significant decrease.
Colorectal cancer is a major problem in the management of cancer in...
The authors declare no conflict of interest.
We would like to express our gratitude and appreciation to everyone who helped us complete this research endeavor.
Name | Company | Catalog Number | Comments |
Agarose Gel Electrophoresis Equipment | Bio-Rad | Mini-Sub Cell GT Systems | Used to check RNA quality |
Ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | E9884 | Used as an anticoagulant for blood samples |
NanoDrop | ThermoFisher Scientific | ND-2000 | Spectrophotometer used to determine RNA purity |
Real-time PCR Machine | Applied Biosystems | A34322 | Used for RT-PCR reactions |
RNA Extraction Kit | Intron Biotechnology Co | #Cat 17061 | Used for RNA extraction from blood samples |
SYBR Green PCR Kit | Thermo Fisher Scientific | 4309155 | Reagents used for RT-PCR experiments |
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