Single nucleotide polymorphism of human colorectal cancer in relation to certain cytokines

number: 
2427
English
Degree: 
Imprint: 
Biotechnology
Author: 
Melad Mumtaz AL-Samak
Supervisor: 
Dr. Hameed M. Jasim Al-Dulaimi
Dr. Anis M. Al-Rawi
year: 
2010
Abstract:

Colorectal cancer (CRC) is an important public health problem. It is one of the leading causes of cancer mortality in the world. Single or combination of different Single nucleotide polymorphisms of certain genes is associated with susceptibility to cancer. The current novel study attempts to discover the influence of single nucleotide polymorphism and expression of cancer associated biological agents mostly cytokines in colorectal carcinoma of Swedish patients. Single nucleotide polymorphisms was conducted using blood DNA sample from colorectal cancer patients and control subjects to investigate the influence of certain polymorphic variant of MHC class II transactivator (CIITA), resistin, CC chemokine 21 (CCL21) and inhibitor of differentiation 1 and 3 (d1 and Id3) genes in colorectal cancer risk, using the 5’-exonuclease allelic discrimination assay (Tag man Real time PCR). Immunohistochemical (IHC) assay was used to study the cell type origin of resistin and CCL21 expression in 4 μm sections from formalin-fixed paraffin- mbedded tissue blocks of CRC samples. Enzyme linked immunosorbent assay (ELISA) was used to measure the protein level of resistin and CCL21in paired mucosal samples (tumour and matched normal mucosa). Western blot was used to detect resistin in paired mucosal samples. Moreover Real-time RT-PCR was used to determine the MMP-2, MMP- 9 and TIMP-1 mRNA expression levels of non-stimulated and resistin stimulated THP-1 monocytes and macrophages tissue cultures. And finally Gelatin zymography was used to document the cleavage of gelatin by MMP-2 and MMP-9 of the culture medium from non-stimulated and resistin stimulated THP-1 monocytes and macrophages. Two hundred seventy four blood samples from colorectal cancer patients were used in this study, mean and median age of CRC patients were 70 years and 71 years respectively (range 29- 93 years). CRC group represents 144 males and 130 females. Clinicohistopathological characteristics which involves; localization (colon and rectum), colon site (left and right) staging (classified according to Dukes’ classification system), differentiation grade (high, moderate and low differentiation), in addition to the age and sex for colorectal cancer patients were recorded. Blood samples were obtained from 278 control subjects, this group was composed of 146 males and 132 females with a mean and median age of 68 years and 70 years respectively (range 50-83 years). Blood samples were used for DNA extraction that was used in genotyping of certain SNP in CIITA, resistin, CCL21, ID1 and ID3 genes. DNA was successfully extracted from blood samples using the QIAamp DNA Blood Mini Kits. One hundred fifty seven Lysates were successfully prepared from paired mucosal samples (tumour and matched normal mucosa) in order to use it in ELISA and western blot. Finally 26 formalin-fixed paraffin embedded tissue blocks were used to prepare it 4 μm sections for IHC analyses. MHC-II transactivator (CIITA), encoded by the MHC2TA gene, is considered to be the master regulator for MHC-II gene expression. Reduced expression of major histocompatibility complex class II (MHC-II) genes in colorectal cancer (CRC) has been reported. A functional single nucleotide polymorphism (SNP) –168A→G in the promoter region of the MHC2TA gene is suggested to have an influence on different autoimmune diseases. This study was performed to evaluate the association between the –168A→G MHC2TA gene variant in patients with CRC versus a control group. It was found that there is no significant difference in genotype distribution or in allelic frequencies between the two groups, nor any association with clinical characteristics. Resistin is work as a potential immunomodulatory functions and mechanistic actions in inflammation as a cytokines, inflammatory cytokines that produced by tumour cells and or stromal cells contribute directly to tumour proliferation, spreed and metastasize to a secondary tumor at a distant site. In current study a single nucleotide polymorphism −420C>G of the resistin gene was screened in colorectal cancer (CRC) patients and controls. The result indicated that there were no significant differences in genotype distribution or in allelic frequencies between the two groups, nor any association with clinical characteristics. However, ELISA test found an upregulation in 92% of the samples in the levels of resistin protein in cancer tissue (n=83). Immunohistochemical analysis revealed heterogenous staining of resistin predominantly in the cancer tissue. Further, resistin induced secretion of MMP-2 and MMP-9 from monocytes. The results of this study suggest that resistin may play a partial role in CRC but that the −420C>G resistin polymorphism is not a potential genetic susceptibility factor. CCL2 plays a major antitumourigenic role but has also been shown to participate in tumour growth and tumour progression. To gain insight into the possible influence of CCL21 on colorectal cancer (CRC) this noval study was done to determine whether the CCL21 is altered in CRC tissue. Collectively, by using ELISA we noted a significant lower CCL21 level in cancer tissue compared with paired normal tissue. Patients with a tumour localized in the rectum revealed significantly lower level of CCL21 than patients with a tumour localized in the colon both compared with paired normal tissue. Immunohistochemistry demonstrated a heterogeneous immunoreactivity predominantly within areas of stromal cells mainly in macrophages. Moreover TaqMan system was also used to investigate two single-nucleotide polymorphisms rs 11574915 and rs 2812377 with supposed effect on CRC. No significant difference was observed between CRC and control subjects regarding genotype and allelic distributions or associations to clinical characteristics or CCL21 tissue levels. Inhibitor of differentiation (ID) proteins are a subclass of helix-loop-helix family of transcription factors. ID proteins have another role as either cooperating oncogene proteins or as dominant oncogene proteins in various contexts. This novel study was achieved to investigate the influence of ID1 gene polymorphism (rs1802548) and ID3 gene polymorphism (rs11574) in CRC susceptibility. No significant difference was observed between CRC and control subjects
regarding genotype and allelic distributions or associations to clinical characteristics for both SNPs. In conclusion the results of this study suggest that all the studied polymorphisms are not associated with susceptibility to CRC. The results of this study suggest that resistin plays a partial role in CRC. However, it implied that lower level of CCL21 in CRC tissue may indicate that it has antitumour roles and this supports the idea that cancer is related to immunodeficiency probably depending on regulatory factors produced by tumour cells which suppress or reduce antitumour agents. Moreover the different levels of CCL21 in rectum and colon may reflect divergent mechanisms in colorectal carcinogenesis.