Identification of membrane biomakers for colorectal cancer using an in-solico and molecular approach
- Authors: Van Vuuren, Larry Peter
- Date: 2014
- Subjects: Cancer -- Research , Colon (Anatomy) -- Cancer , Rectum -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/47970 , vital:40457
- Description: The aim of this study was to identify membrane biomarkers for colorectal cancer using an insilico and molecular approach. Colorectal cancer (CRC) globally accounts for more than half a million deaths. In South Africa alone, approximately one in 97 men is at risk of getting CRC; and for women, it is one in 162. Novel and non-invasive diagnostic tools, such as biomarkers, are needed for early CRC detection. In order to reduce the fatality rate in this disease, Biomarkers are used as indicators of a biological state; and they are measurable in biological media. They can be used to distinguish between a diseased state and a normal state, thus aiding diagnostics, response to specific therapies, and screening for an early diagnosis. A gene list of potential CRC biomarkers was generated by mining two gene databases, namely: Oncomine and Gene Expression Atlas. A total of 44 candidate genes were identified, based on their location on the cell surface, using the Database for Annotation, Visualisation and Integrated Discovery. These 44 genes were then subjected to an in-depth literature mining. The literature search parameters in PubMed, PubMed Central, Google Scholar and Science direct revealed publications showing that 23 genes were validated, while 21 genes were not validated. Nineteen genes were selected for gene validation in human colorectal cancer and healthy tissue of twelve patients. Total RNA was extracted from 12 colorectal cancer and 12 healthy tissue samples. The RNA was then quantified and reverse-transcribed into cDNA for gene expression analysis. The qPCR running conditions were optimized, by running a melting curve, in order to determine the optimum annealing temperatures. Primarily melt curves were run for nineteen of these twenty-one genes. Melt-curve analysis showed that nine genes were poor candidates for further validation studies; and therefore, only ten genes, namely: AGTRAP, ANKRD46, BACE2, CFB, CIAO1, NOMO3, PTDSS1, SLC5A6, TNFRSF12A and ZDHHC9 were validated by qPCR in human resected colorectal carcinoma and the normal tissues of twelve patients. The qPCR results showed that ZDHHC9 and the SLC5A6 genes were the only two statistically significant ones; and they were found to be down-regulated in human colorectal cancer vs healthy tissue samples.
- Full Text:
- Date Issued: 2014
Identification of membrane biomakers for colorectal cancer using an in-solico and molecular approach
- Authors: Van Vuuren, Larry Peter
- Date: 2014
- Subjects: Cancer -- Research , Colon (Anatomy) -- Cancer , Rectum -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/47970 , vital:40457
- Description: The aim of this study was to identify membrane biomarkers for colorectal cancer using an insilico and molecular approach. Colorectal cancer (CRC) globally accounts for more than half a million deaths. In South Africa alone, approximately one in 97 men is at risk of getting CRC; and for women, it is one in 162. Novel and non-invasive diagnostic tools, such as biomarkers, are needed for early CRC detection. In order to reduce the fatality rate in this disease, Biomarkers are used as indicators of a biological state; and they are measurable in biological media. They can be used to distinguish between a diseased state and a normal state, thus aiding diagnostics, response to specific therapies, and screening for an early diagnosis. A gene list of potential CRC biomarkers was generated by mining two gene databases, namely: Oncomine and Gene Expression Atlas. A total of 44 candidate genes were identified, based on their location on the cell surface, using the Database for Annotation, Visualisation and Integrated Discovery. These 44 genes were then subjected to an in-depth literature mining. The literature search parameters in PubMed, PubMed Central, Google Scholar and Science direct revealed publications showing that 23 genes were validated, while 21 genes were not validated. Nineteen genes were selected for gene validation in human colorectal cancer and healthy tissue of twelve patients. Total RNA was extracted from 12 colorectal cancer and 12 healthy tissue samples. The RNA was then quantified and reverse-transcribed into cDNA for gene expression analysis. The qPCR running conditions were optimized, by running a melting curve, in order to determine the optimum annealing temperatures. Primarily melt curves were run for nineteen of these twenty-one genes. Melt-curve analysis showed that nine genes were poor candidates for further validation studies; and therefore, only ten genes, namely: AGTRAP, ANKRD46, BACE2, CFB, CIAO1, NOMO3, PTDSS1, SLC5A6, TNFRSF12A and ZDHHC9 were validated by qPCR in human resected colorectal carcinoma and the normal tissues of twelve patients. The qPCR results showed that ZDHHC9 and the SLC5A6 genes were the only two statistically significant ones; and they were found to be down-regulated in human colorectal cancer vs healthy tissue samples.
- Full Text:
- Date Issued: 2014
Molecular signaling in colorectal carcinogenesis : the roles and relationships of beta-catenin, PPARgamma and COX-2
- Authors: Fredericks, Ernst
- Date: 2013
- Subjects: Colon (Anatomy) -- Cancer , Cyclooxygenase 2 , Peroxisomes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10357 , http://hdl.handle.net/10948/d1021014
- Description: Colorectal cancer (CRC) is a common disease with significant morbidity and mortality. In spite of significant advances in understanding the molecular signaling in this disorder, unanswered questions remain. Cyclooxygenase-2 (COX-2) and β-catenin have established roles in colorectal carcinogenesis, with both being upregulated early in the disease course. The role of peroxisome proliferator-activated receptor γ (PPARγ) is less clear, but has been shown to be downregulated in colon cancer models. Butyrate, a short chain fatty acid, produced by colon microbiota and transported into the colonocyte by transporter proteins, appears to be important in early carcinogenesis. The butyrate concentration is reduced in CRC and so are its transporters. Interleukin-17 (IL-17) plays a role in colitis-associated colorectal cancer (CAC), but its function in sporadic CRC is less clear. Similarly, Protein kinase C (PKC) has proven involvement in many solid tumours, including CRC, but its exact mechanistic role is still speculative. AIM: To investigate the role and possible signaling pathways of the major role players, β-catenin, COX-2 and PPARγ in early CRC. Further, to elucidate the mechanistic pathways of butyrate and its transporters, IL-17 and PKC in CRC. METHOD: Informed consent was obtained for all patients. Patients were recruited in various disease categories, including normal, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) and CRC. Colon biopsy specimens were obtained during colonoscopy and used for immunohistochemistry (IHC) and gene expression analysis of the above genes by quantitative polymerase chain reaction (qPCR). RESULTS: β-catenin mRNA and protein expression was increased in CRC and the IBD groups compared to the normal group, while it was reduced in the IBS groups. COX-2 mRNA expression showed a steady increase from normal, through IBS, IBD and CRC groups to a statistically significant degree. The COX-2 protein expression, however, did not match the mRNA expression with increased COX-2 protein expression in normal and IBS groups and reduced expression in IBD and CRC groups. PPARγ mRNA expression was unchanged in IBD and CRC groups, but significantly increased in the IBS group compared to normal. Butyrate transporter, SLC16A1 mRNA was significantly reduced in CRC, but also in the IBS groups, which was unexpected. In the IBD group, SLC16A1 mRNA was unchanged in Crohn’s disease (CD) but significantly reduced in ulcerative colitis (UC). Similarly, SLC5A8 mRNA expression was significantly reduced in the CRC as well as the IBS groups. In the IBD groups, SLC5A8 was unchanged in UC but significantly increased in CD. IL-17 mRNA expression was significantly reduced in CRC and IBS groups, but unchanged in the IBD groups. PKCε mRNA was significantly increased in CRC as expected. In the IBD groups, PKCε mRNA was unchanged in CD but significantly increased in UC. In the IBS groups, PKCε mRNA in constipation –IBS (C-IBS) was significantly reduced, but unchanged in diarrhoea – IBS (D-IBS). CONCLUSIONS: β-catenin mRNA and protein expression was increased in CRC and the CRC promoting IBD groups. COX-2 protein expression was incongruent with the COX-2 mRNA expression and this may reflect homeostatic control mechanisms. High COX-2 mRNA expression in CRC and CRC promoting IBD groups may be a secondary phenomenon reflecting the inflammatory milieu, rather than a true carcinogenesis-related event. PPARγ does not appear to play a central role in early colon carcinogenesis, in spite of available literature suggesting otherwise. Butyrate transporters showed inconsistent results and for now no firm conclusions can be drawn from this. IL-17 may play a role in CAC as confirmed in this and other studies, but its role in sporadic CRC is tenuous and requires further investigation. Likewise for PKCε, upregulation is associated with increased tumourigenecity as shown in this study, however, the mechanistic pathway(s) involved is still speculative and requires further study.
- Full Text:
- Date Issued: 2013
- Authors: Fredericks, Ernst
- Date: 2013
- Subjects: Colon (Anatomy) -- Cancer , Cyclooxygenase 2 , Peroxisomes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10357 , http://hdl.handle.net/10948/d1021014
- Description: Colorectal cancer (CRC) is a common disease with significant morbidity and mortality. In spite of significant advances in understanding the molecular signaling in this disorder, unanswered questions remain. Cyclooxygenase-2 (COX-2) and β-catenin have established roles in colorectal carcinogenesis, with both being upregulated early in the disease course. The role of peroxisome proliferator-activated receptor γ (PPARγ) is less clear, but has been shown to be downregulated in colon cancer models. Butyrate, a short chain fatty acid, produced by colon microbiota and transported into the colonocyte by transporter proteins, appears to be important in early carcinogenesis. The butyrate concentration is reduced in CRC and so are its transporters. Interleukin-17 (IL-17) plays a role in colitis-associated colorectal cancer (CAC), but its function in sporadic CRC is less clear. Similarly, Protein kinase C (PKC) has proven involvement in many solid tumours, including CRC, but its exact mechanistic role is still speculative. AIM: To investigate the role and possible signaling pathways of the major role players, β-catenin, COX-2 and PPARγ in early CRC. Further, to elucidate the mechanistic pathways of butyrate and its transporters, IL-17 and PKC in CRC. METHOD: Informed consent was obtained for all patients. Patients were recruited in various disease categories, including normal, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) and CRC. Colon biopsy specimens were obtained during colonoscopy and used for immunohistochemistry (IHC) and gene expression analysis of the above genes by quantitative polymerase chain reaction (qPCR). RESULTS: β-catenin mRNA and protein expression was increased in CRC and the IBD groups compared to the normal group, while it was reduced in the IBS groups. COX-2 mRNA expression showed a steady increase from normal, through IBS, IBD and CRC groups to a statistically significant degree. The COX-2 protein expression, however, did not match the mRNA expression with increased COX-2 protein expression in normal and IBS groups and reduced expression in IBD and CRC groups. PPARγ mRNA expression was unchanged in IBD and CRC groups, but significantly increased in the IBS group compared to normal. Butyrate transporter, SLC16A1 mRNA was significantly reduced in CRC, but also in the IBS groups, which was unexpected. In the IBD group, SLC16A1 mRNA was unchanged in Crohn’s disease (CD) but significantly reduced in ulcerative colitis (UC). Similarly, SLC5A8 mRNA expression was significantly reduced in the CRC as well as the IBS groups. In the IBD groups, SLC5A8 was unchanged in UC but significantly increased in CD. IL-17 mRNA expression was significantly reduced in CRC and IBS groups, but unchanged in the IBD groups. PKCε mRNA was significantly increased in CRC as expected. In the IBD groups, PKCε mRNA was unchanged in CD but significantly increased in UC. In the IBS groups, PKCε mRNA in constipation –IBS (C-IBS) was significantly reduced, but unchanged in diarrhoea – IBS (D-IBS). CONCLUSIONS: β-catenin mRNA and protein expression was increased in CRC and the CRC promoting IBD groups. COX-2 protein expression was incongruent with the COX-2 mRNA expression and this may reflect homeostatic control mechanisms. High COX-2 mRNA expression in CRC and CRC promoting IBD groups may be a secondary phenomenon reflecting the inflammatory milieu, rather than a true carcinogenesis-related event. PPARγ does not appear to play a central role in early colon carcinogenesis, in spite of available literature suggesting otherwise. Butyrate transporters showed inconsistent results and for now no firm conclusions can be drawn from this. IL-17 may play a role in CAC as confirmed in this and other studies, but its role in sporadic CRC is tenuous and requires further investigation. Likewise for PKCε, upregulation is associated with increased tumourigenecity as shown in this study, however, the mechanistic pathway(s) involved is still speculative and requires further study.
- Full Text:
- Date Issued: 2013
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