The Role of Stem Cell Regulated miRNAs in Colorectal Cancer
Embargoed until: 2019-12-09
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Currently, colorectal cancer is the third most common cancer worldwide, with an estimation of 1.2 million new cases per year and more than 600.000 deaths. The 5-year survival rate exceeds 90% when colorectal cancer is detected at an early stage. However; most cases are diagnosed at late stages. Colorectal cancer costs a huge amount of money to cover the needs of patients which are range from simple medication to special hospitalization care. In Australia, bowel cancer is the second most common type of cancers. These facts raise the need to find new methods to diagnose the disease in its earlier stages and to seek efficient kinds of treatment. Recently, cancer stem cells proved to have essential roles in initiation, progression and resistance to cancer treatments. Furthermore, it has been reported that miRNAs are the robust regulators of these cells. miRNAs can act as tumour suppressor genes or as oncogenes and they target directly many genes in different types of cancer including colorectal cancer. This research intends to investigate the functional roles of miR-451 and miR-193a in various aspects of colorectal carcinogenesis. Besides their roles in regulation cancer stem cells including colorectal cancer stem cells, miR-451 and miR-193a target important genes which are already proved to have fundamental functions in colorectal cancer development and progression, these genes are MIF and KRAS respectively. Therefore, these two miRNAs will be our focus in this research. The roles of these miRNAs in colorectal cancer are not well understood and currently, there is lack of information about the correlation of these miRNAs with several clinical and pathological parameters of the patient Fresh frozen human tissues from matched colorectal cancer and adjacent non-cancer tissue were prospectively collected with no selection bias. Two colorectal cancer cell lines (SW480 and SW48) and non-cancer colon cell line (FHC) were also used. The expression level of miR-451 and miR-193a-3p was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical staining was used to detect the expression level of MIF and KRAS proteins as predicted downstream targets for miR-451 and miR-193a respectively. Restoration of the miR-451 and miR-193a levels in the cell lines was achieved by permanent transfection. This followed by in vitro studies of both miRNAs behaviours using clonogenic and MTT calorimetric assays for proliferation study; flow cytometry for cell cycle and apoptotic changes. Also, apoptosis was assessed by using DAPI staining under a fluorescent microscope. To determine the ability of miR-451 and miR-193a in cancer invasion, migration assay was performed. Western blot analysis was used to detect the changes in proteins level of MIF, KRAS and several stem cells and epithelial-mesenchymal transition factors including OCT4, SOX2, TWIST and SNAIL proteins after transfection. Tumour spheres assay was carried out to study the effect of miR-451 and miR-193a on the ability of colon cancer cells to grow as spheres in serum-free media. Immunofluorescence study was used to confirm the changes in stem cell markers expressions after transfection. Results confirm downregulation of miR-451 and miR-193a in both samples of the patients and cell lines. The study shows that miR-451 was down-regulated in (n=59/70, 84.3%) and miR-193a was down-regulated in (n= 49/70, 70%). Correlation of miR-451 and miR-193a expression to many clinical and pathological parameters was studied for the first time in this research. miR-451 down-regulation correlated significantly with co-existence adenoma and recurrence of the disease, P values are (0.02 and 0.028) respectively. A significant correlation between miR-193a downregulation and early stages of colorectal cancer (stage I and stage II) was detected in this study (P =0.023). Co existing multiple polyps shows significant correlation with down-regulation of miR-193a (P =0.034). A near considerable relationship between miR-193a and perforation of colorectal cancer was also reported in this study (P =0.05). Significant inverse correlation between both miRNAs and their targets was confirmed in this study by using immunohistochemical analysis. Moreover, the miRNAs-targets correlation was further studied in-vitro by western blot study. The results show that upregulation of miR-451 and miR-193a significantly inhibit the expression of their targets (MIF and KRAS proteins) respectively. In vitro studies confirm that miR-451 and miR-193a can inhibit the proliferation and the migration of colorectal cancer significantly. Significant apoptotic changes were reported in colon cancer cell lines transfected with miR-193a and miR-451 by both flow cytometry and DAPI staining. Also, this research confirms the significant roles of miR-451and miR-193a in colon cancer cell cycle progression. Furthermore, western blot and for the first time demonstrates that miR-193a can play a role in epithelial-mesenchymal transition of colorectal cancer by targeting TWIST. Interestingly, the results detect significant downregulation of OCT4, SOX2 and SNAIL in colon cancer cell lines transfected with miR-451 when compared to control cells indicating its roles in regulating colon cancer stemness. To further study their roles in stem cell regulation, miR-451 and miR-193a were investigated by using tumour spheres assay and the results indicate that miR-451 significantly inhibits the colon spheres. Immunofluorescence assay revealed that miR-451 suppresses the expression of SOX2 and OCT4 in colon spheres. However, miR-193a did not show such effects in colon spheres and that raises the possibility of being targeting other stem cell markers and may cooperate with another miRNAs to exert its effects. This study also correlates between the expressions patterns of both miRNAs and between their target genes. Statistical analysis indicates that downregulation of miR-451 correlated significantly with down regulation of miR-193a (P=.012) and there is a significant correlation between MIF and KRAS in the sample of the patients. The imunohistochemical study revealed an inverse significant correlation between miR-451 and K RAS protein (P=0.035). Western blot supports these finding and shows that miR-451 can inhibit the expression of KRAS significantly in both SW480 and SW48 cell lines. These findings indicate that miR-451 may cooperate with miR-193a to induce their roles in colorectal pathogenesis. To conclude, this research confirms the tumour- suppressor properties of miR-451 and miR-193a in colorectal cancer by targeting different aspects of carcinogenesis like proliferation, migration and apoptosis. In addition, miR-193a can play roles in epithelial-mesenchymal transition by targeting TWIST. Moreover, miR-451 has shown a significant contribution to stem cell formation and epithelial-mesenchymal transition of colorectal cancer. Both miRNAs show that they can play roles in early steps of this disease which raise the possibility to target them in the future for colorectal cancer therapy. Furthermore, KRAS gene can be a new predictive direct target for miR-451 which needs further investigation. This study provided new and valuable information to understand colorectal cancer pathogenesis and the finding of this research may assist in development or modification a new treatment for colorectal cancer.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medicine
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