Investigating the Role of miRNA-34 family in human thyroid cancer
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Thyroid cancer is the most rampant endocrine malignancy accounting for >80% of endocrine malignancy and 1.8% of all recently distinguished cancer reports. The most important risk factors identified so far, are alcohol consumption, tobacco, radiation exposure, iodine deficiency. There are also additional factors which can cause thyroid cancer but are not still understood. Histological and clinical differentiation evidence have shown two categories of carcinoma, papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) that comprise up to 94% total thyroid cancer cases (nearly 80% PTCs and 12-14% are FTCs). Nevertheless, different alteration of papillary thyroid carcinoma and follicular thyroid carcinoma can create two rare subgroups, which are called poorly differentiated thyroid carcinoma (PDTCs) and anaplastic thyroid carcinoma (ATC). PTCs have an indolent behaviour and may recur and disseminate to distant sites such as the lung, as a primarily metastasis site. Life expectancy of patients who have papillary thyroid cancer is age depended and has increased to the eighth position of females’ cancer and shows an increase of 4% annually in the world. Due to the extensive vascular supply of thyroid gland, the large range of lesions and the spectrum of aggressiveness, have made thyroid cancer a suitable pattern for a study on cancer mechanism and angiogenesis. Angiogenesis is an essential process for tumour growth, metastasis, and tumours, which have lost growth regulatory function and therefore proliferate aberrantly. Controlling of tumour-associated angiogenesis is a tactic for inhibition of cancer progression. Angiogenesis is primarily activated when growing tumour creates a little oxygen microenvironment. The cancer cell undergoes an angiogenic switch directly leading to the secretion of angiogenic factors such as Vascular endothelial growth factor (VEGF), also indirectly, activation of proliferating genes such as B-cell lymphoma 2 (Bcl-2) and Notch homolog 1 (Notch1). Since the discovery of microRNAs (miRNAs) in Caenorhabditis elegans, the evidence is emerging that alteration in miRNAs expression may play a key role in cancer development and progression. miRNAs can be described as small non-coding RNAs which are responsible for regulating expression of multiple target proteins. In mammalian, mature miRNAs have been recognised to function at the post-transcriptional level through interaction with the 3' untranslated region (3’ UTR) of the specific target messenger RNA (mRNA). Their primary function is to suppress translation or occasionally induce their degradation in the major cellular pathways such as cell proliferation and differentiation. miRNAs can act as a tumour suppressor or oncogene in a context-depend manner. Therefore, better understanding the molecular mechanisms by which miRNAs play an important function in derailed cellular signalling in the thyroid cancer cell might be helpful to develop better therapeutic strategies for thyroid cancer treatment. In mammalians, the miR-34 microRNAs precursor family were computationally discovered and later verified experimentally. The two distinct precursors are processed into three mature miRNAs: a and b/c. The mature miR-34 family are a part of the p53 tumour suppressor network; therefore, it is hypothesised that miR-34 family dysregulation is involved in the development of some cancers. This family is transcribed from two different sets of genes located on chromosome 1 and 11. Studies have shown a preference in tissue with lower expression of miR-34a in brain and miR-34b and miR-34c in the lungs. Their promoter region has a p53 binding site. Therefore, they are induced by p53 and thus involved in cell proliferation, survival, apoptosis, migration, invasion and angiogenesis. Many controlling genes are regulated through the actions of this family. For instance, ectopic expression of this family increases factors involved in cell cycle regulation and DNA damage response (DDR) and the suppression of cell cycle promoting genes. Recent studies have highlighted the role of miR-34b as a tumour suppressor in a different type of tumours including non-small cell lung cancer (NSCLC), small cell lung cancer cell SCLC, prostate cancer, lung cancer, colorectal cancer also thyroid cancer. However, very little is known about the role and expression state of miR-34b in thyroid cancer. The overall aim of this study was to determine the functional role of miR-34b in thyroid cancer progression. In our first study, it pointed out for the first time that overall expression of miR-34b-5p is much lower than miR-34b-3p miR-34a and miR-34c in all thyroid carcinoma cell lines. Therefore, miR-34b was chosen for further investigation in this study. Interestingly, the expression levels of miR-34b were also significantly downregulated in thyroid carcinoma tissue samples and associated with T-stages of thyroid carcinomas (p=0.042). High protein expression of VEGF-A, Bcl-2 and Notch1 in thyroid carcinoma cells were noted in cells with low expression of miR-34b when further compared to miR-34b transfected carcinoma cell lines (P<0.05). Therefore, from this study, it was clear that there is a link between miR-34b expression and VEGF-A, Notch1 and Bcl-2 expression in thyroid carcinoma. In the second investigation, I transiently transfected thyroid carcinoma cell lines with miR-34b to investigate its effect on dominant genes involved in angiogenesis cell cycle regulation including VEGF-A, Notch1 and Bcl-2, respectively. miR-34b was overexpressed in thyroid cancer cell lines. We found through immunofluorescent and western blot assay that mir-34b overexpression leads to a significant downregulation of VEGF-A, Notch1 and Bcl-2 expression (P<0.05). miR-34b transfection induced significant accumulation of cells in G0-G1 of the cell cycle by blocking of their entry into the S transitional phase as well as increasing the total apoptosis. ELISA confirmed a ≃ 50% decreased expression of VEGF in all cultured media of the thyroid carcinoma cell lines after transfection with miR-34b. So, it is reasonable to assume that miR-34b can regulate proliferation, migration and angiogenesis in vivo as well. Therefore, our third and final investigation was to use a hydration-of-freeze-dried-matrix (HFDM) formulated liposomes (PEGlyated-miR-34b), as a more efficient transfection approach, for systemic delivery of this microRNA to the thyroid cancer in vitro and in vivo. We confirmed the effect of miR-34b on VEGF-A downregulation, as a regulator of proliferation and angiogenesis in vitro and in vivo, using PEGlyated-miR-34b. miR-34b expression was low and significantly (P<0.05) overexpressed following transfection with PEGlyated-miR-34b in thyroid cancer cell lines. Using Western blot and ELISA assay we found that protein level of VEGF-A remarkably reduced in thyroid cancer cell line after transfection of cells with PEGlyated-miR-34b. Furthermore, miR-34b overexpression significantly (P<0.05) reduced proliferation, wound healing potential, cell cycle progression and increased apoptosis in thyroid cancer cell lines. In vivo xenotransplantation mouse model also showed a functional role for miR-34b in thyroid cancer cell biology in response to its overexpression. Xenotransplantation model further indicated that smaller and low-vascularized tumours were formed upon intravenous (IV) liposomal administration. Taken together, miR-34b play a pivotal role as a tumour suppressor via modulation of angiogenesis in thyroid carcinoma and suggest a miR-34b/proliferation axis with potential therapeutic implications. This finding also provides a mechanistic insight into the miR-34b regulation of thyroid cancer proliferation, and angiogenesis and delivery of miR-34b using this cationic liposome may provide a useful therapeutic delivery strategy for thyroid cancer treatment.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medicine
The author owns the copyright in this thesis, unless stated otherwise.
Thyroid cancer treatment