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Title: The role of miR-196a and HOXB9 in head and neck cancer
Author: Darda, Lav Kishor
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2013
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Head and neck cancers are a heterogeneous group of cancers with 90% of them originating from the squamous epithelium. Only 50% of patients survive on being diagnosed with this cancer after five years and this statistic has not improved over the last few decades. The main risk factors for head and neck squamous cell carcinoma (HNSCC) are smoking and alcohol consumption. In recent years, there has been more interest in understanding the function of elements involved in gene expression such as transcription factors and non-coding RNA's and their role in oncogenesis. HOX genes are transcription factors involved in oncogenesis and embryogenesis. In total, there are 39 HOX genes distributed in four clusters across the human genome. HOX genes have been observed to be aberrantly expressed in several cancers. MicroRNAs are non-coding short RNA transcripts which are approximately 21 nucleotides in length and lead to down-regulation of their target genes by acting on their 3'UTR. miR-196a is found in HOX gene cluster upstream of HOX9 paralogous group. Even miR-196a has been found to be aberrantly expressed in different cancers. The expression pattern of 39 HOX genes in cancer cell lines showed that HOXB9 was highly over-expressed compared to normal cells, which was further confirmed by qPCR in wider cell panel consisting of four normal, four oral pre-malignant and five HNSCC cell lines (p < 0.05). miR-196a was also found to be over-expressed in cancer cell lines compared to normal cells when similar sets of cell lines were used (p < 0.05). This data was also found to be replicated in tissue samples and it was observed that HOXB9 and miR-196a were significantly over-expressed in cancer tissue samples compared to normal tissue samples (p < 0.05). HOXB9 siRNA transfection into HNSCC cells showed significant decrease in invasion, migration and proliferation, whereas anti-miR-196a transfection in HNSCC cells showed significant decrease in invasion, migration and adhesion compared to negative control transfected HNSCC cells (p < 0.05). HOXB9 and miR-196a-1 are spatially closely related to each other on chromosome 17. To check if these two are co-transcribed on same primary transcript, nested PCR was performed with appropriate controls consisting of RNaseA treated RNA and no reverse transcriptase control, which suggested that novel primary transcript for HOXB9 and miR-196a-1 co-expression might exist in HNSCC cells which was confirmed by DNA sequencing. Expression microarray analysis was performed using anti-miR-196a in oral pre-malignant and HNSCC cells and pre-miR-196a in immortalized normal cells to assess if there were any novel direct targets of miR-196a in HNSCC. Based on qPCR (p < 0.05), dual luciferase reporter assay (p < 0.001) and site-directed mutagenesis, MAMDC2 was found to be a direct target of miR-196a in HNSCC. This is the first study in HNSCC looking at expression patterns of both miR-196a and HOXB9 and needs further work to validate them into biomarkers for early detection of HNSCC. miR-196a and HOXB9 could also be developed into potential therapeutic targets in HNSCC, particularly the novel primary transcript could be a novel therapeutic target. Further characterization of MAMDC2 is required but could turn into exciting therapeutic target as it is expressed as transmembrane receptor.
Supervisor: Hunter, Keith ; Lambert, Daniel Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available