ABSTRACT
Lung cancer and diabetes continue to be the leading cause of death globally. Earlier in the 20th century lung cancer was a rare malignancy. At the moment, it is occurring in epidemic proportions worldwide. It has become the most common cause of death from malignancy globally. Despite the use of surgery, chemotherapy and radiation in the treatment of lung cancer, the survival rate for patients remains extremely poor. Misregulation of genes that control cell-cycle and cell-fate determination often contributes to cancer. miRNAs are class of short noncoding RNAs that function as a regulator of gene expression via targeting mRNA for degradation or translational inhibition. They regulate expression of genes involved in tumorigenic processes, such as inflammation, cell cycle regulation, stress response, diferentiation, apoptosis, and invasion, and they have been found to play key roles in many cancers.This work was designed to look at the expression profile of two known oncogenic microRNAs, miR-21 and miR-155 in lung cancer, and to see whether re-introduction or inhibition of these would affect progression or aid sensitivities of the lung cancer cells to major Chemotherapeutics used in the management of lung cancer. It also examined zinc finger nucleases mediated expression of GFP in dermal fibroblast, targeting the AAVS1 locus, using galk recombineering scheme in SW102 strains. This work involved cell culture of H358 and A549 lung adenocarcinoma lines, as well as normal lung cells FC 7333 3KT lines (Broncho-aveolar cells). It compared the miRNA expression profile of two known microRNAs (miR-21 and miR-155) in cancer and wild type lung cell lines using real time qPCR (Qiagen miScript PCR system), and then treated with three known chemotherapeutics used in the management of Lung cancer namely Cisplatin, Etoposide and Paclitaxel and concluded by inhibiting over-expressed miRNA by transfecting the cancer cells with microRNA inhibitors called Antagomirs and then measured the level of proliferation with Sulforhodamine-B assay(SRB assay) which is essentially a survival assay. Concerning recombineering workflow, using galk selection scheme, that relies on homology directed repair (via zinc finger nucleases, ZFN) to effect gene modification or genome editing in SW102 strain of E.coli. galk plasmid was successfully amplified using iProof High Fidelity DNA Polymerase kit (BioRad) with primers with homology to the galk gene. The amplified galk gene was then used to transform competent heat-shocked SW102 cell using electroporation. The galk and GFP was then used to co-transform competent cells. Data from this work showed that miR-21 was over-expressed in all the cell lines used relative to the normal lung cell lines which is consistent with the role of miR -21 as an oncogenic microRNA, (oncomiR) while miR-155 was found to be down-regulated relative to the normal lung cells, suggesting that miR-155 could be acting as a tumour-suppressor contrary to the view that miR-155 is a universal oncomir. Furthermore, inhibition of miR-21 function in H358 lines using 50nM Ambion antimiR microRNA inhibitor led to decreased proliferation of H358 cells compared 50nM AntimiR-155 treated group. Inhibition of miR-21 also led to dose dependent apoptosis in H358 compared to negative control group. Downregulation of miR-21 was able to sensitize lung cancer cells to chemotherapeutics (Etoposide) used in this study. The IC50 for Etoposide following 72h incubation was 15.8μM, while after 96h incubation it was 0.1μM. Furthermore, the IC50 for Cisplatin after 72h incubation was given as 10μM and 5.45μM xxii after 96h of incubation in Cisplatin. For Paclitaxel the IC50 after 72h incubation was 0.7μM and 3.0μM after 96h of incubation in Paclitaxel. Result of the recombineering workflow showed that GFP was successfully expressed in the dermal fibroblast targeting the AAVS1 locus, the safe harbor for transgene expression in chromosome -19 of the dermal fibroblast genome using Zinc finger nucleases. Hence this work demonstrated that miR-21 at 50nM can sensitize lung cancer cells to Chemotherapeutics (Etoposide) and that miR-155, a known oncogenic miRNA seem to be acting as tumour-suppressor in lung cancer, which promise to be of immense therapeutic importance. Furthermore, that data from this study indicated that ZFN could target GFP expression into the AAVS1 site of human dermal fibroblast.