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
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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.
TABLE OF CONTENTS
Title page – – – – – – – – – – i Declaration – – – – – – – – – – ii Certification – – – – – – – – – iii Dedication – – – – – – – – – – iv Acknowledgement – – – – – – – – – v Table of Content – – – – – – – – viii List of Tables – – – – – – – – – – xiii List of Figures – – – – – – – – – – xiv List of Plates – – – – – – – – – – xvi List of Appendices – – – – – – – – – xvii List of Abbreviations – – – – – – – – – xviii Abstract – – – – – – – – – – xix Chapter One 1 1.0 Introduction – – – – – – – – 1 1.1 Background of Study – – – – – – – – 1 1.2 Aims and Objectives of Study – – – – – – 5 1.3 Statement of Problem – – – – – – – – 6 1.4 Significance of Study – – – – – – – – 7 1.5 Scope of Study – – – – – – – 9 Chapter Two – – – – – – – – 11 2.0 Literature Review – – – – – – – – 11 2.1.1 RNAi and microRNA – – – – – – – 11 2.1.2 MicroRNAs a New Class of Gene Regulators – – – 12 2.1.3 MicroRNA Biogenesis – – – – – – – 12 2.1.3.1 Genomics and Biogenesis of MIR-21 – – – – – – 15 2.1.4 Pri-miRNA – – – – – – – – – 16 2.1.5 Pre-miRNA-21 – – – – – – – – 16 2.1.6 Mature MIR-21 – – – – – – – – 17 2.2.7 The Argonaute (AGO) Protein Family – – – – – 17 2.2.1 MicroRNAs and cancer – – – – – – – 18 2.2.2 The miRNA-related Genes and Cancer – – – – – 19
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2.2.3 Therapeutic Importance of miRNAs in Cancer Management – – 20 2.2.4 Challenges of miRNA Therapeutics – – – – – – 20 2.2.5 MicroRNA in Solid Cancers – – – – – – – 21 2.2.6 Lung cancer – – – – – – – – – 25 2.2.7 Oncogenic microRNA – – – – – – – 27 2.2.7.1 MiR-21 – – – – – – – – 27 2.2.7.2 miR-155 – – – – – – – – – 32 2.2.7.3 Tumour Suppressor miRNAs – – – – – – – 33 2.3. Chemotherapeutics and Lung Cancer – – – – – – 35 2.3.1 Cisplatin – – – – – – – – – 35 2.3.1.1 Cisplatin and inhibition of miR-21 – – – – – – 36 2.3.2 Etoposide – – – – – – – – 38 2.3.3 Paclitaxel – – – – – – – – – 40 2.4 Recombineering – – – – – – – – 41 2.4.1 What is recombineering – – – – – – – 41 2.4.2 Recombineering Systems – – – – – – 42 2.4.3 Galk recombineering system – – – – – – – 43 2.4.3.1 SW102 strains as a recombining Vector. – – – – – 43 2.4.4 GalK Expression Cassette – – – – – – – 44 2.4.5 Recombineering using ssDNA – – – – – – 45 2.4.6 Strand bias. – – – – – – – – – 45 2.4.7 Cloning DNA by gap repair – – – – – – – 46 2.4.8 Strategies for DNA engineering in E. coli – – – – – 46 2.4.9 Homologous recombination – – – – – – – 47 2.4.10 Bacterial Artificial Chromosome (BAC) – – – – – 48 2.5 DNA Break Repair – – – – – – – – 50 2.5.1 Genome Editing – – – – – – – – 50 2.5.2 Gene Disruption – – – – – – – – 50 2.5.3 Homology-Directed Genome Editing (HDr) – – – – – 51 2.5.4 Zinc Finger Nucleases- – – – – – – – 51 2.5.7 ZFN and Gene Targeting – – – – – – 55 2.6 Adeno associated Virus AAV and AAVS1 locus – – – – 55 2.6.1 Molecular biology of Adeno associated Virus AAV – – – – 56
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2.6.2 Molecular characterization of AAV integration – – – – 56 2.6.3 Strategies for site-specific gene modification – – – – – 56 2.6.4 Human target sequence – – – – – – – 57 2.6.5 AAV as a vector for gene therapy – – – – – – 58 2.7 Towards insulin replacement – – – – – – – 59 2.7.1 Therapeutic targets of Diabetes – – – – – – 59 2.7.2 Novel Drug Delivery Systems for Insulin – – – – – 60 2.7.3 Transdermal Delivery – – – – – – – – 61 Chapter Three 63 3.0 Materials and Methods – – – – – – – 63 3.1 Materials for Recombineering – – – – – – 63 3.1.2 Materials for PCR amplification of galk plasmid – – – – 64 3.1.3 Sequences of primers used – – – – – – – 64 3.1.4 Materials for Nucleofection of Dermal fibroblast – – – – 65 3.1.5 Cell line for Recombineering. – – – – – – – 66 3.2. Methodology – – – – – – – – – 66 3.2.1 Preparation of competent cells – – – – – – 67 3.2.2 Electroporation of competent cells – – – – – – 68 3.2.3 Set up for PCR reaction for the amplification galk gene from galk plasmid using primers with homology to pAAVS-CAGGS-eGFP – – – 68 3.2.4 PCR set up to detect recombined plasmid DNA in miniprep population – 70 3.2.5 Preparation of alga plates – – – – – – – 71 3.2.6 Preparation of MacConkey indicator plates – – – – – 71 3.2.7 Sigma compozr Targeted integration kit. – – – – – 72 3.2.8 Isolation of SW102 DNA using qiagen miniprep kit – – – – 72 3.2.9 PCRs to check for integration of GFP gene into AAVS1 locus – – 73 3.2.10 Determination of transformation efficiency of colonies. – – 74 3.2.11. Calculation of transformation efficiency of the competent cells – – 75 3.3. Cell culture of Human dermal Fibroblast – – – – – 75 3.3 .1 Transfection using Amaza Nucleofection – – – – – 75 3.3.2 Isolation of genomic DNA from nucleofected human dermal fibroblast using Sigma Genelute mammalian DNA prep kit. – – – – – 76 3.4 Targeted integration workflow – – – – – – 78
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3.4.1 Materials for Cell culture — – – – – – – 79 3.4.2 Cell transfection – – – – – – – 79 3.4.3. SRB assay. – – – – – – – – – 80 3.4.4. Renilla Luciferase reporter Assay – – – – – – 81 3.4.5. Isolation of total RNA – – – – – – – – 81 3.4.6 Quantitative real time reverse transcription PCR – – – – 82 3.4.7 cDNA synthesis using miScript II RT Kit (Qiagen, Valencia, CA) – – 82 3.5 Inhibition of MicroRNA Function – – – – – – 84 3.5.1 Chemotherapeutics treatment – – – – – – – 87 3.6 Statistical Analysis- – – – – – – – – 89 Chapter Four 90 4.0 Results – – – – – – – – – – 90 4.1 H358 Cell Culture – – – – – – – – 90 4.2 MicroRNA profiling of H358, A549, using miScript quantitative Real time-PCR Assay (Qiagen). – – – – – – – 92 4.2.1 MiR-21 is up-regulated in H358, A549 lung cancer cell lines – – 92 4.2.2 MiR-155 is Down-regulated in H358 lung cancer Line. – – – 95 4.3 Determination of IC50 of the Chemotherapeutics used – – 98 4.3.1 The IC50 of Cisplatin following incubation of H358 cells in Cisplatin. – 98 4.3.2 The IC50 of Etoposide following incubation of H358 cells in Etoposide. – 100 4.3.3 Determination of IC50 of Paclitaxel following incubation of H358 cells in Paclitaxel.- – – – – – – – – – 102 4.4 Down-regulation of miR-21 Inhibited Survival Capacity of H358 Cells after Chemotherapeutics treatment – – – – – 104 4. 5 Does inhibition of miR-21 and 155 functions in H358 cells lead to reduced survival of cancer cells? – – – — – – – 106 4.6 Luciferase assay – – – – – – – – 109 4.7 Recombineering in SW102 and Zinc finger Nuclease mediated dermal fibroblast Targeting result – – – – – – – 111 4.7.1 Amplification of pGalK gene – – – – – – – 111 4.7.2. Transformation of SW102 with pSA-21-GFP-2P plasmid and PCR products in both control and experimental cells show successful recombineering. – 114 4.7.3 Transformation efficiency of competent cells – – – – 116
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4.7.4 PCR to confirm integration of GFP into the AAVS1 site – – – 118 4.7.5 Diagnostic PCR to verify recombined and GFP expressing clones from miniprep. – – – – – – – – – 120 Chapter Five 122 5.0 Discussion – – – – – – – – – 122 5.1 MicroRNA mediated sensitization of Lung cancer cells to chemotherapeutics 122 5.2 Zinc finger nuclease-mediated expression of GFP into human dermal Fibroblast AAVS1 locus – – – – – – – – 127 Chapter Six 131 6.0 Summary, Conclusion and Recommendation – – – – – 131 6.1 Summary and Conclusion – – 131 6.2 Recommendations- 131 References – – – – – – – – – 133 Appendices – – – – – – – – – 146
CHAPTER ONE
1.0 INTRODUCTION 1.1 BACKGROUND OF STUDY MicroRNAs are commonly believed to be class of small, non-coding RNAs that post-transcriptionally control the translation and stability of mRNAs. MicroRNAs (miRNAs) are known to hybridize to the 3‘ untranslated region of target mRNAs causing repression at post-transcriptional level. Genes encoding miRNA molecules are found either inserted in introns as polycistronic clusters or in isolated regions of the genome (Jerome et al., 2007). Through forward screening genetics the first microRNA was discovered. Even though it is known that miRNAs are dysregulated in various diseases, clear, causal evidence of their role in cancer has only recently come to light (Kasinski and Slack, 2011). According to Stenvang and colleagues,(Stenvang et al., 2012) manipulation of miRNA activity in vivo is of high interest due to the aberrant expression and implication of miRNAs in the pathogenesis of human diseases. The use of antimiR oligonucleotides to target disease-associated miRNAs is the most widely used approach to probe their functions in vivo and shows great promise in the development of novel miRNA-based therapeutics. They further stated that, an increasing number of studies have reported successful therapeutic miRNA silencing in a variety of animal disease models using antimiR oligonucleotides.
Kasinski and Slack noted that the ability of a miRNA to regulate a gene stems from a 6-nucleotide seed region in the miRNA that typically interacts with the 3′ untranslated region of an mRNA, so as to prevent translation or induce degradation of the mRNA. Due to their small which contributes to their pleiotropic role in targeting a large number of critical
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protein-coding genes, misregulation of a single miRNA can give rise to multiple adverse cellular effects (Kasinski and Slack, 2011). Aberrant miRNA levels have been implicated in promoting and maintaining various disease states including cancer. In some cases, overexpression of single miRNA is sufficient to drive tumorigenesis, although its silencing leads to tumor regression (Kasinski and Slack, 2011). They further observed, that based on these findings, and on-going human tumor miRNA profiling studies that have confirmed altered levels of some key miRNAs, it is becoming increasingly attractive to target these master gene-regulators in vivo with cancer therapeutics (Medina et al., 2010a). It is equally important to know that several strains of mice lacking or overexpressing cancer-associated miRNAs have been developed and characterized. These include germline transgenic or knockout mice for: miR.155; miR.21; miR.17~92 and its paralogues; miR.15 and miR.16; miR.146; and miR.29. Additional mouse models are the LIN.28 overexpressing strain (for evaluation of the in vivo loss of mature let.7) and the multiple conditional DICER knockout models (Kasinski et al., 2011). This work would focus on two important microRNAs miR-21 and miR-155 known to be differentially expressed in cancers.
The insertion of exogenous genetic information into the genome of target cells is broadly used in biology. Gene insertion is traditionally achieved via virus-mediated or spontaneous integration of transfected DNA followed by a selection for cells carrying the new DNA. In cell-based therapy, lack of control over the transgene integration site can result in adverse events due to insertional mutagenesis (Hacein-Bey-Abina et al., 2002). Uncontrolled transgene integration results in unwanted phenotypic heterogeneity due to the varying permissivity of integration sites for transgene expression (position-effect variation) (Kwaks and Otte, 2006). In these situations, Cost and co-workers noted that it would be
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advantageous to target DNA insertion to a specific, desirable site in the genome (Orlando et al., 2010). According to Porteus and colleagues,(Porteus, 2010), Homologous recombination is the most precise way to manipulate the genome. It has been used extensively in bacteria, yeast, murine embryonic stem cells, and a few other specialized cell lines, but it has not been available in other genetic systems such as mammalian somatic cells. However, the creation of a gene-specific DNA double-strand break can stimulate homologous recombination by several-thousand fold in mammalian somatic cells. These double-strand breaks can be created in mammalian genomes by zinc finger nucleases (ZFNs), artificial proteins in which a zinc finger DNA-binding domain is fused to a nonspecific nuclease domain. Zinc finger nucleases (ZFNs) have been used successfully to create genome-specific double-strand breaks and thereby stimulate gene targeting by several thousand fold. ZFNs are chimeric proteins composed of a specific DNA-binding domain linked to a non-specific DNA-cleavage domain. By changing key residues in the recognition helix of the specific DNA-binding domain, one can alter the ZFN binding specificity and thereby change the sequence to which a ZFN pair is being targeted. For these and other reasons, ZFNs are being pursued as reagents for genome modification, including use in gene therapy (Pruett-Miller et al., 2009).
Targeted gene transfer is typically performed by transfection of a selectable marker gene flanked by a substantial amount of DNA homologous to the target locus. Spontaneous double stranded breaks (DSBs) are formed at the target locus, likely from stalled DNA replication forks. While normally repaired correctly by homology-directed repair (HDR) templated by the sister chromosome, HDR can instead use the homologous donor DNA to heal the break. When additional DNA sequence is inserted between the two regions of
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homology in the donor plasmid, the cellular DNA repair machinery unwittingly copies this genetic information into the chromosome. As this homology-based targeting relies on the capture of very rare DSBs within the region of donor homology, extensive homology to the target locus is needed to obtain targeted integration at a useful frequency (Porteus, 2010). The initial idea that Zinc finger nucleases, ZFNs could stimulate gene targeting in mammalian somatic cells came from the work of Porteus and colleagues (Porteus and Carroll, 2005a). In these experiments, recognition sites for known zinc finger DNA-binding domains were inserted into a green fluorescent protein (GFP) reporter gene, which was integrated as a single copy into the genome of the human embryonic kidney cell line. In this system, gene targeting is measured by the correction of an integrated mutant GFP target gene by a transfected donor plasmid and the resultant conversion of GFP-negative cells into GFP-positive cells. In addition, this work showed that targeting with ZFN could be induced simultaneously at both the site of the break and at a distance of 400 bp from the break, demonstrating that a single pair of ZFNs can stimulate targeting in a relatively large region surrounding a DSB (Porteus and Carroll, 2005a).
Urnov and workers reported that designed ZFNs can cleave an endogenous human gene in cultured cells and lead to targeted gene replacement in up to 20% of the cells (Urnov et al., 2005). The target was the gene for interleukin (IL) – 2Rγ, a cytokine receptor that is required for T-cell development and the establishment of a functional immune system. However, before ZFN methods become widely used several challenges must be scaled: first, applicability of the ZFN approach needs to be broadened; second, the method for delivering ZFNs and repair substrate to cells requires optimization; and third, the understanding of the process of homologous recombination itself needs to be enhanced. Efforts to broaden the applicability of the approach will require not only the design of
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ZFNs targeting a greater variety of gene targets but also the determination of the conditions for performing gene targeting in different cell types. Until now, ZFNs have been primarily applied to transformed mammalian cell lines that are relatively resistant to apoptotic stimuli. An important advance, therefore, will be to develop ways of using ZFNs in primary cells (like the human dermal fibroblast that this research will address) known to be more sensitive to DNA damage.
1.2 AIMS AND OBJECTIVES OF THE STUDY
The main aim of this study is to sensitize lung cancer cells to chemotherapeutics using two microRNAs (miR-21 and miR-155) by determining the expression profile of these microRNAs via Real time PCR and then inhibiting or over-expressing them using microRNA Inhibitors or Mimics. The second aim of this study is to express fluorescent proteins in human fibroblast targeting the AAVS1 Locus at chromosome 19 using Zinc finger Nucleases. These fluorescent proteins will be used to precisely monitor blood sugar levels in diabetic patients, which will in turn stimulate insulin release from biosensor to be implanted into the skin. The biosensor would be built by Massachusset Institute of Technology. 1.2.1 OBJECTIVES OF STUDY The objectives of the study are to:
I. profile microRNA expression levels in wild type ―WT‖ (FC 7333 3KT) versus ―Cancer‖ (H358) Lung cell lines. II. inhibit microRNAs that would be upregulated in the Lung cancer cells. III. find IC50 for the chemotherapeutics used Cisplatin, Etoposide, and Paclitaxel.
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IV. monitor transfection efficiency of the AntimiR microRNA inhibitors (Ambion) using Renilla Luciferase assay. V. monitor apoptosis and cell survival using Sulforhodamine blue (SRB) assay following microRNA inhibition. VI. use Zinc Finger Nucleases (ZFN), to express Green Fluorescent Protein (GFP), in the human dermal fibroblast at the AAVS1 locus using galK recombineering Scheme in SW102 strain.
1.3 STATEMENT OF PROBLEM
Lung cancer is the most common cause of cancer-related deaths around the world, whereas non-small cell lung cancer (NSCLC) represents the most frequent type of lung cancer. NSCLC is comprised of squamous cell carcinoma, adeno-carcinoma, and large cell undifferentiated carcinoma. Recent studies from genomic and proteomic approaches have changed current view on cancer. From a disease previously perceived as manifested in the alteration of merely several genes, cancer is infact genetically very complex. Tumor cells frequently harbor an array of mutated genes, and each tumor mass can comprise hundreds of cancer cells with distinct cancer genotype.
Resistance to chemotherapeutics is known to be a serious obstacle to effective cancer therapy. Clinically relevant levels of resistance can emerge quickly after treatment. Beside intrinsic resistance, acquired or gradually developing resistance has been observed in tumors under therapy. Several mechanisms underlying resistance have been described, like decreased exposure to the drug, e.g. via reduced drug accumulation/drug-target interaction
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or increased detoxification response, diminished cell-cycle effects, reduced apoptotic responses, or increased DNA-repair Autoimmune destruction of pancreatic beta cells causes insulin-dependent diabetes mellitus or Type I diabetes. As a consequence of partial or complete loss of beta cells, little or no insulin is secreted by the pancreas. Most cells, with the exception of brain cells, require insulin for the uptake of glucose. Inadequate insulin production causes reduced glucose uptake and elevated blood glucose levels. Both reduced glucose uptake and high blood glucose levels are associated with a number of very serious health problems. In fact, without proper treatment, diabetes can be fatal. One conventional treatment for diabetes involves periodic administration of injectable exogenous insulin. This method has extended the life expectancy of millions of people with the disease. However, blood glucose levels must be carefully monitored to ensure that the individual receives an appropriate amount of insulin. Too much insulin can cause blood glucose levels to drop to dangerously low levels. Too little insulin will result in elevated blood glucose levels. Even with careful monitoring of blood glucose levels, control of diet, and insulin injections, the health of the vast majority of individuals with diabetes is adversely impacted in some way. Hence, a need for efficient insulin delivery method that obviates these highlighted challenges.
1.4 JUSTIFICATION/SIGNIFICANCE OF STUDY
Gene targeting provides an important research tool for probing the complex interplay between the genome, the physiologic processes and the environment for manipulating the genome (Porteus and Carroll, 2005b). In gene targeting, an exogenously introduced DNA fragment replaces an endogenous segment of DNA by homologous recombination. This
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process was reported in yeast more than 25 years ago and variations on this technique are now commonly used for evaluating gene function in that organism. ZFN-mediated targeting holds promise of providing useful experimental tool for manipulating the mammalian genome for many of the potential applications. Given further development, the ZFN strategy may be applied in the treatment of human genetic diseases as well as in other disease like diabetes which this present work will be using ZFN to mediate the expression of fluorescent proteins that would be used by a biosensor to regulate blood sugar by triggering off precise release of insulin into blood stream, thereby obviating the use of manual insulin injection in diabetics. The second aspect of this work will elucidate therapeutically important link between microRNA expressions, the prognosis of lung cancer, by demonstrating that anti-miR (microRNA inhibitor) molecules also sensitize lung cancer cells to chemotherapeutics. Studies show that up-regulation of miR-21 and miR-155 was frequently found in lung cancer samples. Again, it is hoped that antisense-mediated down-regulation of these micoRNAs could significantly inhibit migration and invasion and reverse the chemo- or radio-resistance of human lung cancer cells partially by targeting the tumor suppressor genes. This work will reveal novel roles of miR- 21 and miR-155 in the development of lung cancer and targeting miR-21and miR-155 will be a novel strategy for the treatment of lung cancer. The study will also help explain how microRNA becomes elevated or decreased in lung cancers and demonstrate that inhibition of miR-21 may have important therapeutic potential as a means to sensitize lung cancer cells.
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1.5 SCOPE OF STUDY
The work was designed to address two thematic areas -Targeted gene therapy towards using microRNAs to sensitizing lung cancer cell to chemotherapy, which took place at Yale University, Department of Molecular, Cellular and Developmental Biology, USA, as well as ZFN-mediated targeted expression of GFP into AAVS1 site of dermal fibroblast aimed at targeting diabetes, which took place at Harvard Stem Cell Institute, Harvard University, USA. At Yale Unversity, this work involved cell culture of H358 lung adenocarcinoma line, and A549 lung cancer cell lines, as well as normal lung cells FC 7333 3KT lines. 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 and then treated with 3 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.
This work was a part of collaboration between Harvard University, MIT and Boston Children Hospital aimed at producing engineered fluorescent proteins that would monitor blood sugar levels. Three different fluorescent proteins would be produced in total in the entire project, namely Green Fluorescent Protein, GFP, Red Fluorescent Protein, RFP and Yellow Fluorescent Protein, YFP which would change colour at Low blood sugar and normal blood sugar levels respectively. The green fluorescent protein would recognize high blood sugar which would be detected by Biosensor and mediate precise release of
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insulin to control sugar metabolism in diabetics. Here, this work would focus on the building of the GFP and expressing it to target the AAVS1 site in chromosome -19 of the human dermal fibroblast guided by ZFN. This will be a proof of principle that would provide a platform subsequently for replacements with the other two fluorescent proteins, red fluorescent protein and yellow fluorescent protein (RFP and YFP).
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