Investigation of the molecular mechanisms underlying the invasive phenotype in a panel of lung cancer cell lines

The research described in this thesis aimed to increase our knowledge of the molecular mechanisms by which lung cancer cells acquire the capacity to invade. The research can be divided into two main sections a) Development of genetic tools for stable, targetable transgene overexpression, b) Investigation of the role of differential expression of a group of genes, including microRNAs, in cancer invasion. DLKP mildly invasive cell line derived from a non-small cell lung carcinoma and its Adriamycin resistant variant DLKPA highly invasive were used in this study to evaluate the roles of differentially expressed mRNAs and microRNAs. We developed two targetable DLKP cell lines based on Cre-LoxP technology. These were generated by random and homologous recombination (site specific) targeting. These cell lines were developed to overcome challenges one faces with non-targetable systems including large discrepancies in target gene expression, genomic instability, and unpredictable phenotypes. A panel of thirty one DLKP single cell clones were assessed for stable and transgene overexpression, over four and a half months with regular passages and freeze thaw cycles. Many clones were found to display unstable transgene expression over time but several were identified with stable expression and at different levels. Two clones were selected based on stable expression level and on invasive phenotype, i) high expressing non invasive clone DLKP 17 and ii) low expressing invasive clone DLKP 11. Microarray profiling studies done in this lab on a panel of lung cancer cell lines with various invasive phenotypes identified a list of differentially expressed genes. GLP1R, KCNJ8 and TFPI2 genes were stably overexpressed and all induced invasion in non-invasive DLKP 17. Furthermore GLP1-R induced invasion could be reversed through siRNA induced silencing of the transgene. GLP1-R overexpression in non-invasive MCF-7 also induced cellular invasion. These findings are the first time that GLP1-R has been linked to this phenotype. MiRNA expression profiling was performed comparing low invasive DLKP (parent) and invasive DLKPA (Adriamycin selected) cell lines. Three differentially regulated miRNAs were selected for functional validation. Mir-21 and mir-27a were pro-invasive and mir-29a anti-invasive in DLKP and DLKPA cell lines. We later confirmed the impact on invasion and proliferation through stable overexpression studies with mir-29a. Pre-mir-29a overexpression in the invasive PANC-1 (pancreatic cancer line) resulted in reduced invasion suggesting that the effect of mir-29a was not cell line specific. 2D-DIGE proteomic profiling of cells transfected with mir-29a generated a list of differentially regulated protein spots, some of which were identified by MALDI-TOF and LC-MS analysis. Bioinformatic analysis revealed that the majority of these proteins were involved in cellular processes like apoptosis, proliferation, motility and differentiation. We chose GRB2, RAN, MIF and ANXA2 gene targets which were differentially downregulated due to mir-29a overexpression and performed siRNA induced knockdown in DLKPA cell line. Results revealed RAN to be anti-invasive and anti-proliferative in our cell line model. Knockdown of the other three targets did not affect the invasive or proliferative phenotype of DLKPA. GFP-RAN 3’-UTR reporter assay indicated a small (5-6%) but statistically significant reduction in GFP RAN-UTR expressing cell population when transfected with mir-29a. In conclusion we provide evidence that mir-29a may be an anti-invasive microRNA and that this effect is mediated via modulation of the expression of RAN and several other cellular proteins

Transcriptomic alterations in the heart of non-obese type 2 diabetic Goto-Kakizaki rats

BACKGROUND: There is a spectacular rise in the global prevalence of type 2 diabetes mellitus (T2DM) due to the worldwide obesity epidemic. However, a significant proportion of T2DM patients are non-obese and they also have an increased risk of cardiovascular diseases. As the Goto-Kakizaki (GK) rat is a well-known model of non-obese T2DM, the goal of this study was to investigate the effect of non-obese T2DM on cardiac alterations of the transcriptome in GK rats. METHODS: Fasting blood glucose, serum insulin and cholesterol levels were measured at 7, 11, and 15 weeks of age in male GK and control rats. Oral glucose tolerance test and pancreatic insulin level measurements were performed at 11 weeks of age. At week 15, total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 41,012 genes, and then expression of selected genes was confirmed by qRT-PCR. Gene ontology and protein-protein network analyses were performed to demonstrate potentially characteristic gene alterations and key genes in non-obese T2DM. RESULTS: Fasting blood glucose, serum insulin and cholesterol levels were significantly increased, glucose tolerance and insulin sensitivity were significantly impaired in GK rats as compared to controls. In hearts of GK rats, 204 genes showed significant up-regulation and 303 genes showed down-regulation as compared to controls according to microarray analysis. Genes with significantly altered expression in the heart due to non-obese T2DM includes functional clusters of metabolism (e.g. Cyp2e1, Akr1b10), signal transduction (e.g. Dpp4, Stat3), receptors and ion channels (e.g. Sln, Chrng), membrane and structural proteins (e.g. Tnni1, Mylk2, Col8a1, Adam33), cell growth and differentiation (e.g. Gpc3, Jund), immune response (e.g. C3, C4a), and others (e.g. Lrp8, Msln, Klkc1, Epn3). Gene ontology analysis revealed several significantly enriched functional inter-relationships between genes influenced by non-obese T2DM. Protein-protein interaction analysis demonstrated that Stat is a potential key gene influenced by non-obese T2DM. CONCLUSIONS: Non-obese T2DM alters cardiac gene expression profile. The altered genes may be involved in the development of cardiac pathologies and could be potential therapeutic targets in non-obese T2DM

Mycobacterium tuberculosis UvrB Is a Robust DNA-Stimulated ATPase That Also Possesses Structure-Specific ATP-Dependent DNA Helicase Activity

Much is known about the Escherichia colt nucleotide excision repair (NER) pathway; however, very little is understood about the proteins involved and the molecular mechanism of NER in mycobaCteria. In this study, we show that Mycobacterium tuberculosis UvrB (MtUvrB), which exists in solution as a monomer, binds to DNA in a structure-dependent manner. A systematic examination of MtUvrB substrate specificity reveals that it associates preferentially with single-stranded DNA, duplexes with 3' or 5' overhangs, and linear duplex DNA with splayed arms. Whereas E. coli UvrB (EcUvrB) binds weakly to undamaged DNA and has no ATPase activity, MtUvrB possesses intrinsic ATPase activity that is greatly stimulated by both single and double-stranded DNA. Strikingly, we found that MtUvrB, but not EcUvrB, possesses the DNA unwinding activity characteristic of an ATP-dependent DNA helicase. The helicase activity of MtUvrB proceeds in the 3' to S' direction and is strongly modulated by a nontranslocating 5' single-stranded tail, indicating that in addition to the translocating strand it also, interacts with. the 5' end of the substrate. The fraction of DNA unwound by MtUvrB decreases significantly as the length, of theiduplex increases: it fails to unwind duplexes longer than 70 bp. These results, on one hand, reveal significant mechanistic differences between MtUvrB and EcUvrB and, on the other, support an alternative role for UvrB in the processing of key DNA replication intermediates. Altogether, our findings. provide insights into the catalytic functions of UvrB and lay the foundation for further understanding of the NER pathway in k tuberculosis

Genome-wide identification of novel micrornas and their target genes in the human parasite schistosoma mansoni

Mature microRNAs (miRNAs) are small, non-coding regulatory RNAs which can elicit post-transcriptional repression of mRNA levels of target genes. Here, we report the identification of 67 mature and 42 precursor miRNAs in the Schistosoma mansoni parasite. The evolutionarily conserved S. mansoni miRNAs consisted of 26 precursor miRNAs and 35 mature miRNAs, while we identified 16 precursor miRNAs and 32 mature miRNAs that displayed no conservation. These S. mansoni miRNAs are located on seven autosomal chromosomes and a sex (W) chromosome. miRNA expansion through gene duplication was suggested for at least two miRNA families miR-71 and mir-2. miRNA target finding analysis identified 389 predicted mRNA targets for the identified miRNAs and suggests that the sma-mir-71 may be involved in female sexual maturation. Given the important roles of miRNAs in animals, the identification and characterization of miRNAs in S. mansoni will facilitate novel approaches towards prevention and treatment of Schistosomiasis

Computational identification and evolutionary relationships of the MicroRNA gene cluster miR-71/2 in protostomes.

MicroRNAs (miRNAs) are small noncoding RNA molecules which are processed into *20–24 nt molecules that can regulate the gene expression posttranscriptionally. MiRNA gene clusters have been identified in a range of species, where in miRNAs are often processed from polycistronic transcripts. In this study, a computational approach is used to investigate the extent of evolutionary conservation of the miR-71/2 cluster in animals, and to identify novel miRNAs in the miRNA cluster miR-71/2. The miR-71/2 cluster, consisting of copies of the miR-71 and miR-2 (including miR-13) families, was found to be Protostome-specific. Although, this cluster is highly conserved across the Protostomia, the miR-2 family is completely absent from the Deuterostomia species, while miR-71 is absent from the Vertebrata and Urochordata. The evolutionary conservation and clustering propensity of the miR-71/2 family across the Protostomes could indicate the common functional roles across the member species of the Protostomia

Comparative Transcriptome Analysis to Reveal Differentially Expressed Cytochrome P450 in Response to Imidacloprid in the Aphid Lion, <i>Chrysoperla zastrowi sillemi</i> (Esben-Petersen)

The aphid lion, Chrysoperla zastrowi sillemi (Neuroptera: Chrysopidae) is a highly effective beneficial predator of many agricultural pests and has developed resistance to several insecticides. Understanding the molecular mechanism of insecticide resistance in the predators is crucial for its effective application in IPM programs. Therefore, transcriptomes of imidacloprid-resistant and susceptible strains have been assessed using RNA-seq. Cytochrome P450 is one of the important gene families involved in xenobiotic metabolism. Hence, our study focused on the CYP gene family where mining, nomenclature, and phylogenetic analysis revealed a total of 95 unique CYP genes with considerable expansion in CYP3 and CYP4 clans. Further, differential gene expression (DGE) analysis revealed ten CYP genes from CYP3 and CYP4 clans to be differentially expressed, out of which nine genes (CYP4419A1, CYP4XK1, CYP4416A10, CYP4416A-fragment8, CYP6YL1, CYP6YH6, CYP9GK-fragment16, CYP9GN2, CYP9GK6) were downregulated and one (CYP9GK3) was upregulated in the resistant strain as compared to the susceptible strain. Expression validation by quantitative real-time PCR (qRT-PCR) is consistent with the DGE results. The expansion and differential expression of CYP genes may be an indicator of the capacity of the predator to detoxify a particular group of insecticides

Effect of constituents from samaras of Austroplenckia populnea (Celastraceae) on human cancer cells.

Background: Aiming the continuity of the studies of Austroplenckia populnea, Brazilian species of the Celastraceae family, in the present study, it was investigated the effect of crude extracts obtained with ethanol, ethyl acetate and chloroform and two purified constituents, proanthocyanidin A and 4’-O-methylepigallocatechin, both isolated from its samaras, on cancer cell proliferation assays. Materials and Methods: The human cancer cells lines MCF-7 (ductal breast carcinoma), A549 (lung cancer), HS578T (ductal breast carcinoma) and non-cancer HEK293 (embryonic kidney cells) were treated with different concentrations of extracts and constituents and the effect was observed through the acid phosphatase method. The chemical structures of the purified compounds were identified by the respective IR and 1H and 13C nuclear magnetic resonance spectral data. Results: While crude extracts from samaras of the folk medicine A. populnea can trigger cell proliferative effects in human cell lines, the purified compounds (proanthocyanidin A and 4’-O-methyl-epigallocatechin) isolated from the same extracts can have an opposite (anti-proliferative) effect. Conclusion: Based on the results, it was possible to suggest that extracts from samaras of A. populnea should be further investigated for possible cancer-promoting activities; and the active extracts can also represent a source of compounds that have anti-cancer properties

Interaction of Plasma Deposited HMDSO-Based Coatings with Fibrinogen and Human Blood Plasma: The Correlation between Bulk Plasma, Surface Characteristics and Biomolecule Interaction

The success of a biomaterial depends on the nature of interaction and the progressive reaction between the biological components and the surface of the biomaterial. In order to control the interaction between the biomaterial and biological component, it is necessary to understand the factors that influence the protein adsorption and cell proliferation. Surface chemistry plays a crucial role in the success of any blood contacting biomaterial. Plasma enhanced chemical vapour deposition (PECVD) is an interesting commonly used technique for tailoring surface characteristics while retaining bulk material properties. Two different films, namely polymer-like and silica-like coatings, with varying surface characteristics have been deposited from hexamethyldisiloxane, by PECVD, on 316L stainless steel. A correlation between the bulk plasma, interfacial adhesion of the coating to 316L steel, surface characteristics and biomolecule interaction is presented in this work The interfacial adhesion strength analysis demonstrated that silica-like coatings have higher adhesion strength to 316L stainless steel than polymer-like coatings, caused due to the formation of a strong Fe-O-Si and Cr-O-Si bonds. It was observed that the effect of nanoscale surface roughness (dose to 6 nm) was less significant, and that the surface chemistry played a significant role in governing the fibrinogen adsorption. Highest fibrinogen adsorption on plain steel was due to the electrostatic interaction of the metal oxide layer with the protein Hydrophobicity of the polymer like film resulted in a higher fibrinogen binding than the silica-like films