In addition to the well-characterized Induced Pluripotent Stem cells (iPSCs) that closely resemble Embryonic Stem cells (ESCs), a recent study has proven the existence of a stable state, resembling partially reprogrammed cells, termed F-class iPSCs. To study these distinct iPSC states, a reprogramming dataset has been generated, featuring the parallel analysis of multiple molecular platforms. MicroRNAs (miRNAs) are small RNA regulators of gene expression whose critical role in reprogramming is now being realized. In the present study, small RNA deep sequencing data from this novel reprogramming dataset was used to identify miRNAs that are likely to enhance reprogramming by detecting significantly up-regulated miRNAs in ESC-like iPSCs versus F-class iPSCs. These candidate miRNAs were cloned and overexpressed in reprogramming mouse embryonic fibroblasts and their effect on reprogramming efficiency was measured. miR-214 was discovered to increase iPSC generation efficiency, marking the first reprogramming-related role for this microRNA.

Genome-wide association studies have implicated autophagy in Crohn’s Disease (CD) pathogenesis. The functional relevance of autophagy in CD remains unknown. I hypothesized that autophagy is involved in microRNA silencing, another process implicated in CD pathogenesis. MicroRNAs are short non-coding RNAs that are loaded onto RNA-induced silencing complex (RISC) and promote degradation and/or repress translation of target mRNAs. RISC formation and turnover occurs on endosomal membranes. Since autophagosomes and endosomes are closely related and RISC components are downstream effectors of microRNA silencing, I hypothesized that autophagy affects RISC, hence modulates microRNA expression. Using immunoblotting and immunofluorescence, I showed that Ago2, a critical component of RISC, is increased in cells with defective autophagy. Using microarray technology, I discovered 5 microRNAs that are differentially expressed in these cells. Taken together, my results propose a compelling mechanism by which autophagy regulates Ago2, thereby affects miRNA expression, which is implicated in the development of CD.

Genome-wide association studies have implicated autophagy in Crohn’s Disease (CD) pathogenesis. The functional relevance of autophagy in CD remains unknown. I hypothesized that autophagy is involved in microRNA silencing, another process implicated in CD pathogenesis. MicroRNAs are short non-coding RNAs that are loaded onto RNA-induced silencing complex (RISC) and promote degradation and/or repress translation of target mRNAs. RISC formation and turnover occurs on endosomal membranes. Since autophagosomes and endosomes are closely related and RISC components are downstream effectors of microRNA silencing, I hypothesized that autophagy affects RISC, hence modulates microRNA expression. Using immunoblotting and immunofluorescence, I showed that Ago2, a critical component of RISC, is increased in cells with defective autophagy. Using microarray technology, I discovered 5 microRNAs that are differentially expressed in these cells. Taken together, my results propose a compelling mechanism by which autophagy regulates Ago2, thereby affects miRNA expression, which is implicated in the development of CD.

Both microRNAs (miRNAs) and RNA-binding proteins (RBPs) regulate post- transcriptional events, but the post-transcriptional contribution to the global mammalian transcriptomes is still not well understood. In this study we study the synergistic interaction between microRNAs that inhibit gene production, and a special RBP, HuR, that positively regulates mRNA stability. We examined their relationship in terms of spatial, conservational and expressional perspective. We show comprehensive mapping of HuR binding sites by combination of its structural and sequential preferences; and cross-platform normalization method within a process of refining miRNA and HuR binding site mapping. Finally, we observed co-evolution of miRNA and HuR binding sites by looking at their proximity and conservation levels. Collectively, our data suggest that mammalian microRNAs and HuR, with seemingly opposing regulatory effects, cooperatively regulate their mutual targets.

For a long time research has focused on the protein-coding mRNA, but there is a complex world of non-coding RNAs regulating the human body that we yet know very little about. Non-coding RNAs (ncRNAs) are involved in modulation of different cell processes including proliferation, differentiation and apoptosis. In the current study the role of ncRNAs in T cell activation and function was investigated. T cells are important mediators of immune responses, for example upon viral infections. The T helper cells (TH or CD4+ cells) are involved in orchestrating immune processes like aiding the activation of macrophages and enhancement of B cell function. The TH1 cell subtype is generally pro-inflammatory and IFNγ-secreting. There are regulatory T (Treg) cells that are involved in downregulation of TH1 cells, to decrease or terminate the immune response. It has been shown that upon repeated stimulation TH1 cells can switch into a Treg-like IL10-secreting anti-inflammatory phenotype. In the IL10-secreting Treg-like cells the microRNA 150 (miR-150) was found upregulated compared to IFNγ-secreting TH1 cells. Thus, miR-150 was believed to be a candidate in key regulation of the switch between the two phenotypes. Predicted target genes of miR-150 were identified using mRNA arrays investigating down-regulated genes in the IL10-secreting Treg-like subpopulation. In this thesis predicted targets of miR-150 were investigated using luciferase assays. Unfortunately no targets were identified. Upon isolation of IFNγ-secreting TH1 cells and Treg-like IL10-secreting cells, it was found that the ncRNA 886 (nc886) was upregulated in these activated cells, compared to resting TH cells. This indicates that nc886 has an important role in T cell activation. Nc886 has been shown to inhibit PKR activation in other cell types. The effect of nc886 on protein kinase R (PKR) was therefore investigated. PKR shuts down translation upon activation in response to viral double-stranded RNA or cellular stress. We showed that in an activated T cell phenotype nc886 is affecting PKR upon activation by dsRNA from HIV or synthetic origin. The PKR activation pattern is reversed in a resting T cell phenotype.

Introduction: Currently, there are no specific pharmacological treatments for sepsis-induced acute respiratory distress syndrome (ARDS). And mesenchymal stem cells (MSCs) have shown reparative potential in both sepsis and ARDS. Objectives: To determine the role of MSC administration in the modulation of pulmonary host-responses to sepsis via differential regulation of regulatory microRNAs (miRNAs/miRs). Methods: MicroRNA and mRNA profiling was performed to identify differential expression. Quantitative real time polymerase chain reaction (qRT-PCR), trans-endothelial electrical resistance (TEER) measurements, and luciferase activity assay were used. Results: MicroRNA expression was examined in Human Pulmonary Microvascular Endothelial Cells (HPMECs). One miRNA – miR-193b-5p, targets occludin, a tight junction protein associated with endothelial leakage. A specific regulatory relationship between miR-193b-5p and occludin was identified. The loss in endothelial integrity was rescued when miR-193b-5p inhibitor was transfected. Conclusion: miR-193b-5p is a suppressor of occludin. Studying transcriptional changes allows identification of therapeutically relevant mediators for ARDS/ALI treatment.

Cervical cancer is the third most common cancer among women worldwide, and the fourth leading cause of cancer mortality. Despite significant declines in the incidence and mortality rates of cervical cancer in Canada, it remains the 4th most common cancer in women aged 20-29 years. In order to gain novel insights into cervical cancer tumourigenesis and clinical outcome, we investigated and characterized the alterations in microRNA (miRNA) expression in this disease. Firstly, we performed global miRNA expression profiling of cervical cancer cell lines (n=3), and patient specimens (n=79). From this analysis, we identified miR-196b to be significantly down-regulated in cervical cancer, and characterized its role in regulating the HOXB7~VEGF axis. The global miRNA expression data also led to the development of a candidate 9-miRNA signature that was prognostic for disease-free survival in patients with cervical cancer, although we were unable to validate this signature in an independent cohort. This report describes important considerations concerning the development and validation of microRNA signatures for cervical cancer. Our investigations also led us to a comparison of three methods for measuring miRNA abundance: the TaqMan Low Density Array, the NanoString nCounter assay, and single-well quantitative real-time PCR. Our findings demonstrated limited concordance between the TLDA and NanoString platforms, although each platform correlated well with PCR, which is considered the gold standard for nucleic acid quantification. Furthermore, we examined biases created by amplification protocols for microarray studies. Our analysis demonstrated that performing a correction using the LTR-method (linear transformation of replicates) could help mitigate, but not completely eliminate such biases. Overall, this report presents insights into the role of miRNAs in cervical cancer, as well as an evaluation of technical considerations concerning miRNA and mRNA expression profiling studies.

Introduction: Currently, there are no specific pharmacological treatments for sepsis-induced acute respiratory distress syndrome (ARDS). And mesenchymal stem cells (MSCs) have shown reparative potential in both sepsis and ARDS. Objectives: To determine the role of MSC administration in the modulation of pulmonary host-responses to sepsis via differential regulation of regulatory microRNAs (miRNAs/miRs). Methods: MicroRNA and mRNA profiling was performed to identify differential expression. Quantitative real time polymerase chain reaction (qRT-PCR), trans-endothelial electrical resistance (TEER) measurements, and luciferase activity assay were used. Results: MicroRNA expression was examined in Human Pulmonary Microvascular Endothelial Cells (HPMECs). One miRNA – miR-193b-5p, targets occludin, a tight junction protein associated with endothelial leakage. A specific regulatory relationship between miR-193b-5p and occludin was identified. The loss in endothelial integrity was rescued when miR-193b-5p inhibitor was transfected. Conclusion: miR-193b-5p is a suppressor of occludin. Studying transcriptional changes allows identification of therapeutically relevant mediators for ARDS/ALI treatment.

No description available.

Small intestinal neuroendocrine tumors (SI-NETs) originate from serotonin-producing enterochromaffin (EC) cells in the intestinal mucosa. Somatostatin analogs (SSAs) are mainly used to control hormonal secretion and tumor growth. However, the molecular mechanisms leading to the control of SI-NETs are unknown. Although microRNAs (miRNAs) are post transcriptional regulators deeply studied in many cancers, are not well-defined in SI-NETs. We adopted a two-pronged strategy to investigate SSAs and miRNAs: first, to provide novel insights into how SSAs control NET cells, and second, to identify an exclusive SI-NET miRNA expression, and investigate the biological functions of miRNA targets. To accomplish the first aim, we treated CNDT2.5 cells with octreotide for 16 months. Affymetrix microarray was performed to study gene variation of CNDT2.5 cells in the presence or absence of octreotide. The study revealed that octreotide induces six genes, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15. To accomplish the second aim, SI-NET tissue specimens were used to run genome-wide Affymetrix miRNA arrays. The expression of five miRNAs (miR-96, -182, -183, -196a and -200a) was significantly upregulated in laser capture microdissected (LCM) tumor cells versus LCM normal EC cells, whereas the expression of four miRNAs (miR-31, -129-5p, -133a and -215) was significantly downregulated in LCM tumor cells. We also detected nine tissue miRNAs in serum samples, showing that the expression of five miRNAs is significantly increased in SSA treated patients versus untreated patients. Conversely, SSAs do not change miRNA expression of four low expressed miRNAs. Silencing miR-196a expression was used to investigate functional activities in NET cells. This experimental approach showed that four miR-196a target genes, HOXA9, HOXB7, LRP4 and RSPO2, are significantly upregulated in silenced miR-196a NET cells. In conclusion, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15 genes might regulate cell growth and differentiation in NET cells, and play a role in an innovative octreotide signaling pathway. The global SI-NET miRNA profiling revealed that nine selected miRNAs might be involved in tumorigenesis, and play a potential role as novel markers for follow-up. Indeed, silencing miR-196a demonstrated that HOXA9, HOXB7, LRP4 and RSPO2 genes are upregulated at both transcriptional and translational levels.