The effect of miR196a of overall gene expression in oral keratinocytes
ABSTRACT: We wished to assess the effect of miR196a on overall gene expression in reducing it in high expressing epithelial cells (D19 and B16) using antimiR196a and over expressing it in low-expressing cells (OKF4) using pre-miR196a. We transfected anti miR196a in to D19 and B16 oral keratinocytes which express high levels of miR196a in triplicate cultures, harvesting 24h after transfection. We transfected pre-miR196 in okf4 immorualised normal oral keratinocytes in duplicate culture. Successful transfection was ensured by qPCR. 16 Samples in total. 2 high expressing cells: control and anti-miR transfected, in triplicate. 1 low expressing cell: control and premiR transfected, in duplicate
Project description:Microarray analysis was performed to examine potential differences in target gene expression of AE9a expressing low cells compared to AE9a expressing high cells. Potential contributing factors to AE9a induced leukemia were investigated. Embryonic day 13.5 to 14.5 fetal liver cells were harvested from C57Bl6 females. Cells were cultured in StemSpan media with 50ng/ml rat recombinant SCF, 10ng/ml human IL-6, and 10ng/ml murine IL-3. After overnight stimulation, cells were collected and transduced with two rounds of retrovirus (MIG-AE9a). Post transduction, cells were collected, counted and transplanted into lethally irradiated BoyJ recipient mice. Cells were expanded two to three weeks in vivo, mice were sacrificed, and bone marrow (BM) harvested. c-Kit+ cells were sorted (BD FACS Aria) for 30% low and 30% high GFP-expressing populations. Six AE9a low and six AE9a high samples were compared. RNA was isolated via the Qiagen RNeasy RNA isolation kit and submitted to the CCHMC Microarray Core. Samples were hybridized to an Affymetrix Mouse Gene 1.0 ST chip. Data was normalized using Expression Console and was analyzed using Qlucore Omics Explorer software.
Project description:Mitochondrial DNA (mtDNA) encodes essential components of the respiratory chain and loss of mtDNA leads to mitochondrial dysfunction and neurodegeneration. Mitochondrial transcription factor A (TFAM) is an essential component of mtDNA replication and a regulator of mitochondrial copy number in cells. Studies have shown that TFAM knockdown leads to mitochondrial dysfunction and respiratory chain deficiencies. Using gene expression analysis, we aimed to investigate the effects of mtDNA dysfunction in the CNS at the molecular level. We used microarray analysis to investigate gene expression in cases of mitochondrial dysfunction in the CNS. RNA was purified from the late third instar larval CNS from control larvae, or larvae over-expressing mitochondrial transcription factor A (TFAM) in post-mitotic neurons using the neuron specific driver nsyb-Gal4. Three replicates are included for each condition.
Project description:Transcriptome of si-RNA transfected NOK cells and adenovirus-transduced HEK cells Overall design: mRNA profiles of HEK (human epidermal keratinocytes) transduced with adenoviruses and NOK (human normal oral keratinocytes) transfected with siRNAs.
Project description:The intestine harbors more immune cells then the rest of the body together in order to maintain a state of tolerance towards the microbiota as well as food antigen on one side and recognition and immune response to invading pathogens on the other side. At birth the intestine transitions from a sterile organ to a highly exposed environment. At the same time the murine adaptive immune system starts to form and newly generated T and B cells home to the intestine. The present study aims to understand the function of CD4 T cells in the neonatal small intestine (SI). For that the transcriptional profiles of CD4 T cells isolated from the SI of 6- and 11-days-old neonates were compared to their counterparts in mLN and thymus as well as to adult CD4 T cells from the lamina propria (LP) and Peyers patches (PP). 4 true biological replicates in single color mode were analyzed in each group.
Project description:Type 2 diabetes is a complex disease associated with many underlying pathomechanisms. Epigenetic regulation of gene expression by DNA methylation has become increasingly recognized as an important component in the etiology of type 2 diabetes. We performed genome-wide methylome and transcriptome analysis in liver from severely obese patients with or without type 2 diabetes to discover aberrant pathways underlying the development of insulin resistance. We identified hypomethylation of five key genes involved in hepatic glycolysis, de novo lipogenesis and insulin resistance with concomitant increased mRNA expression and protein content. The CpG-site within the ATF-motif was hypomethylated in four of these genes in liver of non-diabetic and type 2 diabetic obese patients, suggesting epigenetic regulation of transcription by altered ATF-DNA binding. In conclusion, severely obese non-diabetic and type 2 diabetic patients have distinct alterations in the hepatic methylome and transcriptome and genes controlling glucose and lipid metabolism are hypomethylated at a regulatory site. Thus, obesity may epigenetically reprogram the liver towards increased lipid production and exacerbate the development of insulin resistance. To better understand the molecular mechanisms underlying the development of hepatic insulin resistance and type 2 diabetes at a molecular level, we performed a genome-wide methylome and transcriptome analysis of liver from non-obese metabolically healthy, obese non-diabetic and obese type 2 diabetic patients. Distinct DNA methylation and gene expression profiles were identified in liver from the obese and type 2 diabetic patients compared with the non-obese participants.
Project description:We investigated the impact of on miR-H1 and miR-K12-3-3p- on host transcriptome focusing on gingival epithelial cells that are target sites for various HHV. Results: More than 1300 genes were altered by miR-H1 and miR-K12-3-3p expressing HOK. Overall design: Primary oral keratinocytes were transfected with miR-H1, miR-K12-3-3p or control mimic for 48 hours. Total RNA isolated from cells (n=4) was assessed for global transcriptome changes using Affymetrix Gene array .
Project description:Pediatric GIST commonly harbors a disabled succinate dehydrogenase complex (SDH), which yields tumors with highly conserved genomes but characteristic epigenomic signatures. Mysteriously, nearly half of such SDH-deficient GIST, including tumors from Carney Triad patients, lack identifiable mutations in SDH component genes and genes required for complex assembly (SDHA, SDHB, SDHC, SDHD, SDHAF, termed SDHx). Genomic sequencing coupled with DNA methylation and transcriptional profiling have exposed SDHC promoter-specific CpG island epimutation and concomitant gene silencing in the majority of SDHx-WT GIST. We performed whole genome expression profiling on 20 FFPE dSDH GIST tumors using Affymetrix U133P2 arrays which contain >54K gene target probesets. Included here were data from 7 SDHx-w.t. and 13 SDHx mutants that passed array QC.
Project description:Prevalence of the members of herpesvirus family in oral inflammatory diseases is increasingly acknowledged suggesting their likely role as etiological factor. However, the underlying mechanisms remains obscure. In our recent miRNA profiling of healthy and diseased human tooth pulpal, elevated expression of HHV encoded v-miRs were identified. Based on the fold induction and significance values, we selected three v-miRs namely miR-K12-3-3p (KSHV), miR-H1 (HSV-1), and miR-UL-70-3p (HCMV) to further examine their impact on host cellular functions. We examined their impact on cellular miRNA profiles of primary human oral keratinocytes (HOK). Our results show differential expression of several host miRNAs in v-miR transfected HOK. High levels of v-miRs were detected in exosomes derived from v-miR transfected HOK as well as the latent KSHV infected cell lines. We show that HOK derived exosomes release their contents into macrophages (Mφ) and alter expression of endogenous miRNAs. Overall design: Total RNA isolated from v-miRs (miR-H1, miR-K12-3-3p and miR-UL70-3p) or control mimic transfected human oral keratinocytes and primary human macrophages. Microarray ananlysis was performed to assess global changes in miRNA profiles using Exiqon miRCury LNA microarray.
Project description:The goal of this study is to identify genes that are differentially expressed by E6 in oral epithelial cells that are stably expressing Bmi-1. Experiment Overall Design: Primary Normal human oral keratinocytes (NHOK) is explanted from normal human gingival tissue. The cells were infected with retroviruses capable of expressing Bmi-1 and being selected. Actively dividing HOK/Bm-1 cells were then superinfected with LXSN-based retorviruses capable of expressing E6 and E6(delta)118-122, and selected. Actively proliferating cells are then harvested to isolate total RNA which was subsequently subjected to microarray analysis.