Project description:H1299 cells were overexpressed miR-138 or silenced AGO2. The expression of 92 genes associated with p53 using the “Human p53 Signaling Pathway PCR Array” qPCR gene expression profiling. H1299 cells were transfected with NC mimics, AGO2 siRNA or miR-138 for 48h. Equal amount total RNA from each group was pooled prior to gene expression analysis.
Project description:We explored the biological effects of AGO2 downregulation on the expression of miRNAs in human NSCLC cells. Using AGO2 siRNA as a positive control, we overexpressed miR-138 in H1299 cells to observe changes in the miRNA profile using an miRNA microarray assay. Total RNAs were extracted from H1299 cells transfected with NC, AGO2 siRNA or miR-138 for 48h. Their microRNAs expression profiles were investigated using LCSciences Human miRNA Array platform (LCSciences, Houston, TX; Sanger mirBase Release 19.0).
Project description:ANGPTL1-overexpressed CL1-5 cells were used to identify miRNAs which regulate EMT, cancer invasion and metastasis. We used microRNA Taqman Low Density Array to identify candidate miRNAs ANGPTL1-overexpressed CL1-5 cells were analyzed compare to vector control (pcDNA3.1) CL1-5 cells
Project description:Transmissible gastroenteritis virus (TGEV) is a member of Coronaviridae family. Our previous research showed that TGEV infection could induce mitochondrial dysfunction and up-regulat miR-222 level. Therefore, we presumed that miR-222 might be implicated in regulating mitochondrial dysfunction induced by TGEV infection. To verify the hypothesis, the effect of miR-222 on mitochondrial dysfunction was detected and showed that miR-222 attenuated TGEV-induced mitochondrial dysfunction. To investigate the underlying molecular mechanism of miR-222 in TGEV-induced mitochondrial dysfunction, a quantitative proteomic analysis of PK-15 cells that were transfected with miR-222 mimics and infected with TGEV was performed. In total, 4151 proteins were quantified and 100 differentially expressed proteins were obtained (57 up-regulated, 43 down-regulated), among which thrombospondin-1 (THBS1) and cluster of differentiation 47 (CD47) were down-regulated. THBS1 was identified as the target of miR-222. Knockdown of THBS1 and CD47 increased mitochondrial Ca2+ level and decreased mitochondrial membrane potential (MMP) level. Together, our data establish a significant role of miR-222 in regulating mitochondrial dysfunction in response to TGEV infection.
Project description:To evaluate involvement of miR-221 and miR-222 in lung cancer, we investigated the effects of miR-221 and miR-222 overexpression on six lung cancer cell lines as well as one immortalized normal human bronchial epithelial cell line. Two cell lines, H3255 and H1299 with no replicates were studied. Cells were transfected with miR-221, miR-222, or miR control. Microarray analysis was done to identify genes differentially expressed in lung cancer cells after the transfection of miR-221 or miR-222.
Project description:mRNA microarray experiments were performed to measure global mRNA expression in the presence of increased or decreased miR-106a-5p levels to to identify the total transcripts regulated by miR-106a-5p directly or indirectly. FASTK was identified as a direct target gene of miR-106a-5p. In order to identify the total transcripts regulated by miR-106a-5p directly or indirectly, we first measured the global mRNA expression change through mRNA microarray by overexpressing or knocking down miR-106a-5p in cancer cells. Next, we combined bioinformatics programs to select candidate miR-106a-5p targets from the differentially regulated genes to refine the number of miR-106a-5p targets. Then we validated the miR-106a-5p target gene through western blot analysis, quantitative real-time PCR and luciferase reporter assay.
Project description:miRNA-mediated gene expression silencing has previously been shown to be important for a variety of physiological and pathological processes. Here, we have explored the role of one bona fide human-specific miRNA (miR-941) in evolution of the human-specific expression and function. Using combination of high-throughput sequencing (GSE26545), miRNA transfection and large-scale PCR of various human populations, we have shown that emergence and rapid expansion of miR-941 might take place on the human evolutionary linage between six and one million years ago. Functionally, miR-941 could be associated with hedgehog and insulin signaling pathways, and thus might potentially play a role in evolution of human longevity. Human-specific effects of miR-941 regulation are detectable in human brain and affect genes involved in neurotransmitter signaling. Furthermore, emergence of miR-941 on the human evolutionary linage was accompanied by the accelerated loss of its binding sites. Taken together, these results strongly implicate the contribution of miR-941 in evolution of the human-specific phenotype. miRNA transfection experiments were conducted in 6 cell lines: two human derived kidney cell lines (HEK and 293T), one human skin fibroblast cell line (HSF2), two macaque derived kidney cell lines (LLCMK2 and FrhK-4), and one macaque skin fibroblast cell line (MSF). Briefly, cells were plated in 0.5 ml of growth medium 24h prior to transfection without antibiotics. miR-941 mimics-Lipofectamine 2000 (Invitrogen) complexes were prepared freshly before transfection based on the manufacturer’s protocol. Cells were transfected in six-well plates using miRNA mimics-Lipofectamine 2000 with a final oligonucleotide concentration of 10nmol/L. In parallel, negative control transfections with mock oligonucleotides were conducted according to the manufacturer’s protocol. After 24h, cells were harvested and total RNA were extracted using Trizol reagent (Invitrogen) and further processed and hybridized to Affymetrix Human Genome U133 Plus 2.0 arrays following the manufacturer’s instructions. The R RMA package was used to quantify gene expression levels.
Project description:Since the early 1980s remarkable progress has been made in understanding the role of the HER2 locus in carcinogenesis, but many details of its regulatory network are still elusive. We recently reported the finding of 367 new human microRNA (miRNA) genes of which one, mir-4728, is encoded in an intron of the HER2 gene. Here, we confirm that the HER2 oncogene is a bi-functional locus encoding the membrane receptor and a functional miRNA gene. We further show that miR-4728-3p has alternative functionalities depending on the region used for interaction with its target; the canonical seed between nucleotides 2-8 or a novel, more internal seed shifted to nucleotides 6-12. Analysis of public data shows that this internal seed region, although rare compared to the far more abundant canonical 2-8 seed interaction, can also direct targeted down-regulation by other miRNAs. Through the internal seed, miR-4728-3p regulates expression of estrogen receptor alpha, an interaction that would have remained undetected if classic rules for miRNA-target interaction had been applied. In summary, we present here an alternative mode of miRNA regulation and demonstrate this dual function of the HER2 locus, linking the two major biomarkers in breast cancer. 6 samples treated with hsa-miR-4728-3p mimic and 6 samples treated with non-targeting control 4 samples treated with hsa-miR-1-3p mimic and 4 samples treated with non-targeting control
Project description:The aim of this experiment was to determine how exposure of Hydrogen sulfide impacts gene expression in Mycobacterium tuberculosis. RNA was isolated from actively growing mycobacterial cells (0.6-0.8 OD600) using Trizol according to established protocols (27). Briefly, cells were exposed to 25 µM GYY4137 for 1 hr under carefully controlled conditions (n=3/group) and RNA isolated. Unexposed cells received spent GYY4137 (without any capacity to produce Hydrogen sulfide).