Effect of Pnpla6 gene silencing in D3 mouse embryonic stem cells
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ABSTRACT: Pnpla6 gene was silenced in D3 mouse embryonic stem cells and the cells were allowed to spontaneously differentiate. Transcription of the whole genome was analyzed 96 days afterwards and compared against control cells differentiated during the same time without previous silencing. Comparimg two experimental conditions: control differentiated cells versus Pnpla6 silenced differentiated cells.
Project description:Neuropathy Target Esterase (NTE) is a protein involved in the development of a polyneuropathy caused by exposure to certain organophosphorus compounds. In vivo and in vitro studies have also associated NTE with embryonic development since NTE null mice embryos are non-viable, and silencing the NTE-codifying gene (Pnpla6) in mouse embryonic stem cells strongly alters the differentiation of vascular and nervous systems. In this paper, human embryonal carcinoma stem cells (hNT2) are used as in vitro neurodifferentiation model to determine whether PNPLA6 gene silencing is able to alter the differentiation into a neuronal phenotype. In controls, the PNPLA6 mRNA levels increased in parallel with other neuroectodermal markers during the neurodifferentiation. PNPLA6 gene silencing with specific interference RNA led to a 97% maximum decrease in gene expression by day 3 after transfection, with a maximum of 50% reduction in NTE enzymatic activity by day 4. Microarray analyses of the PNPLA6-silenced cells showed alterations in several developmental processes, mainly neurogenesis and epithelium tube morphogenesis. PNPLA6 silencing also led to a reduction in electrical activity and an altered neuronal phenotype. This work is the first proof supporting the hypothesis that NTE protein plays a critical role in human early neurodevelopment using a human cell differentiation model. PNPA6 gene silencing in undifferentiatied hNT2 cells was performed, together with a Control (non-gene silencing cells) and a Negative Control (non-specific RNA silencer). Three biological replicates.
Project description:Cholangiocarcinoma is the second most common primary hepatic malignancy worldwide, with intrahepatic cholangiocarcinoma (ICC) a particularly significant public health problem in Southeast Asia, due to its strong association with the food-borne parasite Opisthorchis viverrini (OV). This manuscript represents the first comprehensive miRNA expression profiling by microarray of the three most common histological grades of OV-induced ICC: moderately differentiated, papillary, and well differentiated tumor tissue. No cohort of miRNAs emerged as commonly dysregulated among these histological grades of OV-induced ICC. Instead, each histological grade of ICC tissue showed a distinct miRNA profile. Moderately differentiated tumor tissue showed both the greatest number and the highest magnitude of miRNA dysregulation, followed by papillary ICC tumor tissue, and differentiated ICC tumor tissue. When ICC tumor tissue was compared to adjacent non-tumor tissue, a remarkable similarity in miRNA dysregulation was observed between these samples, indicative of intrahepatic metastasis. These findings indicate the possibility of determining the histological grade of ICC by profiling miRNA dysregulation, which not only would greatly enhance the molecular diagnosis of ICC, but could even lead to the personalized the treatment for ICC by the early classification of histological grade. A total of 46 unique liver tissue samples were analyzed on Agilent human miRNA microarray (miRBase Release 16.0). Of the 46 samples, 13 are from non-cancer healthy patients with gastric bypass surgery. The rest of 33 samples are CCA tumor tissue samples and their paired adjacent normal/necrotic tissue: 12 from well differentiated CCA, 17 from papillary CCA, 4 from moderately differentiated CCA.
Project description:SOX2 is an oncogene and a core pluripotency transcription factor. SOX2 has multiple roles in various malignancies, in the maintainance of pluripotency and during various stages of embryonic development. Human embryonal carcinoma cells express SOX2 and the loss of this results in their differentiation. We silenced SOX2 in two human embryonal carcinoma cell lines and measured whole-genome mRNA expression. Many genes related to embryogenesis and tissue morphogensis were upregulated. Other upregulated genes were markers of mesodermal development and epithelial-to-mesenchymal transition. A specific and validated siRNA against SOX2 was chemically transfected in undifferentiated 2102Ep and NTera-2 embryonal carcinoma cell lines. After three days of incubation under normal growth conditions we used Affymetrix microarrays to measure whole-genome mRNA transcript expression in three biological replicates of each cell line and compared this to whole-gene expression in identical samples transfected with a non-targeting, scrambled control siRNA. SOX2 silencing was validated using qRT-PCR and Western blot prior to whole-genome expression analysis.
Project description:Despite their distinct biology, granulosa cell tumours (GCTs) are treated the same as other ovarian tumours. Intriguingly, a recurring somatic mutation in the transcription factor Forkhead Box L2 (FOXL2) 402C>G has been found in nearly all GCTs examined. This investigation aims to identify the pathogenicity of mutant FOXL2 by studying its altered transcriptional targets. The expression of mutant FOXL2 was reduced in the GCT cell line KGN, and wildtype and mutant FOXL2 were overexpressed in the GCT cell line COV434. Comparisons were made between the transcriptomes of control cells and cells altered by FOXL2 knockdown and overexpression, to detect potential transcriptional targets of mutant FOXL2. Comparisons were made between the transcriptomes of control cells and cells altered by FOXL2 knockdown and overexpression, to detect potential transcriptional targets of mutant FOXL2. Mutant FOXL2 was silenced in KGN cells with the use of siRNA and a non-targetting siRNA control. Wildtype and mutant FOXL2 were overexpressed in COV434 cells with the use of overexpression plasmids and an empty plasmid control. Each experiment was performed in triplicate. Total RNA was harvested at 24h post transfection and hybridised to Affymetrix U133 plus 2.0 arrays.
Project description:We and others previously reported that the miR-106b-25 microRNA cluster is a candidate oncogene in human prostate cancer. Here, we made the novel observation that miR-106b-25 expression is further up-regulated in distant metastasis. Moreover, increased tumor miR-106b expression was associated with early disease recurrence. To identify yet unknown oncogenic functions of the prognostic miR-106b, we overexpressed it in LNCaP human prostate cancer cells to examine miR-106b-induced global expression changes among protein-coding genes. The approach revealed that caspase-7 is a candidate direct target of miR-106b, which was confirmed by Western blot analysis and a 3M-bM-^@M-^YUTR reporter assay. Other analyses showed that caspase-7 is down-regulated in primary human prostate tumors and metastatic lesions across multiple datasets and is by itself associated with disease recurrence. Using bioinformatics, we also discovered that miR-106b-25 may specifically influence focal adhesion-related pathways. This observation was experimentally confirmed using miR-106b-25-transduced 22Rv1 human prostate cancer cells. After infection with a miR-106b-25 lentiviral expression construct, 22Rv1 cells showed increased adhesion to basement membrane- and bone matrix-related filaments and enhanced soft agar growth. In summary, miR-106b-25 was found to be associated with prostate cancer progression and metastasis and may do so by altering apoptosis- and focal adhesion-related pathways. To elucidate the effects miR-106b, we up-regulated miR-106b in LNCaP cells and examined gene expression alterations on a global scale with Affymetrix arrays. LNCaP cells were transfected with pre-miR oligos and 24 hr post-transfection total RNA was collected for microarray anaylsis.
Project description:Aim: to detect genes that are differentially transcribed in neuronal cells(SK-N-SH) over-expressing either of the two MECP2 isoforms, MECP2_e1 or MECP2_e2. Methods: the human neuroblastoma cell line SK-N-SH was stably infected by lentiviral vectors over-expressing MECP2_e1, MECP2_e2, or eGFP, and were then differentiated into neurons. RNA extracted, and used for gene expression microarray analysis 3 replicates for MECP2_e1; 3 replicates for MECP2_e2; 3 replicates for eGFP as control; 3 replicates for MECP2_e1 with 9bp insertion mutation in N-terminal
Project description:Clinical applications of human interferon (IFN)-alpha have met with varying degrees of success. Nevertheless, key molecules in IFN-alpha-induced cell death have not been clearly identified. Our previous study indicated that IFN (alpha, beta and omega) receptor (IFNAR) 1/2- and IFN regulatory factor (IRF) 9-RNA interference (RNAi) completely inhibited the antiproliferative (AP) activity of IFN-alpha in human ovarian adenocarcinoma OVCAR3 cells sensitive to IFN-alpha., followed by transcription of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, IFNAR1/2- and IRF9-RNAi inhibited the gene expression of TRAIL, but not of Fas ligand (FasL), following IFN-alpha treatment. In fact, TRAIL but not FasL inhibited the proliferation of OVCAR3 cells. IFN-alpha notably up-regulated the levels of TRAIL protein in the supernatant and on the membrane of OVCAR3 cells. Following TRAIL signaling, Caspase 8 inhibitor and BH3 interacting domain death agonist (BID)-RNAi significantly abrogated both AP activities of IFN-alpha and TRAIL. Furthermore, BID-RNAi prevented both IFN-alpha and TRAIL from collapsing the mitochondrial membrane potential (Delta Psi m). Finally, we provide important new evidence that BID overexpression led to a major enhancement of both AP activities of IFN-alpha and TRAIL in human lung carcinoma A549 cells resistant to IFN-alpha. Thus, this study suggests that BID is crucial in IFN-alpha-induced cell death, indicating a notable potential to be a targeted therapy for IFN-alpha resistant tumors. Biological replicate samples were created by treating OVCAR3 cells with IFN-alpha2c (n=8); IFNAR1-RNAi and IFN-alpha2c (n=4); IFNAR2-RNAi and IFN-alpha2c (n=5); ISGF3gamma-RNAi and IFN-alpha2c (n=3); and Negative RNAi and IFN-alpha2c (n=3). For analysis, the eight IFN-alpha2c treated OVCAR3 samples were paired with an untreated OVCAR3 control sample. The 15 RNAi treated OVCAR3 samples were paired with a Negative RNAi control sample.
Project description:Transcriptional profiling in HACAT cells using a whole human genome array; HACAT cells treated with si RNA against Keap 1 or a scrambled si RNA sequence (Scram) vs HACAT cells mock transfected with lipofectamine (reference control) Experiment Overall Design: 2 biological replicates, 2 technical (dye swap) replicates per treatment.
Project description:Cumulus oophorus cells play an essential role in oocyte development. CBX4 is a member of the Polycomb complex, which plays a role in regulating cellular differentiation. We used siRNA mediated knockdown of Cbx4 expression followed by Affymetrix array analysis to evaluate the role of CBX4 in cumulus cell differentiated state.