Expression profile of Parathyroid hormone-related protein (PTHrP) induced miRNAs during TGF-beta mediated chondrogenesis of marrow derived human mesenchymal stem cells
ABSTRACT: A miRNA microarray was used to identify candidate miRNAs regulated by PTHrP treatment in chondrogenic hMSC differentiation. miRNA-expression patterns after PTHrP treatment for 1 week (CM-T-Pt-L1) or 3 weeks (CM-T-Pt-3) in chondrogenic culture were compared with reference levels obtained from cell pellets treated with TGF-β only (CM-T). Under identical chondrogenesis conditions (except for PTHrP treatment), miRNA-expression patterns were relatively unchanged when compared with the TGF-β-only control, which simplified the selection of candidate miRNAs. Microarray analysis revealed that only 4 miRNAs in CM-T-Pt-L1 cells and 7 miRNAs in CM-T-Pt-3 cells were differentially expressed following PTHrP treatment. Hsa-miR-590-5p and hsa-miR-892b were commonly down-regulated or up-regulated in both PTHrP-treated groups. Interestingly, hsa-miR-892b expression was not detected in the TGF-β-only control, while hsa-miR-877*, hsa-miR-1288, and hsa-miR-1305 were up-regulated. Therefore, PTHrP treatment induced hsa-miR-892b expression during TGF-β-mediated hMSC chondrogenesis. hsa-miR-892b expression in CM-T-Pt-L1 cells was 2.43-fold higher than that in CM-T-Pt-3 cells. Overall design: PTHrP induced miRNA expression in human MSC was measured at 21 days of chondrogenic induction after exposure for last 1 week or all 3 weeks to dose of 100 ng/ml PTHrP. Four independent experiments were performed depending on PTHrP-exposure times (0, 1, and 3 weeks) using two different donors for each experiment.
INSTRUMENT(S): Agilent-021827 Human miRNA Microarray (V3) (miRBase release 12.0 miRNA ID version)
Project description:Long-term dynamic compression enhanced the mechanical properties of MSC-seeded constructs only when loading was initiated after 21 days of chondrogenic differentiation. This study examined the molecular differences of chondrogenic MSCs compared to undifferentiated MSCs (TGF-beta vs no TGF-beta) and the effects of dynamic loading on MSC chondrogenesis (loading vs free-swelling). Overall design: Free-swelling MSC-seeded constructs were cultured for 21 days in chemically defined media. Chondrogenesis was induced with TGF-beta3. Undifferentiated controls were maintained in parallel. After 21 days of chondrogenic differentiation, a subset of constructs were subjected to 21 days of dynamic compressive loading. On days 21 and 42, construct mechanical properties and biochemical content were assessed. Microarray analysis was carried out on day 3, day 21 and day 42 constructs. 6 arrays.
Project description:Long-term dynamic compression enhanced the mechanical properties of MSC-seeded constructs only when loading was initiated after 21 days of chondrogenic differentiation. This study examined the molecular differences of chondrogenic MSCs compared to undifferentiated MSCs (TGF-beta vs no TGF-beta) and the effects of dynamic loading on MSC chondrogenesis (loading vs free-swelling). Free-swelling MSC-seeded constructs were cultured for 21 days in chemically defined media. Chondrogenesis was induced with TGF-beta3. Undifferentiated controls were maintained in parallel. After 21 days of chondrogenic differentiation, a subset of constructs were subjected to 21 days of dynamic compressive loading. On days 21 and 42, construct mechanical properties and biochemical content were assessed. Microarray analysis was carried out on day 3, day 21 and day 42 constructs. 6 arrays.
Project description:This study aimed to investigate the molecular mechanism responsible for primary open-angle glaucoma (POAG) progression. We analyzed microRNAs (miRNAs) expression profiling in aqueous humor (AH) of both POAG patients and normal controls, using a microarray-based approach. Subsequently, differentially expressed miRNAs (DEmiRNAs) were identified using Bayes moderated t-test. Next, DEmiRNAs target genes were predicted based on miRNA databases, followed by GO analysis and pathway analysis using DAVID. Furthermore, OAG-related genes analysis for target genes was carried out using CTD database, respectively. Finally, verification of DEmiRNAs expression levels was performed by RT-qPCR. A total of 40 significant DEmiRNAs were identified between control and POAG groups, including 24 up-regulated miRNAs and 16 down-regulated miRNAs. Further, the target genes of hsa-miR-206, including BMP2, SMAD4, ID2, and TNF, were mainly enriched in transforming growth factor-β (TGF-β) signaling pathway. While, target genes of hsa-miR-184, hsa-miR-34c-5p, hsa-miR-7-2-3p and hsa-miR-20b-3p, including BCL2, EPHB2, VEGFA, COL4A1, APC, and TGFBR1, were enriched in eye development. Moreover, FNDC3B, CAV2 and VEGF, target genes of hsa-miR-206 or hsa-miR-34c-5p, were the OAG-related genes. Ultimately, RT-qPCR analysis confirmed that mRNA levels of hsa-miR-206, hsa-miR-7-2-3p, and hsa-miR-20b-3p were increased, while those of hsa-miR-184 and hsa-miR-34c-5p were decreased in POAG compared with normal groups (P < 0.05). Hsa-miR-206, hsa-miR-184, hsa-miR-34c-5p, hsa-miR-7-2-3p and hsa-miR-20b-3p might play a significant role in the pathogenesis of POAG and hsa-miR-206 might be associated with the development of POAG by regulating TGF-β signaling pathway. These results might provide insight toward a better understanding of the pathogenesis of POAG. Overall design: Fifteen patients with ocular hypertensive POAG, who needed glaucoma filtrating surgeries, were enrolled in this studydisease group:PA group,PB group,PC group;each group of five patients). Fifteen patients with cataract undergoing cataract surgery were recruited as normal group (normal group:NA group,NB group,NC group;each group of five patients).
Project description:A combination therapy of electromagnetic fields (EMF) and simulated microgravity (SMG) has not been examined in regenerative medicine of cartilage. In the present study, a bioreactor system using extremely low-frequency EMF and SMG was applied during the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). It was hypothesized that a beneficial effect of EMF regarding chondrogenesis (COL2A) could be combined with an avoiding effect of SMG regarding hypertrophy (COLXA1) of cartilage. Pellet cultures of hMSCs formed cartilaginous tissue under the addition of growth factors (FGF; TGF-β3). Pure SMG reduced COLXA1 expression but also COL2A expression of hMSCs. Pure EMF showed no gene expression changes of hMSCs during chondrogenic differentiation. Combining EMF/SMG resulted in a re-increase of COL2A but did not reach control levels. The COL2A to COLXA1 ratio of combined EMF/SMG was not significantly different from control levels. The combination therapy of EMF/SMG did not significantly improve the chondrogenic potential of hMSCs. chondrogenic differentiation, electromagnetic stimulation-control, 1 timepoint with/without stimulation
Project description:Adiponectin is known as a key molecule to ameliorate symptoms of the type 2 diabetes mellitus and disorder of lipid metabolism. In this study, we investigated whether hot water extracts of some livestock by-products induced expression of adiponectin using 3T3-L1 adipocytes. Out of 11 extracts tested, pig testis extracts (PT) enhanced adiponectin mRNA expression and secretion of adiponectin protein from 3T3-L1 cells. Furthermore, simultaneous treatment with PT and daidzein, soy phytoestrogen, enhanced synergistically adiponectin secretion. Moreover, pretreatment with an estrogen receptor β (ERβ) antagonist (PHTPP) diminished adiponectin secretion in daidzein treated cell, but not in PT treated cells. Transcriptome analyses revealed that daidzein and PT commonly regulated PPAR signaling pathway, although gene expression was differently regulated in PT-treated cells and daidzein-treated cells. The expression of 476 and 380 genes significantly up-regulated in daidzein and PT treatment, although commonly regulated genes were only 86 genes. These results suggested that PT may ameliorate lipid metabolic dysfunction via promote adipocytes differentiation and enhance adiponectin secretion through different mechanism from daidzein. Overall design: 3T3-L1 cells were induced into differentiated adipocytes and then treated with daidzein (25μM), PT (100μg/ml), daidzein+PT or PBS for 7 days.
Project description:To explore the regulatory effect between miRNAs and mRNAs and the possibility of miRNAs as specific blood-based biomarkers in the developmental process of severe alopecia areata (AA), human peripheral blood samples from AA patients and healthy donors were obtained for analysis. we identified 36 significantly differentially expressed miRNAs in severe active AA patients. MiRNA target gene prediction and functional annotation analysis showed a significant enrichment in several pathways including the ribosome, cancer, cell cycle, insulin signaling, TGF-β signaling and p53 signaling pathways. Analysis of the three kinds of network showed that miR-185-5p, miR-125b-5p, and miR-186-5p might play important and synergistic roles in the developmental process of AA. According to the receiver operating characteristic curve analysis, several miRNAs were selected for the quantitative real-time PCR validation and further applied as biomarkers to classify severe active AA patients and healthy individuals. Among the miRNAs, hsa-miR-210 and hsa-miR-1246 had high prediction with high accuracy. Overall design: Ten blood samples were analyzed, including 5 severe active AA patients and 5 healthy controls.
Project description:Regulation of chondrogenic differentiation by DNA demethylation is little understood. The ten-eleven-translocation (TET) proteins oxidize methylated cytosines (5mC) to 5hmC, 5fC and 5caC eventually leading to DNA demethylation. However, 5hmC is stable and can potentially act as an epigenetic mark as well. In this study, we report that global changes in 5hmC mark chondrogenic differentiation. Overall design: Progenitor and differentiated chondrocyte gene expression patterns were examined to assess the changing expression profiles over the course of chondrogenesis.
Project description:Introduction: MicroRNAs (miRNAs) in circulation have emerged as promising biomarkers. In this study we aimed to identify a circulating miRNA signature for osteoarthritis (OA) patients. Methods: Serum samples were collected from 12 primary OA patients and 12 healthy individuals and were screened using the Agilent Human miRNA Microarray. Receiver Operating Characteristic (ROC) curves were constructed to evaluate the diagnostic performance of the deregulated miRNAs. Expression levels of selected miRNAs were validated by quantitative Real-time PCR (qRT-PCR) in all serum samples and in articular cartilage samples from OA patients (n=12) and healthy individuals (n=7). Bioinformatics analysis was used to investigate the involved pathways and target genes of the above miRNAs. Results: We identified 279 differentially expressed miRNAs in the serum of OA patients compared to healthy controls. 205 (73.5%) were up-regulated and 74 (26.5%) down-regulated. ROC analysis revealed that 77 miRNAs had area under the curve (AUC)> 0.8 and p<0.05. Bioinformatics analysis in 7 out of the 77 selected miRNAs (hsa-miR-33b-3p, hsa-miR-4284, hsa-miR-671-3p, hsa-miR-663a, hsa-miR-140-3p, hsa-miR-150-5p and hsa-miR-1233-3p) revealed that their target genes were involved in multiple signaling pathways, among which FOXO, mTOR, pI3K/akt, lipid metabolism and TGF-β. A serum miRNA signature including three down-regulated miRNAs (hsa-miR-33b-3p, hsa-miR-671-3p and hsa-miR-140-3p) were also verified by qRT-PCR in OA patients. Furthermore, we found that hsa-miR-140-3p, hsa-miR-671-3p and potentially hsa-miR-33b-3p expression levels were consistently down-regulated in articular cartilage of OA patients compared to healthy individuals. Conclusions: A global miRNA serum signature was revealed in OA patients. We identified a three- miRNA signature in peripheral serum which could be potential osteoarthritis biomarkers. Overall design: Serum samples were collected from 12 patients with primary OA (9 females, 3 males, ages: 69.83 ± 4.83 years) undergoing total knee replacement surgery at the Orthopaedics Department of the University Hospital of Larissa. Radiographs were obtained before surgery and graded using the Kellgren-Lawrence system according to the following criteria: grade 1 (doubtful narrowing of joint space and possible osteophytes), grade 2 (definite osteophytes and possible narrowing of joint space), grade 3 (moderate multiple osteophytes, definite narrowing of joint space and some sclerosis and possible deformity of bone ends) and grade 4 (large osteophytes, marked narrowing of joint space, severe sclerosis and definite deformity of bone ends). All OA patients had a Kellgren-Lawrence grade ≥3. The assessment of the radiographs by two independent expert observers was blinded. Patients with rheumatoid arthritis and other autoimmune disease as well as chondrodysplasias, infection-induced OA and post-traumatic OA were excluded from the study. As controls, serum samples were obtained from 12 healthy individuals (6 females, 6 males, ages: 64,25± 5,04 years) undergoing knee fracture repair surgery, with no history of joint disease, who did not show clinical manifestations compatible with OA when specifically explored by radiography. Consent was obtained from each participant. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the local ethical committee of the University Hospital of Larissa. Articular cartilage samples (n=12) were collected from femoral condyles and tibial plateaus of the same OA patients, while normal articular cartilage was obtained from 7 out of the 12 healthy individuals.
Project description:Post-embryonic development of the nematode C. elegans is governed by nutrient availability. L1-stage larvae remain in a state of developmental arrest after hatching until they feed. This “L1 arrest” (or "L1 diapause") is associated with increased stress resistance, supporting starvation survival. Loss of the transcription factor daf-16/FOXO, an effector of insulin/IGF signaling, results in arrest-defective and starvation-sensitive phenotypes. We show that daf-16/FOXO regulates L1 arrest cell-nonautonomously, suggesting that insulin/IGF signaling regulates at least one additional signaling pathway. We used mRNA-seq to identify candidate signaling molecules affected by daf-16/FOXO during L1 arrest. daf-16/FOXO had overlapping but distinct effects on gene expression in L1 arrest compared to daf-2/InsR adults. Notably, dbl-1/TGF-β, a ligand for the Sma/Mab pathway, and daf-36, which encodes an upstream component of the daf-12/NHR steroid hormone signaling pathway, were up-regulated during L1 arrest in a daf-16/FOXO mutant. Using genetic epistasis analysis, we show that dbl-1/TGF-β and daf-12/NHR steroid hormone signaling pathways are required for the daf-16/FOXO arrest-defective phenotype, suggesting that daf-16/FOXO represses dbl-1/TGF-β and daf-36. The dbl-1/TGF-β and daf-12/NHR pathways have not previously been shown to affect L1 development, but we found that disruption of these pathways delayed L1 development in fed larvae, consistent with these pathways promoting development in starved daf-16/FOXO mutants. Though the dbl-1/TGF-β and daf-12/NHR pathways are epistatic to daf-16/FOXO for the arrest-defective phenotype, disruption of these pathways does not suppress starvation sensitivity of daf-16/FOXO mutants. This observation uncouples starvation survival from developmental arrest, indicating that DAF-16/FOXO targets distinct effectors for each phenotype, and revealing that inappropriate development during starvation does not cause the early demise of daf-16/FOXO mutants. Overall, this study shows that daf-16/FOXO promotes developmental arrest cell-nonautonomously by repressing pathways that promote larval development. Overall design: Two biological replicates of arrested L1 daf-16(mgDf50) larvae. Compare to arrested N2 larvae from GEO accession GSE33023 (Accessions GSM818545, GSM818548, and GSM818552). We used the previously processed data in the analysis of the current study.