Transient transfection of C28/I2 chondrocytes; the human EST DA125942 (CISTR-ACT's lncRNA) was overexpressed and siRNA-depleted
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE39896 | GEO | 2012/08/07
SECONDARY ACCESSION(S): PRJNA172001
REPOSITORIES: GEO
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Project description:Purpose This in vitro study was designed to determine the effect of the pan-Janus kinase inhibitor, Tofacitinib, on basal and interleukin-6 (IL-6)-induced signal transducers and activators of transcription (STAT) phosphorylation and matrix metalloproteinase (MMP) gene expression and MMP production by C28/I2 human chondrocytes. Methods C28/I2 chondrocytes were grown to a confluent high-density and treated either with recombinant human IL-6 (rhIL-6; 10–20ng/mL) or maintained in the basal state for up to 60 min. MMP gene expression was determined using RT-PCR and MMP production by semi-quantitative immunohistochemistry. The effect of IL-6 with or without Tofacitinib on activation of STAT proteins was determined from quantitative Western blots. Results C28/I2 chondrocytes produced STAT1, STAT3 and STAT5AB which were phosphorylated (p) following treatment with rhIL-6 for 30 min. Tofacitinib (2.5nM–100nM) decreased rhIL-6-induced activation of STAT1, STAT3, and STAT5AB as well as decreasing the expression of MMP3 and MMP13 but not MMP9, MMP1 or MMP2. In addition, Tofacitinib (50nM) reduced the number of rhIL-6-induced MMP3-, and MMP13- antibody-positive C28/I2 chondrocytes. However, Tofacitinib did decrease the number of MMP9-antibody-positive C28/I2 chondrocytes. Conclusion Taken together, these data showed that Tofacitinib, a pan-JAK small molecule inhibitor employed for the medical therapy of rheumatoid arthritis was a potent inhibitor of rhIL-6-induced STAT phosphorylation that appeared to be coupled to the inhibition of MMP-3, -9 and -13 production by C28/I2 chondrocytes.
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Project description:FABP4 has been reported to trigger ADAMTS4 overexpression in chondrocytes. Here we described the transcriptional changes of FABP4-treatment.
2023-06-22 | GSE227483 | GEO
Project description:ObjectiveOsteoarthritis (OA) is a degenerative joint disease characterized by deterioration of articular cartilage functions. Previous studies have confirmed the role of circular RNAs (circRNAs) in OA, but the role of mechanical stress-related circRNA (circRNA-MSR) in OA is unknown.DesignThe human chondrocytes C28/I2 were cultured and treated with lipopolysaccharide (LPS) to establish the OA model. The mRNA and protein levels were measured by qRT-PCR or Western blot. Cell viability was analyzed by MTT assay. Flow cytometry was carried out to detect cell apoptosis. The levels of TNF-α, IL-1β, and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA). Pull-down assay was conducted to measure circRNA-MSR-related miRNA. Dual-luciferase reporter gene detection was performed to detect the target relationships between miR-643 and circRNA-MSR or Mitogen-activated protein kinase kinase 6 (MAP2K6). The RNA-fluorescence in situ hybridization (RNA-FISH) assay was conducted to verify the localization of circRNA-MSR and miR-643.ResultsThe expressions of circRNA-MSR were upregulated in LPS stimulated C28/I2 cells. Knockdown of circRNA-MSR can inhibit LPS-induced apoptosis, inflammatory response, and extracellular matrix (ECM) degradation, and promote cell C28/I2 cells proliferation. Moreover, circRNA-MSR directly targeted miR-643. RNA-FISH exhibited that circRNA-MSR may act as a competing endogenous RNA (ceRNA) of miR-643. Over-expression of miR-643 could alleviate LPS-induced C28/I2 chondrocyte injury and promote cell proliferation. Besides, miR-643 directly bound to MAP2K6 mRNA. MiR-643 inhibition or MAP2K6 overexpression can reverse the role of circRNA-MSR knockdown on LPS-treated chondrocytes.ConclusioncircRNA-MSR can upregulate MAP2K6 by targeting miR-643, thereby inhibiting cell proliferation and promoting apoptosis of C28/I2 cells.
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Project description:In order to study the role of piR_019914 in C28/I2 cells, we established piR_019914_mimics to overexpress piR_019914 in C28/I2 cells We then performed gene expression profiling analysis using data obtained from RNA-seq of piR_019914 overexpression C28/I2 cells and mimics_NC C28/I2 cells.
2023-03-27 | GSE227945 | GEO
Project description:Here we described the transcriptional differences of chondrocytes in monolayer and spheroid cultures.
2023-04-18 | GSE224598 | GEO
Project description:We report here the overexpression of DPP4 in C28/I2 chondrocyte cells to investigate the changes induced by elevated levels of this gene. DPP4 is known as a marker of cellular senescence, yet its regulatory mechanisms remain unclear. In this study, we utilized the C28/I2 cell line to overexpress DPP4 and analyzed the resultant effects on gene expression and cellular behavior. Our findings reveal that DPP4 overexpression influences several pathways associated with chondrocyte function and senescence. Specifically, we observed alterations in pathways related to extracellular matrix organization, inflammatory response, and cellular aging. These results suggest that DPP4 plays a significant role in the modulation of chondrocyte senescence and may provide new insights into the mechanisms of cellular aging. Taken together, our data provide a molecular framework for understanding how DPP4 overexpression impacts chondrocyte physiology and offers potential targets for therapeutic intervention in age-related cartilage diseases.
2025-02-19 | GSE273261 | GEO
Project description:SDF-1 has been reported to trigger ADAMTS4,5 overexpression through activating CXCR4 signaling in chondrocytes. Here we described the transcriptional changes of SDF-1-treatment as well as natural products CXCR4 antagonists treatment.
2022-03-09 | GSE197921 | GEO
Project description:the objective of this study was to analyze the role of NGF and BDNF in the inflammatory process of OA and their effects on chondrogenesis, chondrocyte differentiation, cartilage degeneration and pain
2021-01-02 | GSE142823 | GEO
Project description:The development of genetic manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not been established for Babesia microti. Here, we report the first successful transient transfection of B. microti. The plasmids containing the firefly luciferase reporter gene were transfected into B. microti by an AMAXA 4D Nucleofection system. Twenty-four-hour synchronization, the 5'-actin promoter, program FA100, and 50 ?g of plasmid DNA constituted the best conditions for the transient transfection of B. microti. This finding is the first step towards a stable transfection method for B. microti, which may contribute to a better understanding of the biology of the parasite.
| S-EPMC7169379 | biostudies-literature
Project description:BackgroundCalcium phosphate mediated transfection has been used for delivering DNA into mammalian cells in excess of 30 years due to its most low cost for introducing recombinant DNA into culture cells. However, multiple factors affecting the transfect efficiency are commonly recognized meanwhile for years, the low transfection efficiency of this approach on higher differentiated and non-tumor cells such as CHO and C2C12 limits its application on research.ResultsIn this paper, we systematically evaluated the possible factors affecting the transfection rate of this approach. Two categories, calcium phosphate-DNA co-precipitation and on-cell treatments were set for optimization of plasmid DNA transfection into CHO and C2C12 cell-lines. Throughout experimentation of these categories such as buffer system, transfection media and time, glycerol shocking and so on, we optimized the best procedure to obtain the highest efficiency ultimately. During calcium phosphate DNA-precipitation, the transfection buffer is critical condition optimized with HBS at pH 7.10 (P = 0.013 compared to HEPES in CHO). In the transfection step, FBS is a necessary component in transfection DMEM for high efficiency (P = 0.0005 compared to DMEM alone), and high concentration of co-precipitated particles applied to cultured cells in combination with intermittent vortexing is also crucial to preserve the efficiency. For 6-well culture plates, 800 µl of co-precipitated particles (11.25 µg/mL of cDNA) in 1 well is the optimal (P = 0.007 compared to 200 µl). For the highest transfection efficiency, the most important condition is glycerol in shock treatment (P = 0.002 compared to no shock treatment in CHO, and P = 0.008 compared to no shock treatment in C2C12) after a 6 h incubation (P = 0.004 compared to 16 h in CHO, and P = 0.039 compared to 16 h in C2C12) on cultured cells.ConclusionsCalcium phosphate mediated transfection is the most low-cost approach to introduce recombinant DNA into culture cells. However, the utility of this procedure is limited in highly-differentiated cells. Here we describe the specific HBS-buffered saline, PH, glycerol shock, vortex strength, transfection medium, and particle concentrations conditions necessary to optimize this transfection method in highly differentiated cells.
| S-EPMC5372392 | biostudies-literature