ABSTRACT
Objective@#To investigate the role of adenovirus type 36 (Ad36) in the browning of 3T3-L1 cells.@*Methods@#BODIPY staining was performed on the 0, 2nd, 4th, 6th and 8th days of the cocktail induction (control) group and the cocktail plus Ad36 induction (experimental) group to observe the adipogenesis of 3T3-L1 cells.The mRNA and protein expressions of uncoupling protein-1(Ucp1), ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit (Atp5o), cytochrome c oxidase subunit 5B(Cox5b), and perilipin were detected by real-time PCR and Western-blot.@*Results@#The results of BODIPY staining showed that the lipid droplets in the control group gradually became larger with the differentiation of the cells, while the lipid droplets in the experimental group firstly became larger, and then appeared smaller after Ad36 was added on the fourth day. Compared with the control group, the mRNA and protein expression levels of Ucp1, Atp5o, and Cox5b in the experimental group were significantly increased while the expression level of perilipin was significantly decreased (all P<0.05).@*Conclusion@#During the differentiation of 3T3-L1 cells, Ad36 promotes its browning via increasing the expressions of Ucp1, Atp5o, and Cox5b.
ABSTRACT
To explore the effects of miR-101-3p on IL-1β-induced chondrocyte injury and its underlying mechanisms. Methods: Chondrocytes were divided into 4 groups: a control group (NC group), a IL-1β group, a negative control group (IL-1β+miR-NC group), and a miR-101-3p group (IL-1β+miR-101-3p group), which were treated with IL-1β after transfecting with miR-101-3p mimic or negative mimic. The expressions of miR-101-3p-5p and stanniocalcin 1 (STC1) at different concentrations of IL-1β (1, 5, 10 ng/mL)-induced chondrocytes were detected by Western blotting and real-time PCR. MTT assay was used to detect cell proliferation rate, while caspases assay kits and flow cytometry were used to measure the cell caspase and apoptosis level. Western blotting assay was used to detect the expression levels of pro-inflammatory and ECM-related protein, such as matrix metalloproteinase 9 (MMP9) and collagen Type II. In addition, 3'-untranslated regions (UTR) of wild-type STC1 (STC1-3'-UTR-WT) or 3'-UTR of mutant STC1 (STC1-3'-UTR-MUT) were co-transfected with miR-101-3p mimic or miR-NC, respectively, while luciferase reporter assay was used to examine the regulative role of miR-101-3p in STC1. In order to detect whether STC1 was involved in the effect of miR-101-3p on chondrocytes, miR-NC (miR-NC group), miR-101-3p (miR-101-3p group), anti-NC (anti-NC group) and anti-miR-101-3p (anti-miR-101-3p group) were respectively transfected into the cells, and the expression of STC1 protein was detected by Western blotting. Subsequently, the cells were randomly divided into a miR-101-3P group (IL-1β+miR-101-3p group), an over-expression control group (IL-1β+miR-101-3p+ad-GFP group), and an over-expression STC1 group (IL-1β+miR-101-3p+ad-STC1 group) to investigate whether STC1 was involved in the role of miR-101-3p in chondrocyte. Similarly, MTT assay was used to detect cell proliferation rate, caspases assay kits and flow cytometry were used to measure the cell caspase and apoptosis level. Western blotting assay was used to detect the expression levels of pro-inflammatory and ECM-related protein MMP9 and collagen Type II. Results: Compared with the 0 ng/mL IL-1β, the expression of miR-101-3p was decreased in chondrocyte at different concentration of IL-1β (1, 5, 10 ng/mL) (all P<0.05), while the level of STC1 was increased (P<0.05). Compared with the NC group, the chondrocyte proliferation rate was down-regulated (P<0.05), while the apoptosis rate, the levels of caspases, IL-6 and TNF-α were increased in the IL-1β group (P<0.05). Moreover, the MMP9 levels were increased obviously, and the protein levels of collagen Type II were decreased in the IL-1β group compared with the NC group (both P<0.05). Compared with the IL-1β+miR-NC group, the proliferation rate was increased (P<0.05), whereas the apoptosis rates, the caspase-3/9 levels, the IL-6 and TNF-α levels were increased in the IL-1β+miR-101-3p group (all P<0.05). Then MMP9 levels were decreased obviously (P<0.05), and the protein levels of collagen Type II were increased in IL-1β+miR-101-3p group compared with the IL-1β+miR-NC group (both P<0.05). In addition, the double luciferase assay showed that the STC1 levels could be inhibited in the miR-101-3p group compared with the miR-NC group (P<0.05). STC1 levels were decreased in the miR-101-3p group compared with the miR-NC group (P<0.05), and the STC1 levels were increased in the anti-miR-101-3p group compared with those in the anti-NC group (P<0.05). The results of miR-101-3p+ad-STC1 group showed that compared with the miR-101-3p+ad-GFP group, the STC1 could reverse the effects of miR-101-3p on IL-1β-induced proliferation, apoptosis, inflammatory responses and ECM protein of chondrocytes. Conclusion: The regulation of miR-101-3p/STC1 signal pathway may have a role in reducing the IL-1β-induced chondrocyte injury.
Subject(s)
Cell Proliferation , Chondrocytes , Glycoproteins , Metabolism , Interleukin-1beta , Metabolism , MicroRNAsABSTRACT
Objective To investigate the role of adenovirus type 36 ( Ad36) in the browning of 3T3-L1 cells. Methods BODIPY staining was performed on the 0, 2nd, 4th, 6th and 8th days of the cocktail induction (control) group and the cocktail plus Ad36 induction ( experimental) group to observe the adipogenesis of 3T3-L1 cells. The mRNA and protein expressions of uncoupling protein-1 ( Ucp1 ), ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit ( Atp5o), cytochrome c oxidase subunit 5B ( Cox5b), and perilipin were detected by real-time PCR and Western-blot. Results The results of BODIPY staining showed that the lipid droplets in the control group gradually became larger with the differentiation of the cells, while the lipid droplets in the experimental group firstly became larger, and then appeared smaller after Ad36 was added on the fourth day. Compared with the control group, the mRNA and protein expression levels of Ucp1, Atp5o, and Cox5b in the experimental group were significantly increased while the expression level of perilipin was significantly decreased ( all P＜0. 05 ). Conclusion During the differentiation of 3T3-L1 cells, Ad36 promotes its browning via increasing the expressions of Ucp1, Atp5o, and Cox5b.
ABSTRACT
Objective To investigate the role of LncRNA ROR in Ad36-induced browning of human adipose-derived stem cells(hADSC). Methods After hADSC was induced by cocktail and Ad36 for 2,4,6,and 8 days,Oil red O staining was performed for observing the adipogenic status. The mRNA expressions of LncRNA ROR, uncoupling protein 1(UCP1),and PRDM16 were detected by real-time PCR and the protein expressions of UCP1 and PRDM16 were detected by Western-blot. After LncRNA ROR was knocked down by siRNA, UCP1 and PRDM16 mRNA and protein expression levels in the process of Ad36-induced adipocyte differentiation were detected by real-time PCR and Western-blot. Results Oil red O staining showed that fat droplets in the cocktail-induced group were larger than those in the Ad36-induced group. Compared with the cocktail group,the mRNA expressions of LncRNA ROR, UCP1 and PRDM16, and the protein expressions of UCP1 and PRDM16 in Ad36 group were significantly increased(P<0.05). The mRNA and protein expressions of UCP1 and PRDM16 in LncRNA ROR knockdown group were significantly lower than those in control group(P<0.05). Conclusion In the process of Ad36-induced hADSC differentiation,the up-regulation of LncRNA ROR may stimulate UCP1 and PRDM16 expression,and thus promote the browning of hADSC.
ABSTRACT
Objective To investigate the expressions of RNA-binding protein human antigen R(HuR), fatty acid binding protein type 4(FABP4),fatty acid synthetase(FASN),and lipoprotein lipase(LPL)during the differentiation of human adipocytes, and to explore their possible roles. Methods Human adipose-derived mesenchymal stem cells were induced by adipogenic differentiation,and the adipogenesis of cells was observed by oil red O staining. The expressions of HuR,FABP4,FASN,and LPL mRNA and protein were detected by real-time PCR and Western blotting. After HuR was silenced by siRNA, the change of adipogenesis for human adipose-derived mesenchymal stem cells was observed and the expressions of adipogenic genes were detected. Results The expressions of HuR,FABP4,FASN,and LPL mRNA and protein were significantly increased after human adipose-derived mesenchymal stem cells were induced to differentiate into adipocytes(all P<0.01). After HuR expression was down-regulated by siRNA,the adipogenic level of human adipose-derived mesenchymal stem cells was reduced,with decreased protein levels of FABP4,FASN,and LPL(all P<0.05),which were without changes for their mRNA levels. Conclusion HuR promotes the differentiation of human adipocytes mainly via regulating the changes of FABP4,FASN,and LPL protein levels.