Downregulation of miR-34a Promotes Proliferation and Inhibits Apoptosis of Rat Osteoarthritic Cartilage Cells by Activating PI3K/Akt Pathway.
To elucidate the expression and function of miR-34a in rat osteoarthritic cartilage cells, and further to explore its mechanism.
Rat model of osteoarthritis was constructed and knee joint cartilage cells were isolated in vitro. Immunocytochemical staining was used for identification. qRT-PCR was used to detect the expression of miR-34a in cartilaginous tissues and cartilage cells. Cartilage cells were divided into blank control (BC), negative control (NC), miR-34a inhibitor (34aI), osteoarthritis model (OA), osteoarthritis model + negative control (OA + NC) and osteoarthritis model + miR-34a inhibitor (OA + 34aI) groups. Cell proliferation was detected by CCK-8 and colony formation assays. Cell apoptosis was studied by flow cytometry and Western blot. PI3K/AKT-pathway-related proteins were also analyzed by Western blot. To further validate the effect of miR-34a on the PI3K/Akt pathway, the cartilage cells were divided into blank control (BC), osteoarthritis model (OA), osteoarthritis model + miR-34a inhibitor (OA + 34aI), osteoarthritis model + PI3K activator (OA + IGF-1) and osteoarthritis model + miR-34a inhibitor + PI3K inhibitor (OA + 34aI + LY) groups, the experiments above were repeated.
The expression of miR-34a in cartilaginous tissues and cells of osteoarthritis model was significantly higher than that in normal (p < 0.05). After silencing miR-34a gene, the cell proliferation and proteins expression of PI3K/Akt pathway were increased, while the apoptosis rate and expression of apoptosis-related proteins were decreased. Addition of PI3K activator also evidently promoted proliferation and inhibited apoptosis. The protein expression of Bax, Cleaved caspase-3 and Cleaved caspase-9 were dramatically decreased, while the ratios of p-PI3K/PI3K and p-Akt/Akt were increased in OA + IGF-1 group.
Downregulation of miR-34a regulated proliferation and apoptosis of cartilage cells by activating PI3K/Akt pathway, providing a potential therapeutic approach for the treatment of osteoarthritis.
Tao H
，Cheng L
，Yang R
《Clinical Interventions in Aging》
MiR-34a Enhances Chondrocyte Apoptosis, Senescence and Facilitates Development of Osteoarthritis by Targeting DLL1 and Regulating PI3K/AKT Pathway.
Osteoarthritis (OA) is the prevalent degenerative disease caused by various factors. MicroRNAs are important regulators in the inflammation and immune response. The aim of this study was to investigate the effect of microRNA-34a (MiR-34a) on the death of chondrocytes, senescence, as well as its role in OA progression.
A series of experiments involving CCK-8, flow cytometry, β-galactosidase staining and wound healing assays were conducted to determine the cellular capabilities of proliferation, cell apoptosis, senescence and the ability of cells to recover from injury, respectively. Binding sites between miR-34a and delta-like protein 1 (DLL1) were identified using a luciferase reporter system, whereas mRNA and protein expression of target genes was determined by RT-PCR and immunoblot, respectively. OA model was generated via surgery.
We found that miR-34a expression was increased in the cartilage of OA patients. In rat chondrocytes and chondrosarcoma cells, miR-34a transfections noticeably inhibited the expression of DLL1, triggered cell death and senescence, suppressed proliferation, and prevented scratch assay wound closure. However, transfection of a miR-34a inhibitor displayed adverse effects. Additionally, secretion and expression of factors associated with cartilage degeneration were altered via miR-34a. Moreover, miR-34a directly inhibits DLL1 mRNA. Furthermore, concentrations of DLL1, total PI3K, and p-AKT declined in chondrocytes that overexpress miR-34a. DLL1 overexpression elevated PI3K and p-AKT levels, and eliminated cell death triggered by a miR-34a mimic. In vivo, miR-34a remarkably inhibited miR-34a up-regulation, while enhanced the level of DLL1 expression. In the knee joints of surgery-induced OA rats, articular chondrocyte death and loss of cartilage were attenuated via miR-34a antagomir injection.
These findings indicate that miR-34a contributes to chondrocyte death, causing OA progression through DLL1 and modulation of the PI3K/AKT pathway.
Zhang W
，Hsu P
，Zhong B
，Guo S
，Zhang C
，Wang Y
，Luo C
，Zhan Y
，Zhang C
... -
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microRNA-206 is required for osteoarthritis development through its effect on apoptosis and autophagy of articular chondrocytes via modulating the phosphoinositide 3-kinase/protein kinase B-mTOR pathway by targeting insulin-like growth factor-1.
microRNA (miR) has been shown to be involved in the treatment of diseases such as osteoarthritis (OA). This study aims to investigate the role of miR-206 in regulating insulin-like growth factor-1 (IGF-1) in chondrocyte autophagy and apoptosis in an OA rat model via the phosphoinositide 3-kinase (P13K)/protein kinase B (AKT)-mechanistic target of rapamycin (mTOR) signaling pathway. Wistar rats were used to establish the OA rat model, followed by the observation of histopathological changes, Mankin score, and the detection of IGF-1-positive expression and tissue apoptosis. The underlying regulatory mechanisms of miR-206 were analyzed in concert with treatment by an miR-206 mimic, an miR-206 inhibitor, or small interfering RNA against IGF-1 in chondrocytes isolated from OA rats. Then, the expression of miR-206, IGF-1, and related factors in the signaling pathway, cell cycle, and apoptosis, as well as inflammatory factors, were determined. Subsequently, chondrocyte proliferation, cell cycle distribution, apoptosis, autophagy, and autolysosome were measured. OA articular cartilage tissue exhibited a higher Mankin score, promoted cell apoptotic rate, increased expression of IGF-1, Beclin1, light chain 3 (LC3), Unc-51-like autophagy activating kinase 1 (ULK1), autophagy-related 5 (Atg5), caspase-3, and Bax, yet exhibited decreased expression of miR-206, P13K, AKT, mTOR, and Bcl-2. Besides, miR-206 downregulated the expression of IGF-1 and activated the P13K/AKT signaling pathway. Moreover, miR-206 overexpression and IGF-1 silencing inhibited the interleukins levels (IL-6, IL-17, and IL-18), cell apoptotic rate, the formation of autolysosome, and cell autophagy while promoting the expression of IL-1β and cell proliferation. The findings from our study provide a basis for the efficient treatment of OA by investigating the inhibitory effects of miR-206 on autophagy and apoptosis of articular cartilage in OA via activating the IGF-1-mediated PI3K/AKT-mTOR signaling pathway.
Yu Q
，Zhao B
，He Q
，Zhang Y
，Peng XB
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ResearchGate
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钛学术
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