Conclusion
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.
Material and methods
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.
Methods
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.
Objective
To elucidate the expression and function of miR-34a in rat osteoarthritic cartilage cells, and further to explore its mechanism. Material and
Results
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.