LncRNA PVT1 Regulates Proliferation And Apoptosis In Chondrocytes Of Osteoarthritis By Targeting MicroRNA-497/AKT3 Axis

Osteoarthritis(OA)is a very common diereses,also known as a degenerative joint disease.Degeneration of articular cartilage is the main pathological feature of OA.Thickening of subchondral bone and osteophyte formation are also commonly seen in OA tissues.Extracellular matrix degradation is a complex biological process.The imbalance of chondrocyte proliferation and apoptosis as well as the imbalance of anabolism and catabolism of the extracellular matrix can cause progressive destruction of articular cartilage and cartilage loss,ultimately leading to the development of osteoarthritis.The progression of osteoarthritis is closely related to joint injury,decreased immune function,and inflammatory response.However,the specific molecular mechanisms by which articular chondrocytes and extracellular matrix maintain balance have not been unfolded so far,which greatly limits the diagnosis and treatment of osteoarthritis.The human transcriptome includes not only messenger ribonucleic acids(m RNAs)that are involved encoding proteins,but also a large number of transcripts that are related to encoding proteins including micro RNAs(miRNAs)and long non-coding RNAs(lncRNAs).These non-coding RNAs have been said to play vital regulatory roles in a variety of physiological and pathological things to do such as boom and development,cell proliferation and apoptosis,stem cell differentiation,and tumor formation.Previous research have proven that lncRNAPVT1 is differentially expressed in osteoarthritis sufferers and healthful people,and is related with chondrocyte damage in osteoarthritis,suggesting that lncRNA PVT1 is anticipated to be an necessary goal for osteoarthritis treatment,however there are nevertheless many unknowns about the molecular mechanisms worried that stay to be in addition explored.Therefore,extra research are integral to similarly apprehend the mechanism of lncRNAPVT1 in OA progression.In this study,we examined lncRNAPVT1 expression in OA articular cartilage tissue.Chondrocytes had been triggered the use of IL-1β to assemble a cell model of OA.Downstream target genes(micro RNAs and m RNAs)of lncRNAPVT1 had been recognized via database prediction and twin luciferin reporter assays.Cell proliferation and apoptosis and chondrocyte extracellular matrix-associated component(aggrecan,collagen Type II,and MMP-9)modifications had been consequently assessed after knockdown or elevation of PVT1,micro RNA-497,and AKT3 treatments.PART ⅠObjective: To investigate the expression of lncRNAPVT1 in osteoarthritic cartilage tissue and chondrocytes and its effects on chondrocyte proliferation,apoptosis and extracellular matrix.Methods: The expression of lncRNAPVT1 in cartilage tissues of OA patients and non-OA patients was detected by qRT-PCR,and the expression of lncRNA PVT1 in chondrocytes before and after IL-1β induction was also detected by qRT-PCR.Effect of chondrocyte proliferation before and after knockdown of lncRNA PVT1 by MTT assay.Apoptosis assay flow cytometry was performed to detect the effect of chondrocyte apoptosis before and after knockdown of lncRNA PVT1.Westernblot was used to detect extracellular matrix-related gene changes in chondrocytes before and after knockdown of lncRNAPVT1.Results:(1)The expression of lncRNAPVT1 was increased in osteoarthritic cartilage tissues,and the expression of lncRNAPVT1 was increased in chondrocytes after IL-1β induction.(2)After knockdown of lncRNAPVT1 in IL-1β-induced chondrocytes,the cell proliferation ability was enhanced and the apoptosis rate was decreased.Knockdown of lncRNAPVT1 promoted IL-1 β-induced proliferation of chondrocytes and inhibited apoptosis.(3)Aggrecan and collagen Type II decreased and MMP-9 increased in chondrocytes after induction with IL-1β,while knockdown of PVT1 decreased the effect of IL-1β on the three proteins in chondrocytes.Knockdown of lncRNAPVT1 inhibited IL-1β-induced extracellular matrix degradation in chondrocytes.Conclusion: Lnc RNAPVT1 is closely related to the occurrence and development of osteoarthritis and can inhibit the progression of osteoarthritis by down-regulating the expression of lncRNA PVT1.PART ⅡObjective: To investigate the expression of micro RNA-497 in osteoarthritic cartilage and chondrocytes and its effects on chondrocyte proliferation,apoptosis and extracellular matrix.Methods: The expression of micro RNA-497 in chondrocytes before and after IL-1β induction was detected by qRT-PCR.The effect of micro RNA-497 or negative control on chondrocyte proliferation and apoptosis was examined by MTT assay and apoptosis assay.Westernblot was used to detect extracellular matrix-related gene changes in chondrocytes by micro RNA-497 or negative control.Database prediction,dual-luciferase activity assay to validate the regulatory relationship between AKT and micro RNA-497.Assessment,IL-1β + miR-497-mimics group,IL-1 β + miR-497-mimics + sh-AKT3 cell proliferation and apoptosis,and chondrocyte extracellular matrix-associated factor(aggrecan,collagen Type II,and MMP-9)changes.Results:(1)The expression of micro RNA-497 used to be lowered in chondrocytes prompted by means of IL-1β.(2)Micro RNA-497 promotes chondrocyte proliferation,inhibits chondrocyte apoptosis,and inhibits extracellular matrix degradation in osteoarthritis.(3)AKT is a micro RNA-497 target gene.(4)The effects of overexpression of micro RNA-497 on IL-1β-induced chondrocyte proliferation,apoptosis,and extracellular matrix-associated molecules could be reversed by overexpressed AKT3.Conclusion: Micro RNA-497 is closely related to the occurrence and development of osteoarthritis,and micro RNA-497 can inhibit the progression of osteoarthritis by negatively regulating the expression of AKT.PART ⅢObjective:To inspect the interplay between lncRNAPVT1 and micro RNA-497/AKT3 axis in the improvement of osteoarthritis.Methods: Database prediction and dual-luciferase activity assay have been used to affirm the regulatory relationship between micro RNA-497 and lncRNAPVT1.MTT assay and apoptosis assay Western blot assay had been used to inspect the results of transfection with miR-497-inhibitor or transfection with sh-AKT3 on IL-1β-induced chondrocyte proliferation,apoptosis and extracellular matrix-related molecules:(1)Database prediction and dual-luciferase activity assay established that micro RNA-497 used to be a target gene of lncRNAPVT1.(2)lncRNAPVT1 can target and inhibit micro RNA-497 expression in OA chondrocytes.(3)miR-497-inhibitor or sh-AKT3 reversed the effects of si-PVT1 on IL-1β-induced chondrocyte proliferation,apoptosis,and extracellular matrix.Conclusion: The function of lncRNAPVT1 in regulating chondrocytes depends on the regulation of miR-497/AKT3 axis.Conclusions(1)The expression of lncRNAPVT1 was increased in cartilage tissues and cells of osteoarthritis,and after knockdown the expression of lncRNAPVT1 can promote proliferation,anti-apoptosis and anti-cartilage matrix decomposition in chondrocytes.(2)Upregulated the expression of micro RNA-497 can promote proliferation,anti-apoptosis and anti-cartilage matrix decomposition in chondrocytes by negatively regulating the expression of AKT3.(3)The regulation of chondrocyte function by lncRNAPVT1 depends on the regulation of miR-497/ AKT3 axis.