| Part 1 Study on the role of lncRNA MEG3 in animal model of rat osteoarthritisObjective:Osteoarthritis(OA)is a chronic joint disease and there is currently no definitive treatment.Long non-coding RNA(lncRNA)has been shown to play an important role in the development of OA.However,its specific mechanism of action has not yet been clarified.Methods:Rat OA model and interleukin-1?(IL-1?)-induced rat chondrocytes were constructed.Plasmid construction(MEG3 expression plasmid and sh-MEG3)and lentivirus packaging.RT-qPCR was used to detect the expression pattern of lncRNA MEG3 in cartilage tissue of rat OA model.And divided into pcDNA,meg3,si-nc and si-meg3 groups.The expression of MEG3 and knockdown in chondrocytes were detected,and the expression of MEG3 after overexpression was detected.The effect of MEG3 on IL-1?-induced chondrocytes was evaluated based on cell viability and apoptosis.Including MEG3 knockdown and overexpression effects on chondrocyte proliferation and apoptosis.The effect of knockdown and overexpression of MEG3 on the expression of Bax and Bcl2 was detected by Western blot.Results:(1)Expression changes of MEG3 in osteoarthritis rat models and chondrocytes:From the data,it was found that cartilage in rat OA model(n=10)was compared with that of cartilage tissue of pseudogroup(n=10).The expression of MEG3 was significantly reduced in the tissues.(2)Effects of MEG3 on proliferation of chondrocytes:effects of MEG3 proliferation and apoptosis of chondrocytes induced by IL-1? Experiments using a gain function by using MEG3(SI-MEG3)and MEG3 overexpression vectors(small interferences)Loss of RNA Transfection and Exploration of MEG3).The incorporation of MEG3 or SI-MEG3 significantly enhanced or inhibited MEG3 expression compared to the corresponding negative control(pcDNA or SI-NC)(P<0.05).In addition,cell viability assay showed that knockdown of MEG3 significantly inhibited cell proliferation,whereas upregulation of MEG3 significantly promoted IL-1?-induced chondrocyte proliferation(P<0.05).(3)Effect of MEG3 on chondrocytes apoptosis:The effect of MEG3 on apoptosis of chondrocytes induced by IL-1? was evaluated by flow cytometry and Western blot analysis.Data showed that IL-1? triggered chondrocyte apoptosis,MEG3 downregulation.,the IL-1?-induced chondrocyte apoptosis was significantly promoted,while MEG3 upregulation showed the opposite effect(P<0.05).Western blotting assays showed that MEG3 knockdown resulted in promotion of apoptosis,and MEG3 overexpression resulted in the inhibition of apoptosis,which was shown by changes in the expression of apoptosis-related proteins Bax protein and Bcl2.Conclusions:(1)MEG3 expression was significantly reduced in cartilage tissue of rat OA model(n=10).(2)MEG3 knockdown significantly inhibited cell proliferation,while MEG3 upregulation significantly promoted IL-1?-induced chondrocyte proliferation.(3)Overexpression of MEG3 promotes the proliferation of chondrocytes induced by IL-1? and inhibits apoptosis.These results indicate that MEG3 may play an important role in the progression of OA.In conclusion,LncRNA MEG3 is closely related to the occurrence and development of osteoarthritis in rats,suggesting that the expression of MEG3 may be overexpressed,thereby inhibiting the effect of arthritis in rats.Part 2 The role of miR-16/SMAD7 signaling pathway in animal models of rat OsteoarthritisObjective:Osteoarthritis(OA)is a chronic joint disease and there is currently no definitive treatment.Micro-RNA has been confirmed to play an important role in the development of OA.However,its specific mechanism of action has not yet been clarified,especially the miR-16/SMAD7 signaling pathway.Methods:Rat OA model and interleukin-1?(IL-1?)-induced rat chondrocytes were constructed.Plasmid construction(miR-16,anti-miR-16,SMAD7 WT,SMAD7 MUT)and lentivirus packaging.RT-qPCR was used to detect the expression pattern of miR-16 in cartilage tissue of rat OA model.And divided into mir-nc,mir-16,ANTI-MIR-NC with anti-mir-16 Group.The effect of miR-16 on the proliferation or apoptosis of osteoblasts in chondrocytes was detected.The effect of miR-16 on IL-1?-induced chondrocytes was evaluated based on cell viability and apoptosis.The effect of miR-16 on SMAD7 expression was detected by Western blot.The luciferase reporter reaction was used to detect the interaction of miR-16 and SMAD7.Results:(1)Expression changes of miR-16 in rat osteoarthritis models and chondrocytes:The role of miR-16 in OA was evaluated,and miR-16 expression patterns were detected in cartilage tissues of rat OA models.Compared with the sham group,the expression of miR-16 in the cartilage tissue of the rat DMM model was significantly increased(P<0.05).Subsequently,miR-16 mimics and anti-miR-16 were synthesized and then transfected into IL-1?-induced chondrocytes to check their efficiency.By transfecting miR-16 mimics or anti-miR-16,respectively,the expression of miR-16 was significantly increased or decreased(P<0.05).(2)Effect of miR-16 on chondrocytes proliferation:miR-16 consumption significantly inhibited proliferation of chondrocytes induced by IL-1?(P<0.05).On the other hand,miR-16 overexpression significantly inhibited cell proliferation(P<0.05).Overexpression of miR-16 inhibits proliferation of chondrocytes induced by IL-1?.These results suggest that miR-16 may be closely related to the development and progression of OA.(3)Effect of miR-16 on apoptosis of chondrocytes:The effect of miR-16 on apoptosis of chondrocytes induced by IL-1? was investigated using miR-16 mimics and anti-miR-16.These data reveal that miR-16 depletion significantly inhibits apoptosis of chondrocytes induced by IL-1?(P<0.05).On the other hand,miR-16 overexpression significantly promoted apoptosis(P<0.05).In conclusion,overexpression of miR-16 promotes apoptosis of chondrocytes induced by IL-1?.These results suggest that miR-16 may be closely related to the development and progression of OA.(4)SMAD7 is the target gene of miR-16:using online software Target Scan to search for the endogenous target gene of miR-16.The 3'-UTR of miR-16 and SMAD7 were found to have some complementary bases,suggesting that SMAD7 may be the target gene for miR-16.Therefore,a wild-type SMAD7 luciferase vector(SMAD7-WT)and a mutant SMAD7 luciferase vector(SMAD7-MUT)were constructed and introduced into HEK293T cells to verify the interaction between miR-16 and SMAD7.By introducing the miR-16 mimetic into HEK 293T cells transfected with the SMAD7(WT)vector,the relative luciferase activity is significantly reduced,whereas the mutation of the predicted matching site in the 3'-UTR of SMAD7 is associated with miR-16 The luciferase activity after up-regulation had no effect.In addition,Western blot analysis showed that miR-16 overexpression significantly inhibited the expression of SMAD7,whereas miR-16 downregulation significantly promoted SMAD7 expression in chondrocytes induced by IL-1?.A dual luciferase reporter assay was performed by transfecting the SMAD7-WT vector into miR-NC,miR-16,miR-16+pcDNA and miR-16+MEG3 into HEK293T cells.The data shows that overexpression of miR-16 results in a decrease in luciferase activity in HEK 293T cells transfected with the SMAD7-WT vector,which is significantly improved by the introduction of MEG3(P<0.05).On the other hand,MEG3 overexpression significantly promoted SMAD7 expression,and MEG3 low expression significantly inhibited SMAD7 expression.In addition,alterations in the expression of SMAD7 induced by MEG3 or si-MEG3 were significantly reversed by the introduction of miR-16 or anti-miR-16 in IL-1?-induced chondrocytes(P<0.05).In summary,our results indicate that SMAD7 is a target gene for miR-16,and MEG3 is involved in the IL-1?-induced miR-16/SMAD7 pathway in chondrocytes.Conclusions:(1)miR-16 expression was significantly increased in cartilage tissue of rat OA model.(2)Overexpression of miR-16 inhibits proliferation of chondrocytes induced by IL-1?.These results suggest that miR-16 may be closely related to the development and progression of OA.(3)Overexpression of miR-16 promotes IL-1?-induced chondrocyte apoptosis.These results suggest that miR-16 may be closely related to the development and progression of OA.(4)The expression of SMAD7 induced by MEG3 or si-MEG3 was significantly reversed by the introduction of miR-16 or anti-miR-16 in IL-1(3-induced chondrocytes.The results showed that SMAD7 is a target gene of miR-16,and MEG3 is involved in the IL-1?-induced miR-16/SMAD7 signaling pathway in chondrocytes.Part 3 Study on interaction between lncRNA MEG3 and miR-16/SMAD7 pathwayObjective:Osteoarthritis(OA)is a chronic joint disease and there is currently no definitive treatment.Micro-RNA has been confirmed to play an important role in the development of OA.However,its specific mechanism of action has not yet been clarified,especially the interaction between LncRNA MEG3 and miR-16/SMAD7 pathways.Methods:Rat OA model and interleukin-1?(IL-1?)-induced rat chondrocytes were constructed.RT-qPCR was used to detect the expression patterns of miR-16 and MEG3 in cartilage tissue of rat OA model.The effect of miR-16 on the proliferation or apoptosis of osteoblasts in chondrocytes was detected.And divided into mir-nc,Mir-16,ANTI-MIR-NC with anti-mir-16 Group The effect of miR-16 and MEG3 on IL-1?-induced chondrocytes was evaluated based on cell viability and apoptosis.The effect of MEG3 on the expression of SMAD7 was detected by Western blot.The luciferase reporter reaction was used to detect the interaction of miR-16 with MEG3.The expression of miR-16 and MEG3 was analyzed by RNA immuno precipitation.Results:(1)miR-16 and MEG3 are ceRNAs to each otherIt was found that the sequences of miR-16 and MEG3 have some complementary bases,suggesting that miR-16 may interact with MEG3.To verify the binding between MEG3 and miR-16,RNA immunoprecipitation(RIP)assays and dual luciferase reporter gene assays were performed.RIP assays were performed using Ago2 antibodies to confirm potential endogenous interactions between MEG3 and miR-16.The data shows that MEG3 and miR-16 were mostly captured by anti-Ago2 compared to the negative control in chondrocytes induced by IL-1[beta](P<0.05)..(2)MEG3 Regulates miR-16/SMAD7 PathwayFor dual luciferase reporter assays,wild-type(WT)and mutant(MUT)MEG3 luciferase reporter vectors were constructed and combined with miR-NC,miR-16 mimics,anti-miR-NC or anti-miR--miR-16.These results show that the luciferase activity of the MEG3(WT)vector is significantly reduced or enhanced by transfection with miR-16 mimetics or anti-miR-16,respectively(P<0.05).Although the mutation in the putative site in the MEG3 reporter vector had little effect on miR-16 upregulation or downregulation of miR-16 luciferase activity.To further elucidate the interaction between MEG3 and miR-16,the expression of MEG3 and miR-16 was detected in the cartilage tissue of the rat OA model.There was a negative correlation between MEG3 expression and miR-16.In addition,over-expression of MEG3 significantly inhibited miR-16 expression,whereas down-regulation of MEG3 significantly promoted miR-16 expression.(3)miR-16 inhibits MEG3-induced chondrocytes proliferation or apoptosisCell viability assay showed that the introduction of anti-miR-16 significantly reversed the proliferation-promoting effect of si-MEG3-mediated,and MEG3-mediated antiproliferative effects significantly attenuated miR-16 regulation(P<0.05).In addition,the anti-apoptotic effect of si-MEG3-mediated was significantly abolished by the introduction of anti-miR-16,and the induction of MEG3-mediated miR-16 significantly reversed the apoptotic effect.The results showed that changes in the expression of Bc12 and Bax revealed the discovery of apoptosis.The results indicate that miR-16 reverses the pro-proliferation and anti-apoptosis effects of MEG3-mediated in IL-1?-induced chondrocytes.In other words,MEG3 may regulate cell proliferation and apoptosis in OA through miR-16.(4)MEG3 induces chondrocytes proliferation or apoptosis by modulating SMAD7Corresponding negative controls for cell proliferation and apoptosis were detected in chondrocytes induced with si-MEG3+PBS,si-MEG3+ SMAD7,MEG3+ PBS,MEG3+si-SMAD7 or IL-1(3.Interestingly,the data show that after SMAD7 upregulation,the si-MEG3 mediated pro-proliferative effect was significantly reversed,whereas MEG3 mediated anti-proliferative effects were significantly improved by SMAD7 knockdown(P<0.05).Overexpression of SMAD7 significantly improved si-MEG3 mediated anti-apoptotic effects,and MEG3-mediated pro-apoptosis was significantly abolished by the introduction of si-SMAD7.In addition,western blot analysis also verified the finding of apoptosis,(P<0.05)which was shown by changes in the expression of Bc12 and Bax in chondrocytes induced by IL-1?.Taken together,these results suggest that MEG3 may exert its pro-proliferation and anti-apoptosis effects in OA by regulating SMAD7.Conclusions:(1)MEG3 participated in miR-16 pathway,MEG3 inhibited miR-16 expression(2)SMAD7 is the target gene of miR-16,and miR-16 inhibits SMAD7 expression in i1-1?-induced chondrocytes.(3)The expression of SMAD7 induced by MEG3 or si-meg3 is significantly reversed by introducing miR-16 or resisting miR-16.(4)MEG3 through regulating miR-16 and SMAD7 to play its role in promoting proliferation and resisting apoptosis.By up-regulation the MEG3,miR-16 expression lowering in OA model cartilage,the increased of MEG3 may promote proliferation and inhibition of apoptosis of rat chondrocytes induced by i1-1? through miR-16/SMAD7 signaling pathway,suggesting that MEG3 may be useful markers and potential therapeutic targets in OA. |
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