The Mechanism Of Cell Cycle Changes Mediated By Circadian Clock Gene Bmal1 In Knee Osteoarthritis

Objective: To investigate the mechanism of Knee Osteoarthritis(KOA)caused by cell cycle changes mediated by circadian clock gene Bmal1.Firstly,the articular cartilage gene chip data set of KOA was analyzed by bioinformatics methods to screen differentially expressed genes and KOA-related pathways.Secondly,the articular cartilage of patients with clinical knee arthroplasty was collected.According to the sleep quality of patients,the expression differences of circadian clock and cell cycle related genes in KOA cartilage tissue were discussed.Then,the relationship between OA-like changes and cell cycle-related protein expression changes in knee cartilage was explored through the circadian rhythm disorder rat model.Finally,through the cell level,the mechanism of the clock gene Bmal1-mediated cell cycle changes and the mechanism of knee osteoarthritis were studied.Methods:(1)Through the GEO database,the gene chip data and related documents numbered GSE16464,GSE117999 and GSE169077 were downloaded,and the gene expression profiles of knee cartilage samples in 20 healthy control groups and 21 KOA treatment groups were obtained.After merging the data,gene difference analysis and enrichment analysis were performed,and the GSE82107 data set was verified.The Least Absolute Shrinkage and Selection Operator(LASSO)model was used to screen and verify the characteristic genes from key modules and differentially expressed genes(DEGs).(2)Sixty patients with knee osteoarthritis(OA)undergoing knee arthroplasty were selected.The lateral femoral condyle cartilage was collected after operation.According to the Pittsburgh sleep quality score,they were divided into good sleep quality group(OA-well)and poor sleep quality group(OA-bad).H.E staining and safranin-fast green staining were used to observe the pathological changes of cartilage tissue.The relative m RNA expression levels of BMAL1,WEE1,CDK1,CCNB1 and MMP13 were detected by reverse transcriptase polymerase chain reaction(RT-PCR),and their expression differences between OA-well group and OA-bad group were analyzed.The expression differences between OA-well group and OA-bad group were analyzed,and the correlation between them was analyzed.The expression levels of BMAL1,WEE1,CDK1,CCNB1 and MMP13 proteins were detected by immunohistochemistry(IHC)and Western Blot(WB)to further verify their expression differences between OA-well group and OA-bad group.(3)Forty rats were randomly divided into normal circadian rhythm group(Normal),circadian rhythm disorder group(Control),Bmal1 overexpression lentivirus infection circadian rhythm disorder group(LV-Control)and Bmal1 overexpression lentivirus negative infection circadian rhythm disorder group(NLV-Control),with 10 rats in each group.The pathological changes of cartilage tissue were observed by safranin-fast green staining.RT-q PCR was used to detect the m RNA expression intensity of Bmal1,Wee1,Cdk1,Ccnb1,Bax,Bcl-2,Il-1β,Il-6,Tnf-α and Mmp13 in different groups of articular cartilage,and the correlation between Bmal1,Wee1,Cdk1,Ccnb1,Bax and Bcl-2 was analyzed.The expression levels of BMAL1,WEE1,CDK1,CCNB1,BAX and BCL-2 proteins were detected by IHC and Western-blot,and the differences were analyzed.Tunel staining was used to analyze the apoptosis of chondrocytes in different groups.(4)The induced ATDC5 cells were divided into normal group(Normal),Bmal1 knockdown lentivirus transfected cartilage-like ATDC5 cells group(Bmal1-KD);Bmal1 overexpression lentivirus transfected cartilage-like ATDC5 cell group(Bmal1-OE).CCK8 technique was used to detect the cell viability of different groups.RT-q PCR was used to detect the relative m RNA expression intensity and difference of Bmal1,Wee1,Cdk1,Ccnb1,Bax,Bcl-2,Il-1β,Il-6,Tnf-α and Mmp13 in different groups.According to the relative expression of m RNA,the correlation between Bmal1,Wee1,Cdk1,Ccnb1,Bax and Bcl-2 was analyzed.The contents of IL-1β,IL-6,TNF-α and MMP13 in different groups were detected by ELISA,and the differences were analyzed.WB method was used to detect the protein expression levels and differences of BMAL1,WEE1,CDK1,CCNB1,BAX and BCL-2 in different groups.Flow cytometry was used to analyze the effect of BMAL1 on different stages of cell cycle and apoptosis.Results:(1)A total of 34 differentially expressed genes were screened out,of which 6 were up-regulated and 28 were down-regulated.Among them,the circadian clock and cell cycle genes related to KOA were BAML1,WEE1,and CDK1,and the circadian rhythm pathways related to KOA were screened out.(2)Compared with the OA-well group,the OA-bad group had more obvious cartilage surface smoothness,matrix destruction and proteoglycan loss,significantly decreased cartilage thickness,down-regulated BMAL1 and WEE1,and up-regulated CDK1,CCNB1 and MMP13(all P < 0.05).Correlation analysis showed that BMAL1 was positively correlated with WEE1,and negatively correlated with CDK1,CCNB1 and MMP13.(3)Safranin fast green staining showed that the thickness of cartilage matrix in Normal group was normal,showing uniform red,and the subchondral bone was green.The cartilage matrix in Control group and NLV-Control group was destroyed,and the proteoglycan loss was obvious,showing uneven red.The thickness of cartilage matrix in LV-Control group was basically normal,with no obvious loss of proteoglycan and slightly uneven red.Compared with the Normal group,the positive rate of apoptotic cells in the articular cartilage of the Control group and the NLV-Control group was significantly increased,BMAL1,WEE1,and BCL-2 were down-regulated,CDK1,CCNB1,and BAX were up-regulated,and the relative m RNA expression of Il-1β,Il-6,Tnf-α,and Mmp13 was increased(P<0.05).There was no significant change in the positive rate of apoptotic cells in articular cartilage of LV-Control group.There was no significant change in the expression of BMAL1,WEE1,BCL-2,CDK1,CCNB1 and BAX.There was no significant change in the relative expression of Il-1β,Il-6,Tnf-α and Mmp13 m RNA(P>0.05).Compared with the Control group,there was no significant change in the positive rate of apoptotic cells in the articular cartilage of the NLV-Control group.There was no significant change in the expression of BMAL1,WEE1,BCL-2,CDK1,CCNB1 and BAX.The relative expression of Il-1β,Il-6,Tnf-α and Mmp13 m RNA did not change(P>0.05).The positive rate of apoptotic cells in articular cartilage of LV-Control group was significantly decreased,BMAL1,WEE1 and BCL-2 were up-regulated,CDK1,CCNB1 and BAX were down-regulated,and the relative expression levels of Il-1β,Il-6,Tnf-α and Mmp13 m RNA were decreased(all P<0.05).Compared with the LV-Control group,the positive rate of apoptotic cells in the articular cartilage of the NLV-Control group was significantly increased,BMAL1,WEE1 and BCL-2 were down-regulated,CDK1,CCNB1 and BAX were up-regulated,and the relative expression levels of Il-1β,Il-6,Tnf-α and Mmp13 m RNA were increased(all P<0.05).Correlation analysis showed that Bmal1,Wee1 and Bcl-2 were positively correlated,and the three were negatively correlated with Cdk1,Ccnb1 and Bax.(4)The results of CCK8 showed that compared with the Normal group,the cell viability of the Bmal1-KD group was increased,and the cell viability of the Bmal1-OE group was decreased(P < 0.05).Compared with the Bmal1-KD group,the cell viability of the Bmal1-OE group was significantly decreased(P<0.05).The results of PCR and ELISA showed that compared with the Normal group,the relative expression of Bmal1,Wee1 and Bcl-2 m RNA in the Bmal1-KD group decreased,the relative expression of Cdk1,Ccnb1,Bax,Il-1β,Il-6,Tnf-α and Mmp13 m RNA increased significantly,and the contents of IL-1β,IL-6,TNF-αand MMP13 in the cell culture medium increased(P<0.05).The relative expression of Bmal1,Wee1 and Bcl-2 m RNA in Bmal1-OE group increased,the relative expression of Cdk1,Ccnb1,Bax,Il-1β,Il-6,Tnf-α and Mmp13 m RNA decreased,and the content of IL-1β,IL-6,TNF-α and MMP13 in cell culture medium decreased(P<0.05).Compared with Bmal1-KD group,the relative m RNA expression levels of Bmal1,Wee1 and Bcl-2in Bmal1-OE group were significantly increased,the relative m RNA expression levels of Cdk1,Ccnb1,Bax,Il-1β,Il-6,Tnf-α and Mmp13 were significantly decreased,and the contents of IL-1β,IL-6,TNF-α and MMP13 in cell culture medium were decreased(all P<0.05).The results of Western Blot showed that the results of protein expression levels in different groups were consistent with the results of PCR trend.In the results of flow cytometry cycle and apoptosis,compared with the Normal group,the proportion of cells in the S phase and G2/M phase of the Bmal1-KD group decreased,the cycle was shortened,and the proportion of late apoptosis increased(P<0.05).In the Bmal1-OE group,the proportion of cells in the G2 phase and M phase increased,the cell cycle was blocked,and the proportion of early and late apoptosis decreased(P<0.05).Compared with the Bmal1-KD group,the proportion of S and G2/M phase cells in the Bmal1-OE group increased more significantly,the cell cycle was delayed,and the proportion of early and late apoptosis decreased significantly(P<0.05).Conclusion: The results of bioinformatics analysis showed that BMAL1,WEE1,CDK1 and circadian rhythm pathways were correlated with KOA.In patients undergoing knee arthroplasty,KOA cartilage with good sleep quality and KOA cartilage with poor sleep quality have different expression and correlation of circadian clock gene Bmal1 and cell cycle-related genes.The circadian clock gene Bmal1 can cause chondrocyte apoptosis and inflammatory changes by regulating cell cycle-related genes and proteins,and OA-like changes in knee cartilage.