| Objective:Articular cartilage injury and degeneration are the second most common diseases after coronary heart disease.The prevalence rate of them increases with the age of patients and the ages of patients tend to be younger.Articular cartilage has no blood vessels and nerves,and its nutrition mainly depends on the diffusion mechanism of synovial fluid.Therefore,once articular cartilage is damaged,it is difficult to repair itself.Autologous chondrocyte implantation(ACI)is an effective method to treat chronic articular cartilage injury in recent years.This method requires a sufficient number of hyaline chondrocytes to be cultured in vitro for transplantation.Unfortunately,the dedifferentiation of human hyaline chondrocytes often occurs in vitro.At the same time,there is a similar process of dedifferentiation in the process of articular cartilage degeneration.However,the mechanism of dedifferentiation is not clear.Therefore,it is of great significance to clarify the mechanism of dedifferentiation in promoting the application of ACI technology and exploring the mechanism of articular cartilage degeneration.Long non-coding RNA(lncRNA)plays a regulatory role in gene expression in many pathological and physiological processes.However,the role of lncRNAs in the dedifferentiation of human hyaline chondrocytes in vitro is not clear.In this study,we first investigated the expression profiles of lncRNAs in the process of human hyaline chondrocyte dedifferentiation in vitro,and analyzed the overall differential expression of lncRNAs in the process of dedifferentiation.Then,the lncRNA AP001505.9 with obvious differential expression was selected to explore its specific mechanism in the process of dedifferentiation.Methods:1)In this study,we first cultured human hyaline chondrocytes of knee joint in vitro.Compared with P1(passage 1)and P5 passage human hyaline chondrocytes,the morphology of chondrocytes was observed by histology.The expression of COL1A1,COL2A1 and SOX9 was detected by RT-qPCR.The expression of related proteins was detected by Western blot and immunofluorescence to confirm the occurrence of dedifferentiation.2)We selected P1 and P5 chondrocytes for Microarray analysis to obtain the differentially expressed lncRNAs and m RNAs in the dedifferentiation process.At the same time,we made GO and Pathway analysis,and selected interested lncRNAs and m RNAs for RT-qPCR to identify the accuracy of Microarray analysis results.3)We selected the P5 chondrocytes for pellet culture,and detected the occurrence of redifferentiation by means of safranine O staining,Alcian blue staining and immunohistochemistry,and detected whether the expression of selected lncRNAs in the process of redifferentiation was reversed by RT-qPCR.4)We selected lncRNA-AP001505.9,which was significantly down regulated in the process of dedifferentiation.Firstly,we constructed chondrocytes that overexpressed AP001505.9,detected the expression of related markers by RT-qPCR and Western blotting.5)we transplanted the chondrocytes that overexpressed AP001505.9 into the subcutaneous body of nude mice.In the internal experiment,the expression of related markers was detected by safranine O staining and immunohistochemistry.6)Finally,we selected the mi RNA and m RNA that may be regulated by AP001505.9 through CeRNA analysis,and detected their expression in the above process by RT-qPCR and Western blotting,and detected whether there is a direct regulatory relationship by luciferase experiment.Results:(1)The dedifferentiation of human hyaline chondrocytes occurred in vitro passage culture.The expression of the hyaline chondrocyte markers,COL2 and SOX9,was significantly down regulated(P<0.001),while the expression of the fibrocartilage markers,COL1,was significantly up regulated(P<0.001);(2)Compared with P1 chondrocytes,the expression of 49 lncRNAs in P5 chondrocytes was up-regulated(P < 0.05)and the expression of 83 lncRNAs was down regulated(P < 0.05)through microarray analysis;(3)Go analysis showed that in the process of dedifferentiation,protein DNA complex assembly and other biological processes were significantly down regulated;protein DNA complex and other cell components were down regulated;protein heterodimerization activity and other molecular functions were significantly down regulated(P<0.05).However,the expression of nucleoside diphosphate in metabolism and other biological processes was significantly up-regulated;the expression of cell components such as neuron cell body was significantly up-regulated;the expression of molecular functions such as ion channel binding was significantly up-regulated(P< 0.05).(4)In the course of dedifferentiation of human chondrocytes,pathway analysis showed that the down regulated pathways included DNA replication pathway,nitrogen metabolism pathway,Hippo signaling pathway,butyrate metabolism pathway,etc.And the up regulated pathways included p53 signaling pathway,purine metabolism pathway,Erb signaling pathway,HIF1 signaling pathway,pyrimidine metabolism pathway,etc.(P < 0.05).(5)The results of RT-qPCR of 10 significantly changed lncRNAs and 7 m RNAs were consistent with those of microarray analysis.In addition,we found that the expression of AP001505.9,RP6-65G23.3,LINC00473,and LINC162 decreased significantly(P<0.01),the expression of G062245 had no change(P>0.05),while the expression of LINC01021,GS1-600G8.5,AC020594.5,CLYBL-AS1,and G012825 increased significantly(P < 0.01).(6)It was found by safranine O staining,immunohistochemistry and RT-qPCR that the chondrocytes of P5 generation cultured in vitro for 7 days achieved the re differentiation,which showed that the expression of COL1 was significantly down regulated(P < 0.001),while the expression of COL2 and SOX9 was significantly up regulated(P < 0.001).In the course of redifferentiation,the expression of lncRNAs was reversed,that is,the expression of AP001505.9 and LINC162 was significantly up-regulated(P < 0.001),while the expression of LINC01021 and GS1-600G8.5 was significantly down regulated(P < 0.001).(7)The overexpression of AP001505.9 significantly increased the expression of SOX9(P < 0.001),and significantly decreased the expression of COL1(P < 0.001),indicating that AP001505.9 can promote the maintenance of chondrocyte phenotype and inhibit dedifferentiation.(8)The chondrocytes with overexpression of AP001505.9 were detected by safranine O and immunohistochemistry after subcutaneous transplantation in nude mice for one month.It was found that there was a large amount of expression of cartilage matrix,and the expression of COL1 was significantly decreased,while the expression of COL2 and SOX9 was significantly increased,indicating that AP001505.9 could also inhibit the occurrence of dedifferentiation in vivo.(9)Through the analysis of CeRNA and RT-qPCR,we found that AP001505.9inhibited the expression of mi R-495-3p and promoted the expression of SOX9.Luciferase assay showed that AP001505.9 could not sponge mi R-495-3p by CeRNA mechanism,which might indirectly inhibit the expression of mi R-495-3p through other ways.Conclusion:This study is the first to reveal the changes of lncRNA expression profile during the dedifferentiation of human hyaline chondrocytes in vitro.It was found that AP001505.9 could promote the expression of SOX9 by inhibiting the expression of mi R-495-3p,and ultimately promote the maintenance of phenotype of hyaline chondrocytes and inhibit the occurrence of dedifferentiation.This study explored the potential new mechanism of dedifferentiation of chondrocytes from the perspective of lncRNA,and provided a new theory for the development of technology to effectively inhibit dedifferentiation and obtain a large number of phenotypic stable hyaline chondrocytes in vitro. |
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