Studies Of MiR-101a Epigenetic Control Of Cox-2 Gene Expression And Chondrogenesis

Micro RNAs are endogenous non-coding RNAs that interfere with protein synthesis in vivo.Micro RNAs are key regulatory elements for gene expression via affecting the translation and stability of m RNA.As an important family member of micro RNAs,mi R-101 has recently been extensively studied.mi R-101 is essential for different physiological conditions(such as normal angiogenesis and embryo implantation)and also a key regulatory molecule for multiple diseases(such as carinogenesis of solid tumors and during progression of osteoarthritis).In this review,we will focus on how mi R-101 functions via regulation of transcription factors,signaling pathways,proteins of epigenetic controls and key enzymes in vivo.Such literature review will help us to systematically understand how mi R-101 play its key roles under different patho-physilogical conditions,and therefore provide basis for potential clinical applications.Background: Endochondral ossification is considered to be a critical stage of long(such as limbs)bone development.In this process,the resting chondrocytes undergo proliferation and are accompanied by accumulation of type II collagen and aggrecan.The chondrocytes then differentiate into hypertrophic chondrocytes and begin to express type X collagen gene(Col10al),which is under control of multiple transcription factors.A number of hypertrophic chondrocyte related genes such as Cox-2 and Mmp-13 etc.are also expressed during this stage.Cox-2 is a fast induced enzyme when target cells under proper stimulation.Our previous studies showed that Cox-2 promotes chondrocyte hypertrophy via upregulation of Col10 al and other relevant transcription factors.Aims: 1)We aim to investigate the role of mi R-101 a in regulation of hypertrophic chondrocytes related genes expression and to explore its influence on chondrocyte hypertrophic differentiation using MCT and ATDC5 chondrogenic cell lines.2)To determine the effects of mi R-101 a on cell proliferation of MCT and ATDC5 cells.3)To explore the potential mechanism of mi R-101 a regulation of chondrogenesis and related gene expression.Methods: 1),Web-based bioinformatics analysis softwares Targetscan and Mi Randa were used to predict the binding site of mi R-101 in target genes(Cox-2 and Col10a1).We also use a reporter assay system to confirm the real binding site in Cox-2 and Col10a1 3' UTR region.2),We used q RT-PCR to analyze the expression of mi R-101 a,Cox-2,Col10a1 in proliferative and hypertrophic MCT and ATDC5 chondrocytes.3),Overexpression and silencing mi R-101 a in two chondrogenic cell lines,then we used q RT-PCR,immunoblotting,Ed U cell proliferation assay to determine its influence on proliferative,hypertrophic chondrocytes.4),We used a image to show the gene-gene interactions network during chondrocyte differentiation.Results: 1),Bioinformatics analysis suggest that Cox-2 and Col10a1 are target genes of mi R-101 a.Results of luciferase reporter assay suggest that mi R-101 a binds with Cox-2 3'UTR region with a reduction of fluorescence intensity.2),Cox-2 and Col10a1 expression level was significantly increased while mi R-101 a was significantly down-regulated inhypertrophic chondrocytes compared with proliferative chondrocytes by real-time q RT-PCR analysis.3),Overexpression or silencing of mi R-101 a in MCT and ATDC5 cells significant inhibits or increases the m RNA and protein levels of Col10a1,Cox-2,and Mmp-13 by q RT-PCR and immunoblotting assays.No significant change of the expression levels of Runx2 and Sox9 was detected.4),Results of Ed U cell proliferation assay and Alcian blue staining suggest that mi R-101 a inhibits chondrocyte proliferation.Conclusion: There exist mi R-101 binding sites at 3'-UTR of Cox-2 and Col10a1 genes.Further firefly luciferase reporter assay demonstrated only the binding affinity of mi R-101 with the 3'-UTR of Cox-2.mi R-101 a may play an essential role during chondrogenesis via targeting Cox-2 gene expression.