Sox9-Upregulated MiR-322-5p Facilitates BMP2-Induced Chondrogenic Differentiation By Targeting Smad7 In Mesenchymal Stem Cells

Objective: With the development of society and the change of people’s living habits,articular cartilage defects caused by trauma or degeneration are increasing year by year.However,the repair of cartilage defects is still a challenging issue all over the world.Chondrogenic differentiation of mesenchymal stem cells(MSCs)induced by Bone Morphogenetic Protein 2(BMP2)is a widely accepted method of cartilage tissue engineering at present.It induces MSCs to differentiate into chondrocyte by promoting the expression of Sox9.At the same time,BMP2 also induces chondrocyte hypertrophy and endochondral ossification by up-regulating the expression of Smad7,thus destroying cartilage formation.In addition,it has been found that Smad7 can be inhibited by Sox9,but the underlying mechanism is not clear.At present,more and more studies have shown that micro RNAs play an important role in the processes of cartilage formation and pathophysiology.The purposes of this study are:(1)to screen out the micro RNA which is up-regulated by Sox9 and targeting Smad7(2)to verify the effect of differently expressed micro RNA on cartilage hypertrophic differentiation and apoptosis;(3)to verify its promoting effect on chondrogenic differentiation of MSC in vivo and in vitro.Materials and methods:(1)First,three short hairpin RNA sequences of silencing Sox9 labeled with RFP were constructed and conveyed by adenovirus;C3H10T1/2 cells were infected with the adenovirus.The silencing effect of the three sequences on Sox9 was verified by WB and q PCR,and the one with the best silencing effect was used in the follow-up experiment.Then,under the premise of BMP2 induction,the expression of Sox9 was silenced or not,and the expressions of cartilage markers,Sox9,COL2A1 and Smad7 were detected at different time points.At the same time,the induced micromasses were stained with Alcian Blue to observe the effect of silencing Sox9 on proteoglycan synthesis.The samples at a time point with the most significant difference in Sox9 expression were sent for the Next Generation Sequencing.Differently expressed micro RNAs were screened out and analyzed for volcanic map,Veen diagram,scatter plot,heat map,GO enrichment map and KEGG enrichment map.The micro RNAs that were up-regulated by Sox9 and predicted to target Smad7 were screened out by bioinformatic analysis on three websites.Under the premise of BMP2 induction,the expression of the screened out micro RNA was detected among the the three groups of overexpression of Sox9,no interference with Sox9 and silencing Sox9,which confirmed the induction effect of Sox9 on the micro RNA.Under the induction of BMP2,the function of the micro RNA was inhibited and promoted by antagomir and agomir respectively,and the expression of Smad7 was detected by PCR and WB.Finally,the targeting relationship between the micro RNA and Smad7 was confirmed by dual luciferase reporter system.(2)C3H10T1/2 cells were treated with overexpressed or silenced Sox9 and/or selected micro RNA,and the effect of micro RNA on early apoptosis rate was detected by flow cytometry 3 days later.The forelimbs of 18.5-day-old mice fetus were amputated and peeled,followed by treatment with overexpressed or silenced Sox9 and/or selected micro RNA.After 14 days of culture,tissue sections and H&E staining were performed for analyzing the effect of the micro RNA on the length of hypertrophic region.(3)C3H10T1/2 cells were treated with overexpressed or silenced Sox9 and/or selected micro RNA,and cultured in micromasses.After 7 days,the expression of COL2A1,COL10A1 and Smad7 was detected by WB and q PCR,and the micromasses were stained with Alcian Blue.At the same time,the corresponding treated cells were injected subcutaneously into flanks of nude mice.After 3 weeks,the masses were taken out for H&E staining and Masson staining,and the expression of COL2A1 and COL10A1 was detected by immunohistochemistry.Results:(1)We found that all the three sequences can inhibit the expression of Sox9,and the second sequence had the best inhibitory effect.Silencing Sox9 could significantly inhibit the chondrogenesis induced by BMP2.The Next Generation Sequencing and bioinformatis analysis suggested that miR-322-5p was upregulated by Sox9 and may target Smad7.The targeting relationship between miR-322-5p and Smad7 was confirmed by dual luciferase report assay,q PCR and WB.(2)Flow cytometry analysis showed that the early apoptosis rate induced by miR-322-5p overexpression group was significantly lower than that in control group,while that in miR-322-5p silence group was significantly higher than that in control group.The experiment of mouse fetal limb culture in vitro showed that the expression of miR-322-5p was negatively correlated with the length of hypertrophic zone of growth plate stimulated by BMP2.(3)The in vitro experiment showed that the overexpression of miR-322-5p significantly inhibited the expression of Smad7,decreased the markers of hypertrophy and increased the markers of cartilage formation.In vivo experiments also confirmed that miR-322-5p could facilitate BMP2 on the chondrogenesis of MSC.Conclusion: These results suggest that:(1)there is a Sox9/miR-322-5p/Smad7 signal axis in the process of chondrogenic differentiation of MSC induced by BMP2;(2)miR-322-5p can inhibit the hypertrophy and apoptosis of chondrocytes induced by Smad7;(3)miR-322-5p facilitates BMP2 induced chondrogenesis.