The Mechanism Of MiR-145 Targeting SOX9 In Regulating Chondrogenic Differentiation Of Deer Antler Mesenchymal Stem Cells

Antler is the only organ that mammals can regenerate after cutting.Its growth rate is much faster than that of tumors without causing tissue cancer,and it shows a highly orderly state while maintaining rapid growth and differentiation.It has been demonstrated that regeneration of the internal and external tissues of antler depends on the expression of key embryonic stem cell markers by Antler Reserve mesenchymal cells(RMCs),and the proliferation and differentiation of RMCs directly from internal antler components.In conclusion,RMCs are always present in the growth process of velvet antler.They constantly proliferate and renew the stem cell pool,and continuously differentiate into cartilage to maintain the rapid growth of velvet antler without carcinogenesis.Therefore,the molecular mechanism of chondrogenic differentiation of RMCs can provide theoretical support for the rapid growth of antler.In recent years,some reports have found that micro RNAs(miRNAs)play an important role in regulating the dry maintenance and differentiation of mesenchymal stem cells(MSCs).miRNAs can stabilize the phenotype of chondrocytes,prevent them from entering the differentiation process of endochondral osteogenesis hypertrophy and prevent the formation of fibrocartilage,so as to achieve stable repair of cartilage defects.Therefore,studying the regulatory network of chondrogenic differentiation of antler RMCs from the perspective of miRNAs can provide a new perspective for the analysis of the mechanism of chondrogenic differentiation of antler RMCs.In view of this,we carry out the following aspects of research.In the first part,antler RMCs were successfully isolated and identified.Microsomes,a threedimensional model of chondrogenic differentiation of antler RMCs,were established by exogenous TGF-β1.Induced by Small RNA sequencing technology testing different period(0 d,7 d,14 d)microsomal and antler chondrocytes.The results identified 475 known miRNAs and 70 novel miRNAs,of which 288 miRNAs differentially expressed.GO and KEGG analyses identified that differentially expressed miRNAs were enriched in cell proliferation,survival,apoptosis and immune response,AMPK signaling pathway,calcium signaling pathway,m TOR signaling pathway and other pathways.By comparing the expression of differentially expressed miRNAs between CP＿7 vs CP＿0,CP＿14 vs CP＿7,and CC vs CP＿14 groups,74 miRNAs were found to be differentially expressed between these three comparison groups.Combined with our previous set of miRNAs related to deer antler growth in different tissues.Finally confirmed 12 candidate miRNAs,among which miR-145 was closely related to cartilage proliferation,differentiation,and migration.Therefore,we hypothesized that miR-145 may play an important role in the chondrogenic differentiation of antler RMCs.In the second part,to clarify the effect of miR-145 on chondrogenic differentiation of antler RMCs,RMCs transfected with miR-145 mimic and miR-145 inhibitor were induced into microsomes in vitro,and samples were collected on the 14 th day.Alcian blue staining results showed that the positive rate of acid mucopolysaccharide increased significantly in miR-145 inhibitor group,while that in miR-145 mimic group was the opposite.These results suggest that miR-145 may inhibit the chondrogenic differentiation of RMCs.Subsequently,Q-PCR,IFA and Western Blot were used to detect cartilage-related genes.The results showed that miR-145 inhibitor significantly up-regulated the expression of cartilage marker genes COLⅡ and COMP,and down-regulated the expression of cartilage hypertrophy marker gene COLⅩ.The expression of COLⅡ and COMP was significantly down-regulated and COLⅩ was up-regulated in miR-145 mimic group.These results indicated that miR-145 inhibited chondrogenic differentiation of RMCs.In the third part,firstly,in order to identify the downstream regulatory genes of miR-145,bioinformatics software was used to predict that the targeted regulatory gene of miR-145 was the key transcription factor SOX9.The expression of SOX9 was negatively correlated with miR-145 at 0,7,and 14 days of chondrogenic differentiation.The dual luciferase reporter system further confirmed that miR-145 could effectively bind to the predicted target of SOX9 3’UTR and exert inhibitory effect.These results confirmed that SOX9 was the target gene of miR-145.Second,to clarify whether miR-145 plays a role in chondrogenic differentiation of RMCs by targeting SOX9,Q-PCR,IFA,and Western Blot were used to detect SOX9 expression in microsomes induced by RMCs transfected with miR-145 mimic or miR-145 inhibitor at day 14.The expression of SOX9 was significantly increased in the miR-145 inhibitor group,and was significantly decreased in the miR-145 mimic group.The above results indicated that miR-145 had a negative regulatory effect on SOX9.Finally,to investigate the mechanism of miR-145 targeting SOX9 in regulating chondrogenic differentiation of RMCs.In this study,the following 8 groups were transfected into RMCs using the lentivirus system: miR-145 mimic,miR-145 inhibitor,LV-SOX9,LV-SOX9 sh RNA,miR-145 mimic + LV-SOX9,miR-145 mimic + LV-SOX9 sh RNA,miR-145 inhibitor +LV-SOX9,miR-145 inhibitor + LV-SOX9 sh RNA,Microsomal samples were collected on day 14 after chondrogenic differentiation.Alcian blue staining showed that the positive rate of LV-SOX9 group and miR-145 inhibitor + LV-SOX9 group increased,while the positive rate of LV-SOX9 sh RNA group and miR-145 mimic + LV-SOX9 sh RNA group decreased.The positive rate of miR-145 mimic + LV-SOX9 group and miR-145 inhibitor + LV-SOX9 sh RNA group had no significant change compared with the control group.The results of Q-PCR,IFA and Western Blot showed that the expression of COLⅡ,COMP and SOX9 in the LV-SOX9 group and the miR-145 inhibitor+ LV-SOX9 group was significantly up-regulated,and the expression of COLⅩ was significantly down-regulated.The expression of COLⅡ,COMP and SOX9 in LV-SOX9 sh RNA group and miR-145 mimic + LV-SOX9 sh RNA group was significantly down-regulated,and the expression of COLⅩ was up-regulated.The expressions of COLⅡ,COMP,SOX9 and COLⅩ in miR-145 mimic + LV-SOX9 group and miR-145 inhibitor + LV-SOX9 sh RNA group were not significantly different from those in the control group.These results confirmed that miR-145 inhibited chondrogenic differentiation of RMCs by targeting SOX9.To sum up,miR-145 can inhibit RMCs directional differentiation into cartilage cells,the regulation mechanism is an important transcription factor through targeted negative regulating SOX9,thus inhibiting antler RMCs into cartilage differentiation process.This provides theoretical support for the analysis of the regulatory mechanism of chondrogenic differentiation of RMCs,and also provides scientific basis for the elucidation of the rapid growth mechanism of antler.