Molecular Mechanism Of WISP1 Influences On Bovine MDSC Differentiation In Vitro

Skeletal muscle bears important responsibilities in animal life activities.The growth and development of skeletal muscle is always complicated and orderly.In the process of skeletal muscle growth,muscle-derived stem cells(MDSCs)are formed first,then proliferated into myoblasts,and finally differentiated and fused to form multinucleated myotubes,and finally form muscles organization step by step.During this process,myocytes in each period are always regulated by a variety of muscle differentiation genes and related signaling pathways in the environment.The classic Wnt/?-catenin signaling pathway plays an important role in muscle growth and regeneration.WISP1 is a downstream prote in induced by Wnt1,a ligand of the classical Wnt/?-catenin signaling pathway.WISP1,also known as CCN4,is also a member of the CCN family that plays an important role in the differentiation process.In previous studies,WISP1 has been reported to play an important role in migration,adhesion,proliferation,osteogenesis and cancer.In our previous experiments,we found that WISP1 expression is increased in bovine MDSCs,so we speculate that WISP1 may also play an important role in the differentiation process,but its specific mechanism of promoting differentiation is still unclear and has not been reported.In this study,we examined the expression of WISP1 in bovine MDSCs differentiated in vitro.Using software to predict the transcription factor binding sequence related to differentiation function on the WISP1 promoter,a WISP1 gene activation and suppression vector was constructed,and si RAN fragments were used to inhibit WISP1 gene expression,and the effect of WISP1 on bovine MDSCs differentiated in vitro was detected.Next,in order to study the mechanism of WISP1 affecting the differentiation of MDSCs,we used Co-Immunoprecipitation(CO-IP)and ESI mass spectrometry to analyze the protein ANXA1 interacting with it and verified the results using CO-IP technology.Next,we used the same method to examine the expression and function of ANXA1 in bovine MDSCs differentiated in vitro.In order to determine whether WISP1 regulates ANXA1,we activated and suppressed or inhibited the expression of WISP1 and detected the expression of ANXA1 and the expression of ANXA1 regulated transforming growth factorb(TGF-?)signaling pathway-related marker molecules.Finally,we used the method of activating WISP1 while inhibiting ANXA1 to detect the reduction of ANXA1 in bovine MDSCs differentiated in vitro.WISP1 promoted the expression of TGF-? signaling pathway and the effect of promoting differentiation was inhibited.The main research results obtained are as follows:(1)Using immunofluorescence and Western blotting techniques to detect the expression of WISP1 in bovine MDSCs differentiated in vitro.The results showed that with the differentiation of bovine MDSCs in vitro,the fluorescence intensity of WISP1 gradually increased,and the expression level of WISP1 reached its highest level on the fifth day and remained at the corresponding level.(2)By constructing the activation and suppression vector of the WISP1 gene,designing and synthesizing interference fragments that interfere with si RNA to up-regulate and down-regulate the protein expression of WISP1 in bovine MDSCs differentiated in vitro and observe the effect of this gene on the differentiation of bovine MDSCs in vitro.The results show that activating WISP1 expression can significantly increase the protein expression of WISP1,and the myotube fusion rate of bovine MDSCs differentiated in vitro is significantly increased,and the expression of muscle differentiation marker Myo G is significantly increased.Suppress or inhibit the expression of WISP1,the result is opposite to the activation result.It shows that WISP1 can promote the differentiation of bovine MDSCs in vitro.(3)The protein interacting with WISP1 during the in vitro differentiation of bovine MDSCs was detected by CO-IP and ESI mass spectrometry,and the interacting protein ANXA1 was selected through relevant literature reports and data analysis,and verified by the CO-IP method.The results show that WISP1 interacts with ANXA1.(4)Using immunofluorescence and Western blotting technology to detect the expression of ANXA1 in bovine MDSCs differentiated in vitro.The results showed that with the differentiation of bovine MDSCs in vitro,the fluorescence intensity of ANXA1 gradually increased,and the expression level of WISP1 reached the highest on the fifth day and then gradually decreased.(5)Activation and suppression vectors constructed using CRISPR/d Cas9 technology can significantly increase or decrease the expression of ANXA1 protein,si RNA interference fragments can significantly reduce the expression of ANXA1 protein,and myotube fusion rate and the expression of muscle differentiation marker Myo G The amount also increases or decreases accordingly.It shows that ANXA1 gene can promote the differentiation of bovine MDSCs in vitro.(6)Explore the molecular mechanism of WISP1 to promote the differentiation of bovine MDSCs in vitro using the above methods of activating and suppressing or inhibiting WISP1.The results show that activating WISP1 expression can significantly increase the protein expression of WISP1 in bovine MDSCs differentiated in vitro,while the expression levels of ANXA1 and TGF-? signaling pathway-related marker molecules increase significantly,suppressing or inhibiting the expression of WISP1.Contrary to activation result.(7)Using the above WISP1 activation vector and the si RNA interference fragment of ANXA1,activate WISP1 and interfere with the expression of ANXA1 at the same time,detect the expression of TGF-? signaling pathway-related marker molecules and muscle differentiation marker Myo G in bovine MDSCs differentiated in vitro.The results showed that compared with the WISP1 activation alone group,the activation of WISP1 also inhibited the expression of ANXA1,and the expression levels of related proteins decreased significantly.It shows that WISP1 can regulate the expression of TGF-? signaling pathway by regulating ANXA1,thereby promoting the differentiation of bovine MDSCs in vitro.This study revealed a possible way to promote differentiation of the classical Wnt/?-catenin signaling pathway,clarified WISP1's role in promoting the differentiation of bovine MDSCs in vitro and its molecular mechanism,and showed that the classical Wnt/?-catenin signaling Possible connection between the pathway and the TGF-? signaling pathway.In actual production,it can provide a theoretical basis for improving beef output and improving livestock breeds,as well as new ideas for muscle growth and development,muscle repair and regeneration.