Study On The Function And Mechanism Of SEMA3B-AS1 Regulating The Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Objective(s):Osteoporosis is a chronic progressive metabolic disease involving whole body skeletal muscle.compared with normal people,patients often show the characteristics of bone mass loss,high bone brittleness and severe damage of bone tissue microstructure.The onset of the disease is insidious.Its morbidity and prevalence remain high,treatment options are limited,complications are many,and prognosis is poor,which seriously threatens the health of human beings around the world.On the other hand,its pathogenesis is complex and has not been fully revealed until now.Therefore,it is the only way to promote the development of human society and improve people’s livelihood and well-being by continuously exploring the internal factors of osteoporosis and gradually revealing its potential molecular mechanism.In recent years,the imbalance of lineage differentiation of bone marrow mesenchymal stem cells is considered to be one of the important reasons for the occurrence and development of OP.In addition,a large number of studies have also revealed that long non-coding RNA can regulate the osteogenic differentiation process of bone marrow mesenchymal stem cells through various mechanisms.My research group found that the abnormal expression of lnc RNA SEMA3B-AS1 is inseparable from the onset of osteoporosis in the human body.Through the study of SEMA3B-AS1 function gain and loss experiments,alizarin red staining and proteomic analysis,it was preliminarily clarified that SEMA3B-AS1 has the biological function of inhibiting osteogenic differentiation of bone marrow mesenchymal stem cells.Based on this,this project intends to further explore the function and mechanism of SEMA3B-AS1 regulating the osteogenic differentiation of BMSCs.Methods:We pull down the protein that binds to SEMA3B-AS1 through RNA pull down experiments,and identify the specific binding protein of SEMA3B-AS1 through mass spectrometry.From the results,we give priority to proteins around the nuclear membrane,proteins with the function of entering and exiting the nucleus,or transcription factors,etc.RIP experiments were performed to pull SEMA3B-AS1 backward through the target protein to clarify the interaction between SEMA3B-AS1 and the target protein,and then conduct biological function research on the interacting protein.Design and construct the target gene overexpression vector,synthesize the interference sequence and transfect the bone marrow mesenchymal stem cells,and detect the transfection efficiency.After setting the target gene overexpression group,the interference group and the control group corresponding to each group,CCK-8detected the proliferation of cells in each group;after 14 days of induction of osteogenic differentiation of bone marrow mesenchymal stem cells,alizarin red staining was performed in each group for detection Osteogenic differentiation level;q RT-PCR detected the expression changes of osteogenic differentiation-related genes OCN,OPN,ALP,and RUNX2 in bone marrow mesenchymal stem cells,and Western blot further detected the expression of osteogenic differentiation-related proteins.Results:In this project,a large number of potential interacting proteins of SEMA3B-AS1 were extracted from the RNA pull down experiment.Through the silver staining detection of the enriched protein,the difference bands cannot be clearly identified from the results by naked eyes alone.In order to find differential proteins,we removed the Sense and Antisense protein strips and sent them for LC-MS/MS mass spectrometry identification.Finally,a total of 9323 total spectra were identified in the Sense group,3187 secondary spectra were analyzed,and 1705 peptides were identified,485 proteins were identified;Antisense band analysis obtained 9207 total maps,analyzed 3013 secondary maps,identified 1599 peptides,and identified 487 proteins.104 specific binding proteins were identified in the sense band by Venn diagram selection.We selected HNRNPU from them for follow-up experiments,and reversely verified that SEMA3B-AS1 can be significantly enriched in HNRNPU through RIP experiments and q RT-PCR,indicating that there is an interactive relationship between SEMA3B-AS1 and HNRNPU.We set up the HNRNPU overexpression group,the HNRNPU overexpression control group,the HNRNPU interference group,and the HNRNPU interference control group.The results of CCK8 showed that the overexpression of HNRNPU could promote cell proliferation,and the cell proliferation was inhibited after knockdown.Alizarin red staining showed increased calcium deposition after overexpression of HNRNPU,and decreased calcium deposition after knockdown.The results of q RT-PCR showed that when HNRNPU was overexpressed,the expression levels of osteogenesis-related genes OCN,OPN,ALP,and RUNX2 increased;after HNRNPU was silenced,the expression level of osteogenesis gene RUNX2 decreased,and the results of OCN,OPN,and ALP were negative.Western blot further detected the expression of bone protein in each component,and found that when HNRNPU was overexpressed,the expression of osteogenic protein was up-regulated,while the trend was completely opposite after HNRNPU was interfered.Conclusion(s):1.There is an interaction between SEMA3B-AS1 and HNRNPU.2.HNRNPU can promote the osteogenic differentiation and proliferation of bone marrow mesenchymal stem cells.3.SEMA3B-AS1 may affect the occurrence and development of osteoporosis by targeting HNRNPU to regulate the osteogenic differentiation of bone marrow mesenchymal stem cells.