19nt RNA Library Screening Anti-BMP9 Induced MSCs Osteogenic Differentiation Mechanism And BMP9 Expression In Mice During Growth And Development

Objectives: BMP9 is a ligand of the TGFbeta signaling superfamily and one of the most effective osteoinductive factors.In this study,a 19-base random RNA fragment library was constructed,and BMP9 was used to induce the differentiation of mesenchymal stem cells for in vivo and in vitro screening.The enriched 19 nt RNA fragments were obtained through screening,and the patterns of these fragments were further analyzed.The mechanism of miRNA function during osteogenic differentiation is proposed to provide theoretical support for the early diagnosis and treatment of skeletal system diseases.At the same time,previous studies in our laboratory showed that BMP9 is similar to other BMPs and participates in important roles in tissue,organ development and tumorigenesis.C57BL/6J mice were used.By studying the transcription levels of BMP9 and ALK1,ALK2,SMAD6,SMAD7,and Noggin in different tissues at different ages,we further systematically revealed the expression law of BMP9 signaling pathway.Methods:(1)A 19-base DNA fragment randomly arranged by chemical synthesis,and cloned into the pSLOK plasmid.The pSLOK plasmid was transformed into E.coli by electrotransfer,and the plasmid was extractedafter amplification and cultivation of E.coli.The pSLOK plasmid was used as the backbone for packaging retroviruses,and it was co-transfected with the reverse transcription packaging plasmid pCL-Ampho into 293 PA cells to package retroviruses.Collect the retrovirus-containing supernatant and use the retrovirus to infect mesenchymal stem cell lines(BMSC-TA is immortalized bone marrow mesenchymal stem cells and iMADs are fat derived stem cells).Through BSD screening,a cell population containing19 nt RNA library was successfully obtained.(2)In vitro screening: culture mesenchymal stem cells in vitro,add AdBMP9 to infect mesenchymal stem cells,and expand them to passage.After multiple passages,a large number of MSCs undergo osteogenic differentiation and collect the remaining cells.(3)In vivo screening: After 3 weeks of in vitro screening,the remaining MSCs were collected and injected subcutaneously in nude mice for about 2 weeks to collect subcutaneous masses.(4)Obtain anti-differentiated mesenchymal stem cell population by chopping the mass,pancreatin digestion,cell extraction,and culture.Use AdBMP9 as a growth factor again to promote cell differentiation,and repeat the above steps of in vitro and in vivo culture as a cycle.Each round of cell populations was named A,B,C,D,E.After in vivo and in vitro screening,anti-differentiation cell populations at different stages were obtained.(5)Sequencing was performed by extracting genomic DNA from each round of selected mesenchymal stem cells.The enriched 19 nt RNA fragment sequence was obtained for bioinformatics analysis.(6)The RNA profiling data sets were generated by the Mouse ENCODE project PRJNA66167 as described,the data sets included 30 mouse samples derived from embryonic and adult tissues,and are publiclyavailable through NCBI website(https://www.ncbi.nlm.nih.gov/gene/).The acquired reads per kilobase per million reads(RPMKs)for Bmp9,Alk1,Alk2,Smad6 and Smad7 in the 30 mouse samples were graphed by using the Microsoft Excel software.(7)C57BL/6J mice at newborn(NB),2-week,4-week,3-month,8-month and 18-month old(male,n=5)were obtained from The University of Chicago Transgenic Core Facility.For total RNA isolation,mouse brain,fat(inguinal region),heart,kidney,liver,lung,muscle,spleen,femur and parietal bone(PB)at various ages were harvested immediately after sacrificing the animals,The expression of BMP9 and related signaling molecules in tissues and organs of different ages was analyzed using TouchDown qRT-PCR.Results:(1)Based on the pSLOK plasmid,a 19 random base small RNA library was successfully constructed.The library was used to infect mesenchymal stem cells.After multiple rounds of in vivo and in vitro screening,anti-differentiated cell populations were obtained.(2)Conduct in vitro and in vitro differentiation experiments to confirm the difference between the anti-differentiation cell population and the normal mesenchymal stem cell population.(3)The 19 nt RNA library enriched sequences constructed in this study were matched by pubmed and ensembl databases,indicating that the enriched fragments of the 19 nt RNA library almost did not completely match the genomic site,suggesting that the enriched 19 nt RNA fragments may not conform to the mechanism of siRNA;(4)Analyze the MSCs screened at different degrees.As the screening progresses,the enrichment degree of 19 nt RNA fragments increases;for different mesenchymal cell lines,19 nt RNA screening has repeatedsequences;the first to be enriched The base sequence of RNA fragments,starting from 2-7 bases,have a high degree of similarity,which is in accordance with the mechanism of action of miRNA seeds;(5)The 2-7 bases of the 19 nt RNA sequence enriched at different stages of the same cell line are highly repeatable;the 2-7 bases enriched by different cell lines have overlap;(6)Mouse ENCODE transcriptome data indicate that BMP9 is highly expressed in the liver,while BMP9-related BMP type I receptor Alk1 is highly expressed in the lung.(7)BMP9 is only expressed at moderate to low levels during skeletal development and is highly expressed in postpartum liver and lung tissues.(8)The expression levels of ALK1 and ALK2 gradually increased during the skeletal development period.Alk1 is highly expressed in the lungs,while Alk2 is relatively highly expressed in fat and kidney tissues.(9)The negative regulators of BMP9 signaling,Smad6,Smad7 and Noggin,are widely expressed in the tissues of mice after birth.Conclusions:(1)Using BMP9 to induce mesenchymal stem cell differentiation,19 nt RNA library for anti-differentiation screening can obtain a cell population with certain anti-differentiation characteristics.(2)The enriched 19 nt RNA library fragments are regular,and the specific mechanism may be related to the mechanism of miRNA SEEDs.(3)The expressions of BMP9 and related receptors ALK1 and ALK2 are gradually increased during the development of mouse bones,which may play an important role.