Genome-wide Screening Of Mesenchymal Stem Cells For Anti-BMP9-induced Osteogenic Differentiation Using N19 Small Regulatory RNA Library

Object: Studies show that the occurrence of osteosarcoma is closely related to "imbalance of proliferation and differentiation" of mesenchymal stem cells(MSCs),that is also named "anti-differentiation" theory,but the detail mechanism has not been fully clarified.Small regulatory RNAs(sr RNA)are small RNAs that play regulatory roles in human,which are important regulators of life activities and participates in the regulation of osteogenic differentiation of bone marrow mesenchymal stem cells.Bone morphogenetic protein 9(BMP9),belongs to TGF-β family,is multifunctional growth factor which have strong ability to induce osteogenic differentiation in vivo and in vitro.In the early stage,our research group constructed a N19 small RNA regulatory RNA library bidirectionally expressing 19 random bases independently.In order to simulate the process of bone tumor mediated by "imbalance between proliferation and differentiation" of mesenchymal stem cells in vivo,we used n19 small regulatory RNA library to target the whole transcript to screen the cell phenotype of mesenchymal stem cells against BMP9 induced osteogenic differentiation in vitro and in nude mice,identify single n19 small regulatory RNA that mediates anti osteogenic differentiation,and clarify the molecular mechanism of small regulatory RNA against osteogenic differentiation,so as to lay an experimental foundation for further study of the mechanism of bone tumorigenesis.Method:(1)Using the retro-viruse packaging system,i MEF cells with N19 small regulatory RNA library stable expressed were established as the cell models.Meanwhile,the osteogenic differentiation ability of cells was stimulated by BMP9 adenovirus(Ad BMP9)and then the phenotypes of anti-osteogenic differentiation were screened in nude mice to get the anti-osteogenic differentiation cell model.(2)With the help of DNA high-throughput sequencing technology and bioinformatics analysis,we searched for the N19 small regulatory RNA sequence(Don)enriched in anti-osteogenic differentiation cells;Meanwhile,the enriched Don mediated anti-osteogenic differentiation ability was identified in mesenchymal stem cells such as i MEF,MEF and i BMSC in vivo and in vitro,and the Don sequence with the strongest anti-osteogenic differentiation ability was identified.(3)Mechanistically,the exon mutation in anti-osteogenic differentiation cells was detected by DNA exon sequencing technology.Bioinformatics analysis was carried out to find the SNV that tended to be enriched in multiple rounds of in vivo screening.Through functional experiments,it was clarified that the SNV enriched in anti osteogenic differentiation cells was the potential binding site of mi RNA.(4)Taking the construction of si RNA stable expression plasmid in cells as an example,explore the strategy of transient or stable expression of multiple small regulatory RNAs in cells at the same time.(5)Taking plasmid DNA purification as an example,the methods and strategies to improve the transfection efficiency of plasmid DNA in cells were explored.(6)Take the detection of mi RNA expression profile as an example to explore the enrichment and detection methods of short length micro RNAs(especially low abundance micro RNAs).Results:(1)Based on i MEF cells,we successfully constructed a cell model i MEFn19 with of N19 small regulatory RNA library expressed stablely.At the same time,after 4 rounds of in vivo and in vitro screening,anti-osteogenic differentiation i MEFOR cells(including i MEFOR1 A,i MEFOR1 B,i MEFOR1 C and i MEFOR1D)were obtained.(2)Through high-throughput sequencing the library region,we found N19 fragments DON1-7 were enriched in i MEFOR cells.The anti-osteogenic differentiation ability of DON1-7 was elevated in vivo and in vitro,separately.It was found that DON3 and DON6 had strong ability of anti-osteogenic differentiation induced by BMP9.(3)Genomic exon sequencing of i MEFOR cells revealed that 4277 co-enriched SNVs were identified in i MEFOR cells.7 SNVS were randomly selected,and their potential mi RNA binding sites were predicted by bioinformatics analysis.And it was found that all 7 SNVS were potential binding sites of mi RNAs.The binding sites of mi RNA seed regions were affected by the SNVs.The expression of SNV corresponding transcripts and their potential binding mi RNAs is dysregulated in i MEFOR cells.(4)With the help of the characteristic that Bst XI restriction endonuclease can specifically recognize CCANNNNN^NTGG site,we constructed a small RNA expression plasmid with p SEB361-si RNA as vector,which can express multiple small regulatory RNAs or multiple copies of a single small regulatory RNA at the same time.(5)With the help of the characteristic that size selective magnetic beads(SSMBs)can quickly separate DNA / RNA with large molecular size difference,we successfully designed and verified the rapid purification method of plasmid,which greatly eliminates the RNA with small molecular weight polluted in the plasmid,so as to improve the transfection efficiency of macromolecular plasmid in cells.(6)Based on the characteristics of SSMBs for separating DNA / RNA with different lengths,we designed a separation method to remove long transcripts from total RNA and retain small RNA,so as to realize the enrichment of small RNA,and optimized the RT q PCR detection strategy of small RNA(LTMT-Tq PCR).Conclusion:(1)In this study,i MEFOR cells with reduced osteogenic ability and increased tumorigenic ability were successfully obtained by screening in vivo and in vitro using N19 small regulatory RNA library,which confirmed that the occurrence of bone tumor was closely related to "imbalance of proliferation and differentiation".The cells have certain bone tumor cell like characteristics,suggesting that the screening process simulates the occurrence of osteosarcoma to a certain extent.(2)DON3 and DON6 were with the strongest ability to anti-osteogenic differentiation ability in vivo and in vitro,suggesting that DON3 and DON6 play significant roles in anti-osteogenic differentiation.(3)Mechanistically,we found that a large number of SNVs enriched in i MEFOR cells,and the N19 small regulatory RNA is likely to promote anti-osteogenic differentiation through the enrichment of SNVs by targeting mi RNAs,which leads to the occurrence of bone tumors.(4)We optimized the expression system of multiple si RNAs based on Bst XI,which can be used for the bidirectional expression of multiple small regulatory RNAs or the stable expression of a single small regulatory RNA.(5)DNA / RNA separation technology based on SSMBs can remove contaminated RNA in plasmid,purify plasmid DNA and other macromolecules,and improve plasmid transfection efficiency.(6)The LTMT-Tq PCR designed and optimized by us can remove long transcripts and retain small RNAs with short length from total RNA,so as to realize the enrichment of small RNAs.This method can improve the detection efficiency of low abundance small RNAs and can be used for the expression profile screening of small RNAs.