| Objective: Gene-modified stem cells are of great significance in the field of bone regeneration.At present,long non-coding RNA(lncRNA)has been confirmed to regulate the process of osteogenic differentiation of various stem cells,however,the potential functions and regulatory mechanisms of lncRNA associated with osteogenic differentiation of bone marrow mesenchymal stem cells(BMMSCs)remain to be further elucidated.This study aims to apply high-throughput RNA sequencing(RNA-seq)technology to detect the differential expression profiles of lncRNA,mi RNA and m RNA in the process of rat bone marrow mesenchymal stem cells(r BMMSCs)osteogenic differentiation.Bioinformatics methods were used to analyze the differential expressed molecular functions and pathway enrichment.Combined with predicted competing endogenous RNA(ce RNA)regulatory network,the key lncRNA-mi RNA-m RNA axes were screened.Determine target lncRNA,verifying from the levels of cell function and animal effect,as the basis for studying the possible molecular regulatory mechanisms involved.To identify the key molecular in the process of osteogenic differentiation of r BMMSCs driven by lncRNA,providing molecular targets for the management of critical-sized bone defects.Methods:(1)Part I: Isolation and identification of r BMMSCs,which were induced by ordinary and osteogenic differentiation to prepare samples.RNA-seq technology was used to explore the expression profiles of lncRNA,mi RNA and m RNA related to the 14-day osteogenic differentiation of r BMMSCs.Combined with bioinformatics analysis to screen potential lncRNA-mi RNA-m RNA interaction networks.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses were performed to evaluate potential functions.3 lncRNAs,3 mi RNAs and 3 m RNAs were preliminarily verified by Real-time quantitative reverse transcriptionpolymerase chain reaction(q RT-PCR),and key lncRNA-mi RNA-m RNA axes with related enrichment pathways were constructed.(2)Part II: lncRNA Tug1 was screened as the research target,and q RT-PCR was used to verify its expression at different stages of r BMMSCs osteogenic induction.Lentiviral vectors that interfere with the expression of lncRNA Tug1 were constructed and the optimal transfection conditions were explored.The transfection efficiency was measured by flow cytometry and confocal laser,and the best interference target was screened by q RT-PCR.The r BMMSCs models that interfered with the expression of lncRNA Tug1,the negative control and the blank control were constructed.The effects of lncRNA Tug1 on r BMMSCs proliferation,migration and osteogenic differentiation were investigated by CCK-8 test,scratch test,ALP staining and activity detection,Alizarin red staining and semi-quantitative analysis,Von Kossa staining,q RTPCR and Western blot.lncRNA Tug1 expression in the nucleus/cytoplasm of r BMMSCs was detected,and the expression relationship between lncRNA Tug1 and rno-mi R-93-5p was detected.Their binding sites were predicted by bioinformatics,and the binding relationship was detected by double luciferase reporter gene assay,exploring the potential mechanism of lncRNA Tug1 regulating the osteogenic differentiation of r BMMSCs.(3)Part III: Rat femoral distraction osteogenesis model was constructed,and r BMMSCs interfering with lncRNA Tug1,negative control and blank control were injected into the stretch space of the corresponding group at the end of the stretch period.X-ray,Micro-CT,HE staining,Von Kossa staining and Masson Trichrome staining and immunohistochemical staining were used to evaluate the new bone regeneration in the femoral stretch area of rats in each group with different mineralization stages.Results:(1)Part I: r BMMSCs surface antigens were positive for CD29 and CD44,negative for CD34 and CD45,and had multidirectional differentiation potential.The expression profiles of lncRNA,mi RNA and m RNA were obtained by RNA-seq on 14-day osteogenic induction of r BMMSCs.There were 1524 differentially expressed lncRNAs(812 up-regulated and 712 down-regulated),91 differentially expressed mi RNAs(30 up-regulated and 61 down-regulated),and 2453 differentially expressed m RNAs(1272 up-regulated and 1181 down-regulated).Using 10 up-regulated lncRNAs as clues,21 down-regulated mi RNAs and 650 up-regulated m RNAs were screened out,49 pairs of lncRNA-mi RNA and 1515 pairs of mi RNA-m RNA interaction networks were constructed.GO analysis showed that the most important enrichment items in cell component and molecular function were "cytoplasm" and "protein binding",respectively.Biological process related to osteogenic differentiation such as "cell proliferation","wound healing","cell migration","osteoblast differentiation","extracellular matrix organization" and "response to hypoxia" were also enriched.KEGG analysis showed that differentially expressed genes were mainly enriched in "PI3K-Akt signaling pathway","Signaling pathway regulating pluripotency of stem cells","c GMP-PKG signaling pathway","Axon guidance" and "Calcium signaling pathway".q RT-PCR verified that the results of lncRNA Tug1,lncRNA AABR07011996.1,rno-mi R-93-5p,rno-mi R-322-5p,Sgk1 and Fzd4 were consistent with the sequencing results.Based on the verification results,4 lncRNA-mi RNA-m RNA interaction networks were constructed,and the enrichment pathways were closely related to "PI3K-Akt signaling pathway","Signaling pathway regulating pluripotency of stem cells" and "Wnt signaling pathway".(2)Part II: lncRNA Tug1 was screened as the research target.The expression of lncRNA Tug1 at different stages of r BMMSCs osteogenic differentiation was most significant at day 14(P<0.001),followed by day 21(P<0.01)and day 7(P<0.05).lncRNA Tug1 RNAi lentiviral vector were successfully constructed,and the optimal conditions for transfection were MOI=80,infection enhancer solution A and solution P usage.The transfection efficiency was up to 70%~80%,and sh-lncRNA Tug1-2 group had the best interference effect,which could be used to construct a stable r BMMSCs model to inhibit lncRNA Tug1 expression.Inhibition of lncRNA Tug1 expression significantly reduced the proliferation activity,migration ability,ALP activity,calcium deposition,and expression levels on ALP,Osx,OPN and BMP-2 of r BMMSCs(P<0.05).lncRNA Tug1 was expressed in both the nucleus and cytoplasm of r BMMSCs,and the expression in the nucleus was relatively higher.When lncRNA Tug1 was down-regulated,the expression of rno-mi R-93-5p was up-regulated.Double luciferase reporter assay results showed that the double luciferase activity of lncRNA Tug1-WT + rnomi R-93-5p group was significantly decreased compared with that of lncRNA Tug1-WT + rno-mi R-NC group(P<0.001).(3)Part III: The rat femoral distraction osteogenesis model was successfully constructed.When compared with local injection of r BMMSCs with normal lncRNA Tug1 expression,injection of r BMMSCs with inhibited lncRNA Tug1 expression slowed down the bone mineralization rate and delayed the bone healing process.Conclusion:(1)Based on RNA-seq and bioinformatics analysis,this study screened and preliminatively verified 3 lncRNAs,3 mi RNAs and 3 m RNAs,and constructed lncRNAmi RNA-m RNA interaction networks and enrichment related pathways,providing reference for screening lncRNA target and studying its function and regulatory mechanism.(2)Lentiviral vector that best interfere with the expression of lncRNA Tug1 was screened out,and r BMMSCs models that inhibit the expression of lncRNA Tug1 were successfully constructed,laying a preliminary foundation for the implementation of functional experiments in vitro.(3)Inhibition of lncRNA Tug1 expression weakened the proliferation activity,migration ability and osteogenic differentiation of r BMMSCs.(4)lncRNA Tug1 was negatively correlated with the expression of rno-mi R-93-5p and there was a targeted interaction between them.lncRNA Tug1 may regulate the osteogenic differentiation of r BMMSCs through the adsorption of rno-mi R-93-5p.(5)A reasonable,simply-operated and repeatable rat femoral distraction osteogenesis model was successfully established by using the external fixator,which laid a solid foundation for further exploring the method of promoting the repair and reconstruction of large bone defects in animal models.(6)Inhibition of lncRNA Tug1 expression slowed down the bone mineralization rate during distraction osteogenesis and inhibited bone formation. |
