Linc01238 Regulates Osteogenesis Of MSCs By Accelerating ELAVL1 Degradation And Enhancing RUNX2 MRNA Stability

ObjectivesBone defect is the damage of bone tissue integrity due to factors such as trauma,disease,aging and heredity.At present,bone repair is mainly performed through surgical bone transplantation,which is accompanied by a long healing period and the appearances of complications.Instead,stem cell therapy is effective and safe,and it is more minimally invasive.More importantly,it can stimulate the osteogenic differentiation of osteoprogenitor cells in the organism to accelerate the process of bone repair and bone regeneration.The directed differentiation of stem cells into osteoblasts is the key for stem cells to repair bone tissue.Studies have shown that long non-coding RNA(Lnc RNA)play an important regulatory role in the osteogenic differentiation of stem cells.In our study,Linc01238 regulates osteogenesis of mesenchymal stem cell(MSCs)by accelerating ELAVL1 degradation and enhancing RUNX2 mRNA stability,which will provide a new clue for clinical bone defect repair and bone regeneration.Materials and methodsHuman umbilical cord-derived mesenchymal stem cells(UC-MSCs)and dental pulp-derived mesenchymal stem cells(DP-MSCs)were used as the study objects.Lentivirus was used to establish stable overexpression and stable knockdown MSCs cell lines of Linc01238.Alizarin red S(ARS)staining and alkaline phosphatase staining(ALP)were applied to identify the osteogenic differentiation.The subcellular localization of Linc01238 was confirmed by the nucleocytoplasmic separation assay and the fluorescence in situ hybridization assay(FISH).Bioinformatics prediction combined with RNA pull down and RIP experiments were used to explore RNA binding proteins(RBP)interacting with Linc01238.The regulation relationship between Linc01238 and ELAVL1 is clarified through western blotting and q PCR.Results1.Linc01238 is up-regulated in the osteogenic differentiation of MSCsIn this study,RNA was extracted and detected from the umbilical cord or dental pulp MSCs after osteogenic induction.And the results showed that Linc01238 was significantly up-regulated during the osteogenic differentiation of MSCs.Furthermore,Linc01238 is an intergenic Lnc RNA consisting of 6 exons,which is located on human chromosome 2.It does not encode a protein and has 2170 bp in length from bioinformatics database(NCBI and CPAT).2.Linc01238 promotes osteogenic differentiation of MSCsTo explore the role of Linc01238 in osteogenic differentiation of MSCs,the MSCs with stable overexpression and knockdown of Linc01238 were established by lentivirus expression system.The MSCs were induced with osteogenic induction medium after Linc01238 overexpression or Linc01238 knockdown.The Linc01238 overexpression group had darker and larger staining area after 14 days of induction by ALP staining;on the contrary,the Linc01238 knockdown group had lighter and smaller staining area.And the Linc01238 overexpression group had darker and larger red areas by ARS staining after induction for 28 days.Conversely,the Linc01238 knockdown group had lighter and less staining range than the control group.These results indicated that Linc01238 could up-regulate alkaline phosphatase activity,increase the content of mineralized nodules and enhance MSCs osteogenic differentiation.3.Linc01238 regulates the transcriptional factor RUNX2 and OSX expressionRNA was extracted and reverse transcribed from the Linc01238 overexpression or the Linc01238 knockdown MSCs.And the mRNA levels of osteogenic marker genes were presented by q PCR assay.The results showed that the mRNAs expression of RUNX2 and OSX were upregulated in the Linc01238 overexpression MSCs.Meanwhile,the total protein was extracted.And the protein levels of osteogenic marker genes were performed by Western blotting assay.We observed that the expressions of RUNX2 and OSX were obviously up-regulated in the Linc01238 overexpression MSCs,while they were keeping a low expression in the knockdown Linc01238 cells.The results indicated Linc01238 may promote the osteogenic differentiation of MSCs by regulating the expression of RUNX2 and OSX.4.Linc01238 specifically interacts with ELAVL1 to regulate the osteogenic differentiation of MSCsIn order to explore the molecular regulation mechanism of Linc01238 promoting the osteogenic differentiation of MSCs,the subcellular localization of Linc01238 was determined by FISH assay and RNA nucleoplasmic separation assays.The results showed that Linc01238 was mainly located in the nucleus and a small amount in the cytoplasm.Immunofluorescence assay and protein nucleoplasmic separation assay confirmed that ELAVL1 was distributed in both nucleus and cytoplasm.Then,we found that Linc01238 and ELAVL1 co-localized in the nucleus of MSCs through using FISH and immunofluorescence experiments.Meanwhile we found that Linc01238 interacts with ELAVL1 in HEK293 cells through using bioinformatic CLIP data(the ENCORI database).And we verified that Linc01238 could pull down ELAVL1 protein after osteogenic induction of MSCs by RNA pull down and Western blotting.We also confirmed that ELAVL1 antibody could enrich Linc01238 via RIP experiments.In conclusion,Linc01238 specifically bound to ELAVL1.Then we found that neither knockdown nor overexpression of Linc01238 changed the mRNA level of ELAVL1 by q PCR assay;the knockdown of Linc01238 up-regulated the protein level of ELAVL1 by Western blotting,whereas overexpression of Linc01238 down-regulated ELAVL1 protein level.The results showed that Linc01238 regulated the protein level of ELAVL1.The expressions of osteogenic transcriptional factor RUNX2 and OSX were significantly up-regulated in the MSCs with transient knockdown of ELAVL1 by q PCR assay.Osteogenic induction was performed after knockdown of ELAVL1 in MSCs.And the results showed that ELAVL1 knockdown increased the activity of alkaline phosphatase and enhanced the osteogenic differentiation ability of MSCs through using ALP staining.The above results demonstrated that Linc01238 regulated the osteogenic differentiation of MSCs by specifically binding to ELAVL1 and down-regulating the expression of ELAVL1.5.Linc01238 promotes the proteasome-dependent degradation of ELAVL1To further determine how Linc01238 down-regulates the protein level of ELAVL1,the effect of Linc01238 on the protein level of ELAVL1 was examined by treating MSCs with knockdown Linc01238 and control group with the protein synthesis inhibitor(CHX).The ELAVL1 protein level did not change after the CHX treatment compared with the control group by Western blotting.That is,the protein level of ELAVL1 was still upregulated after knockdown of Linc01238 after inhibiting protein synthesis.Meanwhile,the MSCs of Linc01238 overexpression was treated with proteasome inhibitor(MG132).The down-regulated ELAVL1 protein level was restored by overexpression of Linc01238 compared with the control group.This indicated that Linc01238 did not affect the protein synthesis process of ELAVL1,but down-regulated the protein level of ELAVL1 by promoting the proteasomal-dependent degradation of ELAVL1.6.ELAVL1 specifically enriches RUNX2 mRNA and affects its stabilityTo further clarify the mechanism of ELAVL1 regulating the osteogenic differentiation of MSCs,we found that ELAVL1 protein could interact with the mRNA of the osteogenic transcription factor RUNX2 in HEK293 and Hela cells from the CLIP data(POSTAR database).And the mRNA levels of RUNX2 and OSX were significantly up-regulated in MSCs after ELAVL1 knockdown through q PCR assay compared with the control group.It was speculated that ELAVL1 might regulate the osteogenic differentiation of MSCs by affecting the mRNA stability of the osteogenic transcriptional factor RUNX2.Then we found that ELAVL1 antibody could enrich RUNX2 mRNA by RIP and q PCR assays.It indicated that ELAVL1 could bind to the mRNA of RUNX2.The MSCs of knockdown ELAVL1 were treated with actinomycin D(Act D)in a time-dependent manner,and it showed that the mRNA stability of RUNX2 in ELAVL1 knockdown MSCs was enhanced compared with the control group.It was confirmed that ELAVL1 inhibited the osteogenic differentiation of MSCs by attenuating the mRNA stability of RUNX2.Conclusions(1)Linc01238 was up-regulated during the osteogenic differentiation of MSCs and promoted the osteogenic differentiation of MSCs.(2)Linc01238 specifically bound to RNA-binding protein ELAVL1 and promoted the degradation of ELAVL1 protein.(3)Linc01238 promoted the osteogenic differentiation of MSCs by down-regulating the expression of ELAVL1.(4)ELAVL1 down-regulated the RUNX2 mRNA stability and reduced RUNX2 expression by binding to RUNX2 mRNA.