Profiling LncRNA Alterations During TNF-? Induced Osteogenic Differentiation Of Dental Pulp Stem Cells

The multipotent and easily accessible characteristics of dental pulp stem cells(DPSCs)make them a promising target for bone tissue engineering.Long non-coding RNAs(IncRNAs)have an important role in the osteogenic differentiation of mesenchymal stem cells.Nevertheless,whether lncRNAs are involved in the osteogenic differentiation of DPSCs remains unclear.The present study examined the expression alterations of IncRNAs in tumor necrosis factor-a induced osteogenic differentiation of DPSCs.Following identification of differentially expressed IncRNAs at different time points by RT-qPCR,profiling analysis was performed and a profile was further validated,in which IncRNA expression levels demonstrated significant upregulation.The next generation sequencing analysis identified 77(58 upregulated and 19 downregulated)and 133 differentially expressed IncRNAs(73 upregulated and 60 downregulated)at 7 and 14 days post-treatment,respectively.In addition,34 IncRNAs were predicted to be strongly associated with 336 mRNA transcripts that underwent significant alterations during osteogenic differentiation.The present data demonstrated that one IncRNA,X inactive specific transcript,is essential for efficient osteogenic differentiation of DPSCs by ALP staining.In summary,the present findings provide insight for the understanding of how non-coding RNAs are involved in regulating the osteogenic differentiation of DPSCs,which may further advance the translational studies of bone tissue engineering.ResultsTNF-apromotes osteogenic differentiation of DPSC.On day 7 and day 14,10 ng/mL of TNF-awas added to the medium in which the dental pulp stem cells were cultured,respectively.On day 7,after stimulation with TNF-a,ALP staining showed strong positive expression during osteogenic differentiation(Fig.1A),indicating that DPSCs are undergoing osteogenic differentiation.While TNF-?was intervened on day 14 of cell culture,ALP staining showed an increase in positive expression to 85-90%(Fig.1A).These results indicate that osteoblast differentiation of DPSC is promoted by TNF-?induction during differentiation of dental pulp stem cells.The Gene expression changes during osteogenic differentiation.In order to verify the changes of the gene expression of osteogenic differentiation in dental pulp stem cells,we extracted RNA from the cells collected on days 7 and 14 and constructed a biological analysis.The results showed that the gene sequencing quality was good(Figures A?B and C).The Mapping statistics show that the alignment results are consistent with the sequencing quality(Table D).The above results show that there are differences in genes during osteogenic differentiation.Alterations of lncRNA expression during osteogenic differentiation of DPSCs.As previously demonstrated,DPSCs were treated using an osteogenic differentiation medium containing 10 ng/ml TNF-?.In the present study,RNA was collected after 7 and 14 days of treatment with TNF-?,when DPSCs were undergoing and completing osteogenic differentiation.The next generation sequencing analysis identified 77 and 133 lncRNAs,with 30 lncRNAs overlapping,which were differentially expressed at days 7 and 14 post-treatment,respectively(Fig.3A).In addition,58 and 73 were upregulated,and 19 and 60 were downregulated at day 7 and 14,respectively.These results demonstrated that lncRNAs underwent transitional alterations during osteogenic differentiation of DPSCs.Profiling of differentially expressed lncRNAs.Subsequently,expression pattern analysis was performed on the differentially expressed lncRNAs during osteogenic differentiation of DPSCs.The bioinformatics analysis identified 16 different profiles(Fig.2B).Among them,six demonstrated statistical significance(Fig.3B).Subsequently,Profile 3 was analyzed for the following reasons:?)This profile demonstrated the highest statistical significance;and ?)notably,the expression levels of all lncRNAs within this profile were increased at day 7 and their expression was maintained at relatively high expression levels until day 14 post-treatment with TNF-?(Fig.3C).Association between differentially expressed lncRNAs and mRNAs during osteogenic differentiation of DPSCs.In a previous study,mRNA alterations at day 7 and 14 post TNF-? induction were observed.Therefore,the present study aimed to investigate how lncRNAs alterations were associated with mRNA alterations during osteogenic differentiation of DPSCs.By bioinformatics analysis,34 lncRNAs were predicted to be associated with 336 mRNA transcripts that underwent significant alterations during osteogenic differentiation(Fig.4).A Kyoto Encyclopedia of Genes and Genomes analysis was performed on these mRNA transcripts(Table ?).Notably,the 'PI3K-Akt signaling pathway' and 'MAPK signaling pathway,,which have key roles in osteoblast differentiation,were identified.To further validate the RNA-Seq results,the expression alterations of four predicted IncRNAs in Profile 3 were measured at 7 and 14 days post TNF-? induction by RT-qPCR.The results demonstrated a concomitant increase of all four IncRNAs at day 7,with different expression alterations at day 14 post-TNF-? induction,which was consistent with RNA-Seq data(Fig.5).IncRNA XIST is required for efficient osteogenic differentiation of DPSCs induced by TNF-?.At present,whether and how IncRNAs are involved in osteoblast differentiation remains unclear.To investigate this,a validated IncRNA,XIST(Fig.5),was selected and it was examined to determine how it may affect osteoblast differentiation.RT-qPCR confirmed that a specific siRNA was able to downregulate XIST expression in DPSCs(Fig.6A).A total of 14 days after TNF-? induction,inhibition of XIST by siRNA in primarily cultured DPSCs significantly decreased the presence of alkaline phosphatase positive osteoblast cells(Fig.6B and C;P<0.01).Therefore,XIST,an IncRNA is required for efficient osteogenic differentiation,possibly through a regulatory role in a group of mRNAs associated with this process.Conclusion(1)The expression of IncRNA was changed during osteogenic differentiation of dental pulp stem cells stimulated by TNF-?.(2)After stimulation with TNF-?,the expression level of lncRNAs increased.(3)The expression of IncRNA and mRNA are closely related in the process of osteogenic differentiation of dental pulp stem cells.(4)LncRNA XIST is required for efficient osteogenic differentiation induced by TNF-?.