LncRNA GAS5 Inhibits Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells Through MiR-382-3p/TAF1 Axis

Background: Bone marrow mesenchymal stem cells（BMSCs）,often referred to as the "seed cells" in bone tissue engineering,have garnered enormous interest from researchers due to complete self-renewal throughproliferation and differentiation,low immunogenicity,multi-directional differentiation ability,and excellent transplantation ability.Under specific conditions,BMSCs can be differentiated into chondrocytes,osteoblasts,and adipocytes,among others.The specific directional induction of BMSCs into osteoblasts can be employed for bone remodeling,fracture repair,and osteoporosis.Typically,the long non-coding RNAs（lnc RNAs）,a class of endogenous transcriptional molecules witha lengthof more than 200 nucleotides,are involved in cell biology at the transcriptional,post-transcriptional,and epigenetic levels.The regulation of miRNAs as molecular sponges is one of the essential biological functionalities of lnc RNAs.Recently,a study demonstrated that the lnc RNAs were significantly correlated withthe osteogenic differentiation of hBMSCs.A total of over200 lnc RNAs were differentially expressed during the osteogenic differentiation of hBMSCs.Among various lnc RNAs available,the growtharrest-specific transcription factor 5（GAS5）plays a crucial role in the proliferation and differentiation of stem cells.The latest researchobserved that GAS5 substantially promoted the osteogenic differentiation of human periodontal ligament stem cells by regulating the GDF5 and p38/JNK signaling pathways.In another instance,the upregulated expression of GAS5 in BMSCs significantly promoted their osteogenic differentiation by regulating the miR-135a-5p/FOXO1 pathway.Notably,the up-regulation of GAS5 expression in patients withosteoporosis promotes the osteogenic differentiation of hBMSCs throughup-regulation of RUNX2 expression by miR-498,thereby alleviating the osteoporosis development.Our previous report predicted the potential target miRNAs of GAS5 throughthe star Base V2.0（http://starbase.sysu.edu.cn/index.php）.It was observed that various miRNAs（miR-382-3p,miR-205-5p,miR-223-3p,miR-455-5p,and miR-433-3p）could be used as targets of lnc RNA GAS5.The miR-382-3p levels in hBMSCs were most significantly upregulated after silencing GAS5 in the osteogenic induction of hBMSCs.Therefore,in this study,miR-382-3p is selected as a target gene of GAS5 to explore the role and possible mechanism of GAS5/miR-382-3p in the process of osteogenic differentiation of hBMSCs.Objective: We firmly believe that these investigations exploring the occurrence and development of the disease provide experimental design and theoretical basis.Methods:1.Construction of osteogenic differentiation model of BMSCs Human BMSCs（hBMSCs）were incubated in the osteogenic growthmedium（GM）supplemented with0.05 mmol/L of ascorbic acid,100 mmol/L of dexamethasone,and 10 mmol/L of β-glycerophosphate.Further,the expression levels of three critical osteogenic gene markers（RUNX2,OSX,and ALPL）in hBMSCs were detected on 0,3,7,14,and 21 days,respectively,to evaluate their effect on osteogenic differentiation.2.Evaluation of the GAS5 effect on the osteogenic differentiation of hBMSCs To explore the effects of lnc RNA GAS5,reverse transcription-quantitative polymerase chain reaction（RT-q PCR）,and Western blot analyses were used to detect the expressions of osteogenesis-related genes（RUNX2,OSX,and ALPL）and the proteins（RUNX2 and OSX）after silencing or overexpressing GAS5 in hBMSCs.The alkaline phosphatase（ALP）assay kit was utilized to detect the ALP activity.The Alizarin red staining was used to detect calcium nodule formation in cells,and ALP staining was used to detect ALP expression.3.Role of lnc RNA GAS5 in regulating the osteogenic differentiation of hBMSCs by targeting miR-382-3p Initially,the potential miRNAs of GAS5 were predicted by employing the star Base V2.0 database（http://starbase.sysu.edu.cn/index.php）.Further,the effect of GAS5 silencing on the expression levels of candidate miRNAs was detected by RT-qPCR analysis.The relative expression level of target gene miR-382-3p during osteogenic differentiation,RNA pull-down experiment,and RNA immunoprecipitation assay（RIPA）was recorded to verify the relationship between GAS5 and miR-382-3p.Then,the gene transfection experiment was used to detect the effect of GAS5 silencing or overexpression on the miR-382-3p expression.Further,the intervention of the applied miR-382-3p inhibitor on the expression of osteogenic differentiation-related genes（RUNX2,OSX,and ALPL）was explored.4.Role of lnc RNA GAS5 in regulating the osteogenic differentiation of hBMSCs by targeting miR-382-3p/TAF1 Initially,the target gene of miR-382-3p was predicted online by a star Base database,and then the Dual-Luciferase reporter gene assay was performed.The RIPA experiment was used to verify the relationship between miR-382-3p and TAF1.Further,the Western blot and RT-q PCR analyses were used to detect the effect of the miR-382-3p expression on TAF1.The effect of TAF1 overexpression on RUNX2 and OSX protein expression levels in hBMSCs was observed.Then,the effect of knockdown of TAF1 on ALP activity,calcium nodule formation,and ALP content was explored;Finally,the detected promoter sequence of GAS5 was screened（Q < 0.01,Score > 20）for potential GAS5 promoting TAF1 binding site on the Human TFBD website（http://bioinfo.life.hust.edu.cn/Human TFDB/#!/）.The ChIP experiment was used to verify the targeting relationship between the two,and RT-q PCR to detect the effect of TAF1 on GAS5 expression.Results:1.Osteogenic differentiation of hBMSCs is successfully induced.The osteogenic differentiation of hBMSCs was successfully induced by applying an osteogenic differentiation medium.Further,RT-q PCR analysis indicated that the expression levels of three vital osteogenic genes（RUNX2,OSX,and ALPL）showed an increasing trend withthe incubation time（0,3,7,14,and 21 days）.2.Overexpression of GAS5 significantly inhibited the osteogenic differentiation of hBMSCs.Further,the expression levels of all the transcript variants（T1^T15）of GAS5 in hBMSCs were examined on day-0 and 7 in the osteogenic differentiation medium.It was observed that the expressions of transcript variants T6,T11,and T13 were significantly upregulated,while the expression of body T2 was significantly down-regulated in hBMSCs incubated on day-7.Moreover,the mRNA expressionlevels the RUNX2,OSX,and ALPL were upregulated after GAS5 silencing and vice versa.However,the expressions of RUNX2 and OSX protein levels were upregulated after GAS5 silencing but not after GAS5 overexpression.Several interesting characteristics were observed,in whichknockdown of GAS5 significantly increased ALP activity,calcium nodule formation,and ALP content in hBMSCs.Contrarily,the overexpression of GAS5 resulted in their significant inhibition,indicating the negative regulatory effect on the osteogenic differentiation of hBMSCs.3.miR-382-3p is a compelling target gene of GAS5.It was observed from the potential miRNA prediction results of GAS5 throughthe star Base V2.0 website（http://starbase.sysu.edu.cn/index.php）that miR-382-3p,miR-205-5p,miR-223-3p,miR-455-5p,and miR-433-3p could substantially act as potential target genes of GAS5.The miR-382-3p levels in cells were significantly upregulated after silencing GAS5.Therefore,miR-382-3p was determined as a potential target gene of GAS5 for subsequent experiments.Further,the RNA pull-down and RIPA experimental results indicated that GAS5 showed a substantial targeted relationship withmiR-382-3p.In addition,the RT-q PCR analysis displayed that the overexpression of GAS5 could inhibit the expression of miR-382-3p and transfection of miR-382-3p mimics.Nonetheless,the application of miR-382-3p inhibitor displayed a significant effect on the GAS5 expression in hBMSCs.4.miR-382-3p mediated the regulation of GAS5 in the osteogenic differentiation of hBMSCs.Initially,it was observed that the miR-382-3p level in the osteogenic medium was significantly higher than that in the GM medium.Further,the gene transfection-assisted improved expression of miR-382-3p could significantly upregulate the expression levels of RUNX2,OSX,and ALPL.After inhibiting the expression levels of the above indicators,it was suggested that miR-382-3p could promote the osteogenic differentiation of hBMSCs.On the contrary,the knockdown of GAS5 significantly upregulated the expressions of RUNX2,OSX,and ALPL,indicating that GAS5 expression was negatively correlated withosteogenic differentiation.To verify the function of the GAS5/miR-382-3p axis in the osteogenic differentiation of hBMSCs,the rescue experiment was performed,indicating that the co-transfection of miR-382-3p inhibitor could partially inhibit the osteogenic differentiation by knockdown of GAS5.In addition,the luciferase activity assay demonstrated that the overexpression of miR-382-3p was reduced by GAS5,which,however,could not be reversed by the overexpressed GAS5（Mut）.5.TAF1,as a downstream target gene of miR-382-3p,is involved in the osteogenic differentiation of hBMSCs.First,the star Base software was used to predict the relationship between TAF1 and miR-382-3p.Further,the luciferase reporter gene assay showed that the luciferase activity of TAF1-Wt transfected cells was significantly higher than miR-382-3p mimics transfected withTAF1-Wt.It was significantly decreased after miR-382-3p knockdown and vice versa.Further,the Western blot and RT-q PCR analyses showed that the protein and gene expression levels of TAF1 were inhibited withthe overexpression of miR-382-3p,whichremained ineffective in the case of miR-382 overexpression.Further,RIP assays were utilized to determine the roles of the GAS5 and miR-382-3p/TAF1 axis in osteogenic differentiation.It was observed that GAS5 could substantially bound to miR-382-3p and released TAF1 inhibition.Moreover,the silencing of TAF1 could significantly upregulate the expression levels of RUNX2 and OSX proteins in hBMSCs,while TAF1 overexpression could inhibit the above expressions.In addition,TAF1 overexpression could significantly inhibit the ALP activity,calcium nodules,and ALP content,suggesting TAF1 as the target gene of the GAS5/miR-382-3p axis in the osteogenic differentiation of hBMSCs.Finally,our findings indicate that TAF1 shows a great potential to bind to the GAS5 promoter in hBMSCs,in whichTAF1 activates GAS5 transcription and regulates GAS5 expression.Moreover,GAS5,miR-382-3p,and TAF1 could form a positive regulatory feedback loop in hBMSCs and negatively regulate osteogenic differentiation.Conclusion:1.In summary,the lnc RNA GAS5 showed a negative regulatory effect on the osteogenic differentiation of hBMSCs.The expressions of GAS5 transcript variants 6,11,and 13 were significantly upregulated,while the expression of transcript variant 2 was significantly down-regulated.2.It was observed that miR-382-3p was the downstream target gene of Lnc RNA GAS5.Increased expression of miR-382-3p could significantly upregulate the RUNX2,OSX,and ALPL expression levels and promote the osteogenic differentiation of hBMSCs.Together,the binding of lnc RNA GAS5 to miR-382-3p significantly inhibited the osteogenic differentiation of hBMSCs.3.As TAF1 is a downstream target gene of miR-382-3p,overexpression of miR-382-3p could significantly inhibit the expression of TAF1 mRNA and protein.Moreover,the overexpression of TAF1 could significantly inhibit the osteogenic differentiation ability of hBMSCs.4.In general,TAF1 activates GAS5 transcription by binding to the GAS5 promoter,and feedback regulates the expression of GAS5.Thus,GAS5,miR-382-3p,and TAF1 could form a positive regulatory feedback loop in hBMSCs and negatively regulate osteogenic differentiation.