Lnc-SNHG4 Promotes Osteoblast Differentiation And Alleviates Osteoporosis

Osteoporosis(OP)is a systemic bone metabolic disease characterized by reduction of bone mineral density and deterioration of bone microstructure,which often occurs in middle-aged and elderly people.Considering the aging of population,osteoporosis has increasingly become a public health problem of global concern.More and more studies have shown that long non-coding RNAs(lncRNAs)can regulate gene expression at different levels through a variety of mechanisms,and further involve in many physiological and pathological processes.However,lncRNAs present with low sequence conservation,and few studies have been done on its function in OP.This study aims to find lncRNAs that are dysregulated in the pathogenesis of OP by gene microarray technology,and subsequently to study the mechanism in the pathogenesis and progression of the disease,thus providing new ideas for the diagnosis and therapy of OP.Methods:1.Specimen collection: Bone tissues of OP patients and patients without OP were collected in orthopedic surgery.RNA was extracted and cDNA was reverse transcribed by RT-PCR,and stored at-20℃.2.Screening of dysregulated lncRNA in osteoporotic bone tissue: we have previously identified differentially expressed lncRNAs in ossified ligamentum flavum(OLF)tissue through microarray.Based on the fold change and the lncRNA-mRNA co-expression network,lnc-SNHG4,which is significantly up-regulated in OLF and closely related with osteoblastic differentiation,was identified.The level of this molecule expression in bone tissue of OP patients was detected by qRT-PCR.3.Expression profile of lnc-SNHG4: in NCBI database,lnc-SNHG4 harbors the human,mouse and rat.Ovariectomized-(OVX),tail suspension-(TS),and aginginduced OP mouse models were constructed.RNA from bone tissue was extracted and the transcript in the bone tissue of OP mouse models was detected by qRT-PCR.Human and mouse mesenchymal stem cells(MSCs)and mouse pre-osteoblast MC3T3-E1 cells were induced to osteoblastic differentiate,and total RNA was extracted from the cells at different time points.The level of lnc-SNHG4/lnc-Snhg4 was subsequently detected by qRT-PCR analysis.4.Cellular localization: The cellular localization of lnc-SNHG4/lnc-Snhg4 was determined by the method of nucleoplasmic separation and extraction of cellular RNA.5.Functional study(in vivo): CRISPR/Cas9 assay was used to construct lnc-Snhg4 gene knockout mouse model,micro-CT and histological experiments were used to evaluate the changes of bone mass in mice.At the same time,lnc-Snhg4 gene knockout mice were used to construct OVX and TS mouse models followed by micro-CT and histological experiments to confirm the role of lnc-Snhg4 in the pathogenesis and progressive of OP.6.Functional study(in vitro): siRNA and overexpression plasmid of lnc-SNHG4 were constructed to silence and overexpress lnc-SNHG4 during the osteoblastic differentiation of h MSCs,and qRT-PCR analysis and Western blotting were used to detect the changes of osteogenesis-related genes.In addition,alkaline phosphatase activity and mineral deposition were also detected by alkaline phosphatase staining(ALP)and alizarin red staining(ARS).Lentiviral vectors expressing or silencing lncSnhg4 were constructed and used to infect MC3T3-E1 cells.qRT-PCR analysis and Western blotting were used to detect the changes of osteogenesis-related genes.ALP staining and ARS staining were also used to detect the changes of alkaline phosphatase activity and mineral deposition.7.Mechanism study: Through the transcriptome sequencing with lnc-SNHG4 knock-down h MSCs,lnc-SNHG4 was found to regulate Wnt signaling.The Wnt signaling related components were detected through overexpressing or silencing lncSNHG4/lnc-Snhg4 by qRT-PCR and Western blot technique.Results:1.In this study,we have identified a homogenous lnc-SNHG4,which is significantly up-regulated in OLF by microarray.The level of lnc-SNHG4 was downregulated in bone tissue of OP patients,lnc-Snhg4 was down-regulated in bone tissue of osteoporosis model mice,and up-regulated in MSCs and mouse preosteoblasts during osteogenic differentiation.2.The length of lnc-SNHG4 is 1100 bp,and the length of lnc-Snhg4 is 1151 bp,respectively,both of which have no poly A tails.RNA extracted by nucleoplasmic separation,revealed that lnc-SNHG4/lnc-Snhg4 is localized in both the nucleus and the cytoplasm.3.Lnc-Snhg4 gene knockout mice were constructed,and homozygous knockout mice were obtained during the process of propagation and identification,RNA was extracted from liver,lung and femur tissues and confirmed that lnc-Snhg4 is effectively knocked out.4.Knockout of lnc-Snhg4 induces bone loss in mice through analysis of the bone phenotype of lnc-Snhg4 knockout mice.5.Lnc-Snhg4 promotes osteoblastic differentiation in m MSCs and MC3T3-E1cells;lnc-SNHG4 promotes osteoblastic differentiation in h MSCs.6.Knockout of lnc-Snhg4 aggravates OP induced by ovariectomy(OVX)and tail suspension(TS)in mice.7.Transcriptome sequencing with the lnc-SNHG4 knockdown h MSCs suggests that the genes regulated by lnc-SNHG4 are associated with Wnt pathway.8.lnc-SNHG4/lnc-Snhg4 promotes osteoblastic differentiation by regulating β-catenin,a component of Wnt pathway.Conclusion:Taken together,we have found a homogenous lnc-SNHG4,which is downregulated in bone tissue of OP patients.In vivo study has confirmed that knockout of lnc-Snhg4 induces bone loss in mice and promotes the pathogenesis and progression of OP models induced by OVX and TS.In vitro study shows that lnc-SNHG4 promotes the osteoblastic differentiation of mesenchymal stem cells and osteoblasts.Mechanically,lnc-SNHG4,localized in both the nucleus and the cytoplasm,regulates the expression of CCND1、TCF1、C-MYC、OPG,all of which are the target genes of Wnt pathway.Additionally,it also regulates β-catenin,an important component of Wnt pathway,thus playing a role in promoting OP.This study provides a possible marker and new target for the diagnosis and therapy of OP.