Whole-Transcriptome Analysis Of Ovariectomized Osteoporotic Rat Femur

Postmenopausal osteoporosis（PMOP）is a common type of osteoporosis（OP）.It is a systemic metabolic disease characterized by low bone mass,microstructural degeneration of bone tissue,increased bone fragility,and increased fracture susceptibility,owing to the decline of ovarian function in postmenopausal women and hence the estrogen level declines,which leads to bone resorption greater than bone formation.With the deepening of research,more and more evidences show that non-coding RNA（ncRNA）,especially miRNA,lncRNA and circRNA,can participate in a variety of biological processes of OP and play important regulatory functions.Competing endogenous RNA（ceRNA）means that lncRNA/circRNA can competitively bind to miRNA with the mRNA encoding protein,so as to realize mutual communication and regulation,and then form a huge ceRNA network.Until now,there are no studies of ceRNA network construction based on the whole-transcriptome sequencing of bone tissue.Therefore,the purpose of this study was to screen differential RNAs and construct ceRNA network through high-throughput whole-transcriptome sequencing of femurs of ovariectomized osteoporotic rats and sham-operated rats,and to verify the results.Methods:1.1 Three-month-old healthy female Sprague-Dawley rats（n=50）were randomly divided into ovariectomized group（OVX group,n=25）and sham-operated group（SHAM group,n=25）.Bilateral ovariectomies were performed in OVX group,and the adipose tissue around the ovary was removed in SHAM group.Twelve weeks after operation,all rats were killed and bilateral femurs were obtained.Ten rats in OVX group and ten rats in SHAM group were randomly selected for micro-CT to verify the effectiveness of the models.1.2 Three femur tissues of the each groups were randomly selected for high-throughput whole-transcriptome sequencing.The differential mRNAs,lncRNAs,circRNAs and miRNAs were screened by bioinformatics analysis and data visualization was performed.1.3 Two up-regulated and down-regulated mRNAs,lncRNAs,circRNAs and miRNAs were selected respectively in the sequencing results for further verification by quantitative real-time PCR（qRT-PCR）.2.1 Differential mRNAs,lncRNAs,circRNAs and miRNAs were integrated through bioinformatics analysis to construct PMOP-associated networks of lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA.2.2 The target mRNAs in ceRNA networks were analyzed by Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）.The first 10 items of GO analysis at each domains and the first 30 items of KEGG analysis were obtained,and data visualization was performed.2.3 Protein-Protein Interaction（PPI）networks were constructed based on the target mRNAs in ceRNA networks,and hub genes were screened and visualized.3.1 The lncRNA NR₀37614/miR-29b-3p/Col3a1 pathway in the lncRNA-miRNA-mRNA network was selected for further study.Total RNA was extracted from osteoblasts and bone marrow mesenchymal stem cells of ovariectomized osteoporotic rats and sham-operated rats.qRT-PCR was used to detect the RNA levels and compared with sequencing results.3.2 The levels of miR-29b-3p and Col3a1 were detected in osteoblasts after NR₀37614 silenced.3.3 Dual luciferase reporter assay was conducted:osteoblasts were transfected with miR-29b-3p NC or miR-29b-3p mimics,respectively,and the luciferase activity of wild-type/mutant NR₀37614 and wild-type/mutant Col3a1 were detected.3.4 The levels of miR-29b-3p and Col3a1 were detected in osteoblasts after miR-29b-3p overexpressed or silenced.3.5 Rescue experiment was conducted:osteoblasts were treated with different protocols（blank control,transfected with shNC,transfected with shNR₀37614,transfected with shNR₀37614 and miR-29b-3p NC,transfected with shNR₀37614 and miR-29b-3p inhibitors）,and the levels of Col3a1 were detected.Results:1.1 Micro-CT test results showed that there were statistical differences in bone microstructure parameters between the OVX group and the SHAM group,and the micro-CT scan showed that the femoral trabeculae of the OVX group was significantly sparser than that of the SHAM group,indicating that the model of the ovariectomized osteoporotic rats with was successfully constructed,which could be used for subsequent studies.1.2 Whole-transcriptome sequencing results showed that there were 528 differential mRNAs,368 differential lncRNAs,17 differential circRNAs,and 202 differential miRNAs between the OVX group and the SHAM group.1.3 Two mRNAs,lncRNAs,circRNAs,and miRNAs that were up-regulated and down-regulated in the sequencing results were selected respectively and tested by qRT-PCR.The results showed that the corresponding RNA expression trends were consistent with the sequencing results.2.1 Differential mRNAs,lncRNAs,circRNAs,and miRNAs were integrated through bioinformatics analysis to construct PMOP-associated lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA networks.2.2 In the lncRNA-miRNA-mRNA network,the main items enriched by GO analysis were response to mechanical stimulus,cellular calcium ion homeostasis,response to gravity,proteinaceous extracellular matrix,collagen trimer,extracellular matrix,extracellular matrix structural constituent,sodium ion binding,cation channel activity,etc.The main items enriched by KEGG analysis were protein digestion and absorption,focal adhesion,PPAR signaling pathway,etc.The hub genes were Acta2,Mylpf,Myll,Myocd,Agt,Col3a1,Klhl41,Tpm2,Trdn,Eno3,etc.2.3 In the circRNA-miRNA-mRNA network,the main items enriched by GO analysis were response to mechanical stimulus,cellular calcium ion homeostasis,mesenchymal migration,proteinaceous extracellular matrix,junctional membrane complex,extracellular matrix,extracellular matrix structural constituent,extracellular matrix binding,calcium ion binding,etc.The main items enriched by KEGG analysis were protein digestion and absorption,calcium signaling pathway,insulin signaling pathway,etc.The hub genes were Tnni2,Myl1,Acta2,Tpm2,Col3a1,Atp2a1,Pvalb,Klhl41,Trdn,Pygm,etc.3.1 The qRT-PCR results of rat osteoblasts and bone marrow stem cells showed that the expression trends of NR₀37614,miR-29b-3p and Col3a1 were consistent with the sequencing results.Owing to the difference of the related RNA in osteoblasts is more significant,the subsequent experiments are based on osteoblasts.3.2 After silencing NR₀37614,the level of miR-29b-3p in osteoblasts was significantly up-regulated,and the level of Col3al was significantly down-regulated.3.3 The dual-luciferase reporter assay confirmed that miR-29b-3p could specifically bind to NR₀37614 and Col3a1:overexpression of miR-29b-3p significantly inhibited the luciferase activities of wild-type NR₀37614 and Col3a1,while the luciferase activities of mutant NR₀37614 and Col3a1 did not significantly change.3.4 After overexpression of miR-29b-3p,the level of miR-29b-3p in osteoblasts was significantly up-regulated,and the level of Col3al was significantly down-regulated.After the silencing of miR-29b-3p,the level of miR-29b-3p was significantly down-regulated and the level of Col3al was significantly up-regulated.3.5 The results of rescue experiment showed that the Col3al level in osteoblasts was significantly down-regulated after NR₀37614 silencing,while the down-regulation of Col3a1 was significantly reversed by simultaneously silencing NR₀37614 and miR-29b-3p.Conclusion:1.Whole-transcriptome sequencing was performed on the femur tissues of the ovariectomized osteoporotic rats and the sham-operated rats,and 528 differential mRNAs,368 differential lncRNAs,17 differential circRNAs and 202 differential miRNAs were screened out.These differential RNAs are helpful to further explore the underlying mechanism of osteoporosis.2.After further analysis of differential RNA in the whole-transcriptome sequencing results,the PMOP-associated lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA networks were constructed.These data are helpful to further explore the mechanism of lncRNA and circRNA in osteoporosis from the perspective of competitive endogenous RNA.3.In osteoblast,lncRNA NR₀37614 acts as a ceRNA to competitively bind miR-29b-3p and regulate the expression Col3a1.