Long Non-Coding RNA-ANRIL Regu Lates Proliferation, Apotptosis And Osteogenesis In MSCs And Its Involvement In Osteoporosis

Osteoporosis is clinically regarded as a common metabolic bone disease with increased bone fragility,leading to increased fracture risk observed in patients,which in turn seriously affects the quality of life of patients and increases the social medical and economic burden.Because of its complex pathological mechanism,although a variety of Chinese and Western medicine prevention and treatment measures have been widely used,it is still a major clinical orthopedic problem and challenge.In recent years,advances in molecular biology of osteoporosis,such as long-chain non-coding RNA,lay a solid foundation for disease cognition and treatment.Therefore,this study proposes the hypothesis that "regulation of PRC2-CDKN2B by long chain non-coding RNA-ANRIL" might play an important role in the pathogenesis and treatment of osteoporosis.This study confirms the involvement of IncRNA-ANRIL in osteoporosis,and has d opened up new ideas for its treatment,which has important clinical significances.1 Long-chain non-coding RNA-ANRIL combines with PRC2 to regulate CDKN2B methylation in human BMSCss and its effect on bone remodeling related-genes expression.2 To analyze the effect of LncRNA-ANRIL on osteogenesis in human BMSCs.3 Analysis and correlation value of ANRIL-PRC2-CDKN2B in osteoporosis.Objectives 1To construct shRNA-ANRIL transfected human BMSCs model and to explore its biological effects.Objectives 2To study the effect of shANRIL and icariin on human BMSCs osteogenesis.Objectives 3To analyze the expression and correlation of ANRIL and CDKN2B in bone samples of patients with osteoporosis.Experimental methods 1The recombinant plasmid of shRNA-ANRIL was constructed and verified in vitro.The BMSCs was transfected into human BMSCs.The methylation level of CDKN2B gene was detected by BSP.The binding of PRC2 complex to ANRIL and CDKN2B was detected by RIP and CHIP respectively.Activity-changes;qPCR and western blot detection of genes and proteins expression.Experimental methods 2To obtain BMSCs and induce differentiation:firstly,the effect of ANRIL on human BMSCs differentiation was verified,and the group was divided into control group and shANRIL group.The mechanism of icariin was detected in the control group and icariin group.Finally,the above two groups are divided into four groups.The test contents were:cck-8 experiment,alizarin red staining and other analysis of BMSCs proliferation activity and differentiation degree;Gene and protein expression were detected by qPCR and western blot.Gene chip technology is used to screen differentially expressed genes.BSP technology detects methylation changes of OPG and RNAKL promoter fragments.Experimental methods 3The clinical acquisition of postmenopausal osteoporosis patients group(SOP),in patients with postmenopausal osteoporosis(PMOP)and control group in vertebral cancellous bone samples,after the analysis of cases of disease information,using qPCR,immunohistochemistry and Western blot analysis ANRIL PRC2-CDKN2B and osteoporosis related gene and protein expression level,statistical analysis of the correlation conclusion.Experimental results 1The recombinant plasmids were constructed by silencing ANRIL by shRNA technology.Plasmid sequencing confirmed the nucleotide sequence of the inserted fragment was in accordance with the presetting conditions.QPCR showed that the transcriptional level of ANRIL was significantly reduced after shRNA silencing.After silencing ANRIL by shRNA,the methylation level of CDKN2B gene was decreased.And the demethylation sites were screened out.The combination of ANRIL and PRC2 complex was reduced as demonstrated by RIP technique.And the binding of CDKN2B and PRC2 complex was found to be increased by CHIP technique.The decreased CCK-8 absorption rate of BMSCss after shANRIL treatment suggested the proliferative activity was inhibited.The expression levels of ALP,Runx2,Osterix,OPG,OCN,TGF ?1 and CDKN2B were decreased after ANRIL silencing.The levels of Smad2/3,MAPK JNK and ERK phosphorylation were decreased in gene transcription and protein expression.BMSCsExperimental results 2shRNA-ANRIL was used to induce BMSCs in different stages of osteoblastic differentiation.Cell proliferation activity decreased only in the early stage and recovered in late stage.shANRIL can promote ALP peaks,the promotion of OPG gene expression,but inhibit protein expression in the late differentiation,promote the OCN,Runx2,Osterix gene and protein expression,TGF? 1 gene transcription,middle-late promote protein expression and improve Smad2/3 phosphorylation proportion,and reduce MAPK ERK and JNK protein phosphorylation.Gene-Chip assay screened 9548 target genes such as Macfl that bind to CDKN2B in human BMSCs with good reliability.Significant structures such as zinc finger domain were identified by I PR gene structure annotation system;MAPK signal pathway and other related enrichment pathways were obtained through annotation analysis of KEGG pathway enrichment;signaling mediation and other related functions were identified through GO functional annotation.Gene function network analysis map was depicted.BSP technology was used to detect shANRIL role,OPG promoter site 1 and 5 two pieces and RANKL promoter site 7 exist methylation change,suggests that ANRIL may through regulating OPG CDKN2B methylation and RNAKL segments of the promoter methylation,affect the osteogenic differentiation.Icariin in concentrations' of specific time to promote BMSCs proliferation,improve the activity of ALP in the early BMSCs osteogenetic differentiation process,FCM and alizarin red staining results showed that icariin promote calcium deposition in the cell.RIP technology to detect icariin inhibits ANRIL combined with PRC2,icariin inhibits ANRIL and improve CDKN2B expression,increased ALP,Runx2,Osterix,OPG,OCN,TGF ?1 gene and protein expression,improve Smad2/3 phosphorylation proportion,differentiated early,mid-improve the MAPK ERK and JNK protein phosphorylation.Icariin promotes BMSCs proliferation and shRNA-ANRIL inhibits cell proliferation,and its mechanism is associated with non-anril mediated E2F1,CDk4 and CDk6 transcription factor regulation.Osteogenesis differentiation,icariin with shRNA-ANRIL effect similar to inhibit ANRIL transcription,thus raising CDKN2B gene expression,promote into osseous transcription factor ALP,Runx2,OPG and OCN,TGF ?1 protein expression,the access mechanism may be related to Smad2 3 and MAPK/ERK phosphorylation and JNK regulation,in different period in specific time to improve the activity of BMSCs osteogenetic differentiation.Using shANRIL and icariin after intervention osteogenesis in the process of BMSCs differentiation at the same time,the osteogenesis iconic gene and protein expression and CDKN2B collaborative expression of gene transcription,showed that icariin by ANRIL BMSCs differentiation effect into full play.Specifically,ANRIL gene silence,icariin CDKN2B genes transcription in promoting effect enhancement,but cycle regulation factor E2F1 and decreased expression of CDk4,shANRIL promote icariin of ALP secretion peak in advance,and promote the OPG,OCN,Runx2 and Osterix enhanced effect of gene and protein expression,at the same time,TGF ?1 and Smad2/3 phosphorylation also appear similar changes,prompt its possible mechanism.Experimental results 39 cases of vertebrae were collected,and 3 cases were treated in the control group,SOP group and PMOP group.The results of basic information analysis of clinical cases showed that the age of PMOP group was higher than that of control group,while BMD index of PMOP group and SOP group was lower than that of control group.In the four and five tests of bone markers,only PMOP group's 25-hydroxy vitamin D and type I collagen amino terminal prolongation peptide were higher than the control group,and the serum magnesium ion was lower than the SOP group.The overall results showed that the cases included in the study were representative and were suitable for further genetic protein analysis.QPCR gene expression analysis result shows that the ANRIL PMOP group and SOP transcription activity is stronger than the control group,but PMOP ANRIL transcriptional expression below SOP group,the above trend also found in bone metabolic gene ALP,RANKL,RANK,AP-1,Scr,and expressed the nf-kappa B p50 detection.The immunohistochemical staining of Runx2,OPG and RANKL protein was performed after the biopsy,and the results showed that the Runx2 and OPG in the control group were strongly positive and widespread,while the RANKL was weakly positive.The expression and distribution of Runx2 and OPG in SOP and PMOP group were decreased,and RANKL was strongly positive.The expression distribution of OPG and RANKL in PMOP group was lower than that in SOP group.In Western blot test results,the protein expression of ALP,RANKL,RANK,ap-1,Scr,and NF of the PMOP group was also lower than that of SOP group.The results of correlation statistical analysis showed that the transcriptional level of ANRIL was negatively correlated with OPG transcriptional activity,while the transcription level of CKDN2B was positively correlated with OPG translation activity.Transcriptional levels of ANRIL were positively correlated with the gene transcription and protein expression levels of ALP,RANK,RANKL,Scr,NF-kappa B p50,and there was a positive correlation with the expression level of ap-1 protein.Experimental conclusion 1The shANRIL recombinant plasmid was constructed successfully in vitro and successfully transfected into human BMSCss.ANRIL plays a regulatory role in the "ANRIL combines with PRC2 to inhibit CDKN2B methylation";ANRIL can promote cell proliferation and inhibit apoptosis;ANRIL up-regulates cell cycle factor expression,inhibits osteoblast differentiation factor expression and activates Smad2/3,MAPK ERK/JNK cell pathway.Experimental conclusion 2Icariin through regulating the ANRIL-CDKN2B way a specific time to promote BMSCs osteogenetic differentiation,specific to promote into osseous transcription factor ALP,Runx2,OPG and OCN gene protein expression and its distribution mechanism may be related to TGF?1-Smad2/3 and MAPK ERK phosphorylation and JNK regulation.Experimental conclusion 3higher clinical ANRIL transcription activity in patients with osteoporosis and menopause,inhibit the table it was ANRIL RNA gene transcription and OPG,ALP,RANK,RANKL,AP-1,Scr,the nf-kappa B p50 transcription factor has significant correlation,show ANRIL control significance in the clinical bone osteoporosis pathogenesis,has further understand the mechanism of osteoporosis and the potential development of drugs.