Function And Mechanisms Of Lnc-NR?045553/miR-17-5p/FGD5 In Regulating Ossification Of Ankylosing Spondylitis

BackgroundAnkylosing Spondylitis(AS)is a genetically related autoimmune disease.It is characterized by the inflammation on the early stage.With progression of the disease,spine joint,sacroiliac joint,and hip joint are affected and deformed spine,ankylosed hip,and bony ankylosis occurred,resulting in a bamboo spine,which extremely affects life and motor function and disables AS patients.Researches have demonstrated that the pathological inflammation of AS is involved with bacterial infection,activation of macrophage,certain cytokines,misfolding of HLA-B27 protein,and autophagy.Owing to the difficulty of obtaining lesion tissue,chronic ossification,and large individual varieties,the pathological new bone formation is poorly understand.Inhibition of this chronic progress is still a challenge for scientists.Therefore,exploring the mechanism of abnormal bone formation in AS and developing targeted drugs to intervene and inhibit this mechanism is of greatly clinical significance.It could effectively avoid patients suffering from spinal and articular rigidity and completely improve motor function and quality of life.The non-coding RNA(nc RNA),which were considered to be with little biological function,accounts for 98% of the total hereditary substance.Deep researches indicated large numbers of nc RNAs could regulate gene expression within enormously physiological and pathological activities,of which long non-coding RNA(lnc RNA)and micro RNA(mi RNA)have received more and more attention.Combing with 3' untranslated region of the targeted m RNA,mi RNA restrains translation or directly degrades m RNA to regulate gene expression,including gene of ossification.It has been suggested that lnc RNA is of great importance in physiological activities such as regulation of epigenetic,cell cycle,and cell differentiation.So is the regulation of ossification.Meanwhile,lnc RNA could cross-talk with m RNA mediated by mi RNA in the mode named as competing endogenous RNA.To our best knowledge,there was no research about the mechanism of lnc RNA in AS pathological osteogenesis.We hypothesized that whether lnc RNA could regulate mi RNA/m RNA expression and led to pathological osteogenesis in AS.We mainly focused on lnc-NR?045553/mi R-17-5p/FGD5 and studied the function and molecular mechanism in regulating bone marrow mesenchymal stem cell(MSC)ossification.ObjectivesPart ?: We aimed to comprehensively interpret the gene expression of AS hip joint ligament with gene expression profiling and to determine the differentially expressed(DE)genes for further analysis.Meanwhile,validation of certain DE gene expression was conducted with tissues.Part ?: To obtain the ce RNA affecting ossification of bone marrow MSC and to study the function and mechanism of lnc-NR?045553/mi R-17-5p/FGD5 in ossification.Part ?: To explore the downstream signal pathway involving in osteogensisregulation of FGD5 within bone marrow MSC.Part ?: To study the effect of mi R-17-5p on pathological ossification in animal model of AS.Methods and MaterialsPart ?:(1)The hip joint ligaments were obtained from patients undergoing total hip arthroplasty(THA).There were cut into pieces and stored in liquid nitrogen.(2)After ground into powder and extracted RNA,microarray analysis was conducted to get data.(3)Then the data were deeply explored.The databases on the internet were taken to predict the targeted m RNA of mi RNA and construct mi RNA-m RNA network.Gene ontology(GO)categories and KEGG pathway enrichment analysis were employed to seek pathways related to AS and further to establish mi RNA-GO network.We also built a signal transduction network and lnc RNA-m RNA co-expression network.Finally,according to the correlation and combination of lnc RNA,mi RNA,and m RNA,the ce RNA network in AS was constructed.(4)Part of the DE genes were validated with Real-Time Quantitative Polymerase Chain Reaction(RT-q PCR).Part ?: The bone marrow MSCs were separated on the basis of adherent characteristic.Ficoll was used to extract bone marrow MSCs derived from marrow cavity during the THA.These cells were cultured and amplified until identification purity with morphology and flow cytometry detecting cell marker.(2)The bone marrow MSCs from AS and normal control were induced for ossification.To compare the osteogenesis ability of each group,Alizarin red staining and quantification of Calcium nodules were applied.(3)Validation of lnc-NR?045553,mi R-17-5p,and FGD5 expression levels were conducted with PCR among AS and control groups.(4)The effect of si FGD5 were evaluated with PCR and Western Blot.Then sh FGD5 were constructed.The lentivirus of sh FGD5 and FGD5 were synthetized and validated for effectiveness.(5)We proceeded the gain-and loss-of-function of FGD5 by overexpression and knockdown in bone marrow MSCs from AS.The m RNA expression of ALP,Runx2,and COL1A1 were detected with PCR.The ALP staining and activity quantification,Alizarin red staining and Calcium nodules quantification were used to determine the variety of ossification.(6)The gain-and loss-of-function and rescue experiments of mi R-17-5p by overexpression and knockdown in bone marrow MSCs from AS were conducted.The FGD5 expression was detected with PCR and Western Blot as well.The ALP staining and activity quantification,Alizarin red staining and Calcium nodules quantification were used to determine the variety of ossification.Luciferase reporter assay was used to analyze whether mi R-17-5p could combine with FGD5 m RNA.(7)The location of lnc-NR?045553 was firstly detected with Fluorescence In Situ Hybridization(FISH).Then the gain-and loss-of-function and rescue experiments of this lnc RNA were conducted by overexpression and knockdown in bone marrow MSCs from AS.Lnc-NR?045553,mi R-17-5p,and FGD5 expression were evaluated with PCR and FGD5 protein was determined with Western Blot as well.The ALP staining and activity quantification,Alizarin red staining and Calcium nodules quantification were used to determine the variety of ossification.To determine the combination of mi R-17-5p and lnc-NR?045553,luciferase reporter assay was conducted.Part ?:(1)The experiment was studied among control group,ossification-inducing group,and sh FGD5+ossification-inducing group.Cells were collected after ossification-inducing for three days.Then transcriptome sequencing was performed.(2)To explore the signal pathway involved in osteogenic differentiation,GO and KEGG enrichment analysis of DE gene was compared between ossification-inducing group and sh FGD5+ossification-inducing group.(3)As the key factor in canonical Wnt signaling pathway,the expression of ?-catenin was evaluated with Western Blot in bone marrow MSCs.Furthermore,Wnt-C59,a specific pathway inhibitor for Wnt/?-catenin,was added to perform the rescue experiments.The experiment was divided into six groups,including sh-NC group,sh FGD5 group,LV-NC group,LV-FGD5 group,LV-NC+C59 group,and LV-FGD5+C59 group.(4)The m RNA expression of ALP,Runx2,and COL1A1 were detected with PCR.(5)The ALP staining and activity quantification,Alizarin red staining and Calcium nodules quantification were used to determine the variety of ossification.Part ?:(1)The articular cartilage was obtained from patients undergoing total knee replacement and then extracted for proteoglycan.(2)The proteoglycan induced spondylitis model(PGISp)was constructed and identified with X ray and histological staining.(3)After intervention with mi R-17-5p,histological staining was detected for intervened effectiveness.ResultsPart ?:(1)There DE 611 lnc RNAs,574 m RNAs,and 22 mi RNAs in hip joint ligament between AS and normal control.(2)GO analysis indicated that DE m RNAs and targeted m RNAs of DE mi RNAs were mainly involved in inflammation,activation of cell,angiopoiesis,and ossification which were related to AS pathology.The KEGG analysis suggested AS related signal pathways including cytokine-cytokine receptor,ubiquitin-mediated proteolysis,JAK-STAT signal pathway,TGF-? signal pathway,BMP signal pathway,PI3K/Akt signal pathway,Hippo signal pathway,and HIF-1 signal pathway.(3)The two genes with the highest betweenness centralities,ATM and ITGA2,were derived from signal transduction network.(4)The ce RNA network in AS was composed of six lnc RNAs,eight m RNAs,and six mi RNAs.Part: ?:(1)The morphological and flow cytometry results identified extracted cells as bone marrow MSCs.(2)Compare with normal control,the ossification ability of AS bone marrow MSCs was stronger.(3)The ce RNA axis of lnc-NR?045553/mi R-17-5p/FGD5 was preliminary proved in bone marrow MSC with PCR.(4)The most effective si FGD5 was obtained for constructing sh FGD5.So was the FGD5 overexpression.(5)After overexpression or knockdown of FGD5,PCR results revealed ALP,Runx2,and COL1A1 expressions were positively identical with FGD5.So were the ALP staining and activity quantification,Alizarin red staining and Calcium nodules quantification.These results suggested FGD5 could accelerate ossification of AS bone marrow MSCs.(6)After overexpression or knockdown of mi R-17-5p,FGD5 was negatively expressing.So were the osteogenic genes,ALP staining and activity quantification,Alizarin red staining and Calcium nodules quantification.Furthermore,Luciferase reporter assay confirmed mi R-17-5p could combine with FGD5 m RNA.(7)FISH demonstrated that lnc-NR?045553 was located in cytoplasm.After overexpression or knockdown of lnc-NR?045553,mi R-17-5p negatively expressed while FGD5 positively expressed.The osteogenic genes,ALP staining and activity quantification,Alizarin red staining and Calcium nodules quantification were positively changed as well.Finally,mi R-17-5p was confirmed to combine with lnc-NR?045553.Part: ?:(1)After transcriptome sequencing,GO and pathway enrichment analysis identified that Wnt signaling pathway,which was related to ossification,might involve in regulation osteogenesis of FGD5.(2)Western Blot results revealed that ?-catenin expression was positively identical with FGD5.The Wnt-C59 could rescue ?-catenin against the effect of FGD5.Further experiments such as the osteogenic genes detection,ALP staining and activity quantification,Alizarin red staining and Calcium nodules quantification confirmed FGD5 could regulate ossification through downstream Wnt/?-catenin signal pathway.Part: ?:(1)The X ray and histological staining suggested PGISp was successfully constructed to model bamboo spine and intervertebral fusion well.(2)The overexpression of mi R-17-5p alleviated the destruction of intervertebral disc and pathological bone formation.ConclusionsIn present study,with the above four parts of experiments,we demonstrated lnc-NR?045553 regulated osteogenesis of bone marrow MSC through mi R-17-5p/FGD5 in a ce RNA manner and explored the mechanism during pathological new bone formation in AS.FGD5 could promote the osteogenic differentiation of bone marrow MSC via canonical Wnt/?-catenin.Mi R-17-5p might combine with FGD5 m RNA and negatively regulate FGD5 to inhibit the osteogenesis of MSC.As proved to be located in cytoplasm,lnc-NR?045553 functioning as ce RNA competed with FGD5 for biding mi R-17-5p and then alleviated the inhibitory effect of mi R-17-5p to positively regulate FGD5 and osteogentic differentiation.Meanwhile,our results indicated mi R-17-5p showed slowing-down effect for abnormally spinal bone formation in animal model.These findings could provide theoretical basis in deeply understanding the mechanism leading to pathological ossification in AS and exploring novel therapeutic targets.