The Effects Of Long Non-coding RNA Linc-01135 On The Osteogenic Differentiation Capability Of HPDLSCs From Healthy And Periodontitis Microenvironment Under Static Mechanical Strain Loading

Periodontitis,a highly prevalent inflammatory disease,is characterized by the progressive destruction of tooth-supporting tissues,including alveolar bone,cementum,and periodontal ligament.At present,recent approaches for reconstituting lost periodontal tissue have typically relied on anti-infective measures and regenerative therapies.Periodontal ligament stem cell(PDLSCs)is one of the most important cell sources for periodontal regeneration,and also being highly sensitive to mechanical stimulus.Therefore,they play important role in positive reaction to mechanical stimulation and ensuing tissue remodeling under mechanical loading.However,many studies have proved that inflammatory microenvironment of periodontitis can jeopardize the osteogenic differentiation capability of them and also have negative effect on the reaction to mechanical stimulation.Nowadays,an increasing number of adults pursue esthetic improvement through orthodontic treatment,while this group of people are vulnerable to periodontitis.Therefore,it is of great importance to study PDLSCs’ response to mechanical force in periodontitis microenvironment and guide the orthodontic force loading in clinical practice.Long non-coding RNA(lncRNA)is a type of transcripts that are longer than 200 nt in length.Even though they do not possess the capabilities for protein coding,their indispensable role in regulating biological and pathological processes of diseases have been proved by multiple studies.Furthermore,they were also proved as the promising regulators in stem cells differentiation,but their function in osteogenic differentiation of stem cells are only in inception of studies and their regulation in mechanical response of PDLSCs remain unknown.Our previous finding indicated that the static mechanical strain(SMS)of 12% induced optimal effect in PDLSCs in normal situation.Based on these background,this study aimed at studying the effect of 12%SMS on the oestogenic capability of PDLSCs obtained from healthy periodontal tissues(H-PDLSCs)and PDLSCs obtained from periodontal tissues of periodontitis patients(P-PDLSCs),and studying the function of lnc RNA in regulating osteogenic differentiation of PDLSCs under 12%SMS loading via identifying the differently expressed lnc RNAs in H-PDLSCs and P-PDLSCs through lnc RNA microarray and studying the function of the target one.Part I The effects of 12%SMS loading on the osteogenic capability of H-PDLSCs and P-PDLSCsObjective 1.Isolation and culture of H-PDLSCs and P-PDLSCs.2.Establishment and detection of SMS loading device.3.Study the effects of 12%SMS loading on the osteogenic differentiation capability of H-PDLSCs and P-PDLSCsMethods 1.H-PDLSCs and P-PDLSCs were isolated and cultured by low density inoculation,the expressions of mesenchymal stem cell markers were detected by flow cytometry,proliferative activity was determined by colony-forming assay,the capacity of osteogenic differentiation and adiogenic differentiaion was tested by Alizarin Red staining and Oil Red O staining namely.2.The osteogenic capability of H-PDLSCs and P-PDLSCs after 12%SMS loading was determined by q PCR and Alizarin Red staining.Results 1.H-PDLSCs and P-PDLSCs can be successfully isolated and cultured,both of them presented with long spindle shape under microscopy and strongly expressed the mesenchymal stem cell markers CD29 and CD105,negatively expressed the hematopoietic markers CD45 and CD34.Alizarin Red staining showed that both HPDLSCs and P-PDLSCs generated mineralized nodules,but the osteogenic abilities of H-PDLSCs were stronger than P-PDLSCs.Oil Red O staining showed that both HPDLSCs and P-PDLSCs generated lipid droplets,but the adiogenic abilities of HPDLSCs were stronger than P-PDLSCs.2.The expression of osteogenic gene Runx2 and ALP of H-PDLSCs were significantly improved after 12%SMS loading whereas that of P-PDLSCs did not show significate increase.Alizarin Red staining showed the same trend.Conclusions 1.Both H-PDLSCs and P-PDLSCs expressed mesenchymal stem cell markers and showed osteogenic and adiogenic potentials,but periodontitis microenvironment negatively impacted the osteogenic and adiogenic potentials of P-PDLSCs.2.12%SMS had positive effect on osteogenic abilities of H-PDLSCs but that of PPDLSCs have not been observed.Part II Identification of differently expressed lnc RNAs in H-PDLSCs and P-PDLSCs under 12%SMS loading and osteogenic related target lnc RNAObjective 1.Identify the lnc RNAs that differently expressed lnc RNAs in H-PDLSCs and PPDLSCs under 12%SMS loading.2.Functional analysis and q PCR of differently expressed lnc RNAs in H-PDLSCs and PPDLSCs under 12%SMS loading.3.Identify the target lnc RNA that related to osteogenesis of H-PDLSCs and P-PDLSCs.Methods 1.Arraystar human lnc RNA microarray was employed to indentify the differently expressed lnc RNAs in H-PDLSCs and P-PDLSCs under 12%SMS loading.2.The cells were collected after 12%SMS and RNA of which were extracted by TRIzol.RNA purification and quality testing was performed before inverse transcription.Axon Gene Pix 4000 B was used to array scan and Nimble Scan softwar and Agilent Gene Spring GX software were used to data analysis.3.The database Kyoto Encyclopedia of Genes and Genomes and the Gene Ontology project were used to analyze the function of lnc RNAs.4.q PCR was used to verify the microarray results.5.Osteogenic inductions were performed in both H-PDLSCs and P-PDLSCs,and the expression level of target gene and Runx2 in day 1、3、7、14 in Osteogenic inductions were examined by q PCR.Results 1.Quality testing showed that RNAs of all samples were qualified with the absorbance value in 1.8-2.0.Boxlot diagram indicated that all cell samples had almost the same fluorescence intensity.Scatter diagram and volcano plot indicated that abundant differently expressed lnc RNAs existed in H-PDLSCs and P-PDLSCs after 12% SMS was loading.2.Under 12%SMS loading,the number of up-regulated lnc RNAs in H-PDLSCs was 3920,down-regulated lnc RNAs was 5634;the number of up-regulated m RNAs in HPDLSCs was 7169,down-regulated m RNAs was 5072.Furthermore,the number of up-regulated lnc RNAs in P-PDLSCs was 4079,down-regulated lnc RNAs was 5817;the number of up-regulated m RNAs in H-PDLSCs was 6847,down-regulated m RNAs was 5629.All the aforementioned results were statistically significant and the fold change>2 times.3.GO analysis showed that the involved biological process of differently expressed m RNA in H-PDLSCs and P-PDLSCs included growth and development,response to stimulation and signal communication between cells,etc.,and major part of them located in cytoplasm,cell nucleus and cytomenbrane with the biological functions of cells binding and enzyme binding.KEGG analysis indicated that differently expressed m RNA in H-PDLSCs and P-PDLSCs were mainly involved in cancer regulation and osteogenic pathways such as MAPK signaling pathway and Fox O signaling pathway.4.q PCR results showed that the changing trends of expressed level were almost the same with microarray results but with the difference of change fold.5.linc-01135 significantly improved after osteogenic inductions in both H-PDLSCs and P-PDLSCs,and the expressed level gradually increased in day1 、 3 、 7 、 14 of osteogenic inductions and highly related with the expression of Runx2.Ce RNA analysis indicated that linc-01135 had potential osteogenic mi RNA like mi R-17-5p、mi R-22-3p、mi R-211-5p、mi R-106b-5p、mi R-320 e.Conclusions 1.Abundant differently expressed lnc RNAs existed in H-PDLSCs and P-PDLSCs after 12% SMS was loading.2.linc-01135 had great change during osteogenic differentiation of H-PDLSCs and PPDLSCs,and was considered as the target lnc RNA of the following studies.Part III The effects of long non-coding RNA linc-01135 on the osteogenic differentiation capability of H-PDLSCs and P-PDLSCs under static mechanical strain loadingObjective Determined the regulated effect of linc-01135 on osteogenic differentiation capability of HPDLSCs and P-PDLSCs after 12% SMS loading.Methods 1.Lentivirus were constructed to stably up regulate and down regulate the gene expression of linc-01135 and the transfected conditions of which were examined by fluorescence microscope and q PCR.2.q PCR was used to examine the expression of linc-01135 in H-PDLSCs and P-PDLSCs after 12% SMS loading.3.When the cells were successfully transfected with lentivirus and 12%SMS loading,q PCR was used to examine the expression of osteogenic gene in in H-PDLSCs and PPDLSCs.The osteogenic ability of H-PDLSCs and P-PDLSCs was examined by Alizarin Red staining and ALP staining.4.IL-1β and TNF-α were used to formed inflammatory microenvironment in vitro.q PCR was used to examine the change of linc-01135 expression in stimulated inflammatory microenvironment.Results 1.Under fluorescence microscope,H-PDLSCs and P-PDLSCs that were successfully transfected by lentivirus showed green fluorescence.q PCR showed that up-regulated lentivirus can significantly increase the expression of linc-01135,conversely the downregulated lentivirus decreased the expression of linc-01135.2.Overexpression of linc-01135 in P-PDLSCs can lead to the significant increase of expression level of osteogenic gene Runx2,ALP and OPG.On the contrary,downregulation of linc-01135 decreased the osteogenic gene expression in H-PDLSCs.3.The expression of linc-01135 significantly increased in H-PDLSCs after 12%SMS.Before 12%SMS loading,the expression of lin-01135 in P-PDLSCs was significantly lower than H-PDLSCs,and it did not show obvious increase when 12%SMS was loading.4.When H-PDLSCs was interfered by IL-1β and TNF-α,the expression of linc-01135 significantly decreased and exhibited less increased level than control group after 12%SMS loading.Conclusions 1.linc-01135 can promote the osteogenic ability of H-PDLSCs and P-PDLSCs under 12%SMS loading.2.Inflammatory microenvironment suppressed the expression of linc-01135 and its response to 12%SMS.