The Effect Of LncRNAHHIP-AS1 On The Osteogenic Differentiation,Migration,and Chemotaxis Of Periodontal Ligament Stem Cells And The Regulatory Mechanism

PurposeAlveolar bone remodeling under orthodontic force can be achieved by periodontal ligament stem cells(PDLSCs).How to regulate the function of PDLSCs is a key issue in bone remodeling during orthodontic tooth movement.This study aimed to investigate the expression of long non-coding RNA(lncRNA)Hedgehog-interacting protein antisense RNA 1(HHIP-AS1)under compressive pressure,the roles of HHIP-AS1 in the osteogenic differentiation,migration,and chemotaxis of PDLSCs,and the regulatory mechanism.Materials and Methods1.Human PDLSCs were isolated and cultured.Flow cytometry was used to identify the mesenchymal stem cell surface markers.2.Glass panes were used to apply continuous compressive pressure on PDLSCs and cell morphology was observed under a microscope.The expression of HHIP-AS1 under continuous compressive pressure was detected by real-time reverse transcriptase polymerase chain reaction(real-time RT-PCR).3.HHIP-AS1 was overexpressed by infecting PDLSCs with a lentiviral construct expressing HHIP-AS1,and a shRNA was designed to target HHIP-AS1 and introduced into PDLSCs with lentiviral infection to knock down HHIP-AS1.HHIP-AS1 expression was confirmed using realtime RT-PCR.4.HHIP-AS1-depleted PDLSCs and HHIP-AS1-overexpressed PDLSCs were respectively induced to osteogenic differentiation,and the osteogenic differentiation indicators were detected.The alkaline phosphatase(ALP)activity assay,Alizarin Red staining,and quantitative calcium analysis were conducted.Osteogenic markers,including bone sialoprotein(BSP)and osteocalcin(OCN),and a key transcription factor osterix(OSX)were detected using Western blot.5.The scratch migration assay,transwell chemotaxis assay,and quantitative analysis were performed to assess migration and chemotaxis abilities.6.HHIP-AS 1-depleted PDLSCs were used for RNA sequencing(RNA-seq).The bioinformatic analysis was performed to detect downstream genes,biological processes,and key signaling pathways.Results1.The PDLSCs expressed mesenchymal stem cell marker CD90 and were negative for CD34 and CD45.2.The morphology of PDLSCs was changed and HHIP-AS1 was downregulated in PDLSCs at 2,4,and 6 h under continuous compressive pressure compared to that in PDLSCs under no pressure.3.HHIP-AS1 was efficiently silenced or overexpressed in PDLSCs.During the osteogenic differentiation,the ALP activity was enhanced and the mineralization of PDLSCs was promoted by HHIP-AS1.The expressions of BSP,OCN,and OSX were significantly upregulated in PDLSCs by HHIP-AS1.4.HHIP-AS1 inhibited the migration of PDLSCs at 24 h and 48 h after scratching.HHIP-AS1 inhibited the chemotaxis of PDLSCs at 48 h after PDLSCs were added to Transwell chambers.5.The RNA-seq data showed differentially expressed mRNAs including ROR2,CXCL12,and FGF5,and differentially expressed lncRNAs including NEAT1 and LINC00973,in HHIPAS1-depleted PDLSCs.Then,ROR2 and CXCL12 were upregulated and FGF5 was downregulated in PDLSCs at 2 h under continuous compressive pressure compared to that in PDLSCs under no pressure.Furthermore,bioinformatic analysis revealed several biological processes and signaling pathways related to HHIP-AS1 functions,including the PI3K-Akt signaling pathway and JAK-STAT signaling pathway.Conclusions1.HHIP-AS1 was downregulated in PDLSCs under compressive pressure.2.HHIP-AS1 promoted the osteogenic differentiation potential and inhibited the migration and chemotaxis abilities of PDLSCs.3.HHIP-AS1 may be a candidate target for accelerating tooth movement during orthodontic treatment.