The Function Of Primary Cilia In Proliferation And Osteogenic Differentiation And Sensing Static Pressure Of Human Periodontal Ligament Cells

Background:Periodontal ligament is a special and dense connective tissue that contract cementum and alveolar bone.Human periodontal ligament cells(hPDLCs)is a group of cells mainly composed of fibroblasts in periodontal ligament.It has the ability of multidirectional differentiation and self-renewal,which plays an important role in periodontal tissue regeneration and orthodontic tooth movement(OTM).Primary cilia are highly conserved organelles protruding from cell surface in many eukaryotic cells.Defects of cilia leads to developmental abnormalities and human diseases,referred to"cilipathies".The formation and maintenance of primary cilia depends on the Intraflagella Transport(IFT).IFT88 is a core component of EFT-B complex.Depletion of IFT88 inhibits the formation of primary cilia.Studies show that primary cilia play an important role in sensing mechanical stress and reguling bone homeostasis.Objective:Part 1:Knockdown IFT88 to inhibit the formation of primary cilia to explore the function of primary cilia in the process of proliferation and osteogenic differentiation of hPDLCs.Part2:Explore the related ciliary genes and regulation mechanism of primary cilia in the process of hPDLCs under compression by transcriptome analyses.Methods:Part 1:Human periodontal cells(hPDLCs)were cultured and identified.Passage 4-7 hPDLCs were used for experiment.We construct Control shRNA(NC group)and shIFT88(shIFT88 group)successfully and infected them into hPDLCs.The expression of IFT88 was detectecd by qRT-PCR and Western blot.The formation of primary cilia was detected via immunofluorescence.The proliferation of NC group and shIFT88 were examined by plate clone and MTT assay.The expression of osteogenic-related genes COL1A1,ALP,Runx2,BMP2 were detected by qRT-PCR.Part 2:We constructed static pressure model in vitro by simulating orthodontic stress.NC group and shIFT88 group were seeded on 6-well plate.Experiment group loaded with 2g/cm2 static pressure for 4h while control group without static pressure.Total RNA was extracted and used for transcriptome analyses.Then differential expression genes(DEGs)were screened for analysis.Results:1.hPDLCs possess primary cilia.Cilia ratio was 60.27± 1.83%.2.The PKLO.1-shIFT88 was stably expressed in hPDLCs and inhibited the expression of IFT88 effectively(P<0.01).The formation of primary cilia was partly suppressed(P<0.01).3.The proliferation rate and the expression of osteogenic-related genes like COLIA1,ALP,Runx2,BMP2 were decreased significantly(P<0.01)in shIFT88 group.4.Transcriptome analyses showed that 840 DEGs in NC group and 498 DEGs in shIFT88 group were found after 4h compression compared to unloaded cells relatively.5.According to SYSCILIA Gold Standard protein set,we found 8 up-regulated ciliary genes and 2 down-regulated ciliary genes in NC group,no ciliary genes were detected in shIFT88 group.Then we performed KEGG pathway analysis of these DEGs and found that TGF-beta signaling pathway and osteoclast differentiation only enriched in NC group.qRT-PCR were used to verify the expression of partial DEGs,and the change trend was consistent with transcriptome analyses.Conclusion:hPDLCs possess primary cilia.Knockdown IFT88 blocked the formation of primary cilia and inhibited the proliferation and osteogenic differentiation of hPDLCs.Transcriptome analyses showed that only in NC group,ten ciliary genes were differentially expressed.TGF-beta signaling pathway and osteoclast differentiation were enriched in NC group instead of shIFT88 group.These results indicate that primary cilia may participate in the process of hPDLCs under compression by these ciliary genes and pathways.