| Objectives:Porphyromonas gingivalis,which is one of the main pathogens of periodontal diseases,can invade into human gingival epithelial cells(HGECs),disrupting periodontal homeostasis and inducing immuno-inflammatory responses.Previous study demonstrated that P.gingivalis infection can increase autophagy in epi4 cells,which is an immortalized HGECs line.Long non-coding RNA(lncRNA)TGFB2OT1 plays a role in autophagy regulation,which may also be involved in P.gingivalisinduced autophagy in epi4 cells.This study detected the expression of TGFB2-OT1 in epi4 cells infected by P.gingivalis and its effect on P.gingivalis-induced autophagy in epi4 cells,aiming to preliminarily investigate the possible mechanism of P.gingivalis affecting autophagy of HGECs and provide a new direction for autophagy-related bacteriostatic treatment of periodontal diseases.Methods:1.Epi4 cells were infected by P.gingivalis(MOI 100:1)for 0,2,6,12,24,48 h.QRT-PCR was employed to detect the expression of TGFB2-OT1.2.Transient transfection of siRNA was used to obtain the low expression of TGFB2-OT1 in epi4 cells.Cells were divided into three groups:the blank control group,the negative control group(si-scrambled)and the experiment group(si-TGFB2-OT1).The transfection efficiency was examined by flow cytometry after 6 h of transfection.The RNA levels of TGFB2-OT1 were measured by qRT-PCR after transfection for 24 h and 48 h,and P.gingivalis infecting transfected cells 24 h.3.The expressions of autophagy-related proteins LC3 and P62 were examined using western blot after P.gingivalis(MOI 100:1)infected each group cells for 24 h.4.The LncBook and miRWalk databases were used to predict the miRNAs interacting with TGFB2-OT1 and LC3,respectively,and the ceRNA network map was constructed.The interaction between LC3 and P62 protein was analyzed by STRING database.Results:1.After P.gingivalis infection,the expression of TGFB2-OT1 in epi4 cells enhanced significantly after 12 h and eached to a peak at 24 h,which is 2.31 times of uninfected control group(P<0.01).And then,the TGFB2-OT1 level declined.2.The transfection efficiency was 76.9%when epi4 cells were transfected with 100 nM siTGFB2-OT1.qRT-PCR showed that TGFB2-OT1 in epi4 cells of the experiment group showed a remarkable decrease after 24 h and 48 h of transfection,compared to the blank control and the negative control groups(P<0.01).The inhibition efficiency after 24 h and 48 h of transfection are 75%and 70%respectively,with no obvious difference between them.The RNA expression of TGFB2-OT1 in epi4 cells of the experiment group showed no significant change after P.gingivalis infection for 24 h.3.After exposure to P.gingivalis(MOI 100:1)for 24 h,western blot manifested that LC3Ⅱ/LC3 Ⅰ was obviously increased in the blank control or in the negative control groups(P<0.05),while it barely changed in the experiment group,the levels of P62 protein was reduced in either control groups but was not in the experiment group(P<0.05).Meanwhile,the level of LC3 Ⅱ/LC3 Ⅰ in infected experiment group was substantially lower than that of the blank control or the negative control groups(P<0.05),and the relative expression of P62 was obviously higher than that of either control groups(P<0.05).4.34 miRNAs that may mediate TGFB2-OT1 regulating LC3 through ceRNA mechanisms were predicted by bioinformatics methods,and the changes of autophagic flux caused by LC3 protein will further affect the level of P62 protein.Conclusions:P.gingivalis infection can promote the autophagy of epi4 cells by increasing LncRNA TGFB2-OT1,which may provide a new perspective for autophagy-related bacteriostatic therapy of periodontal diseases. |
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