A Preliminary Study On The Regulation And Mechanism Of LncRNA-TNFRSF13C On B Cell Function

Objective:Our previous research found that the expression of long non-coding RNA(lnc RNA)tumor necrosis factor receptor superfamily 13C(TNFRSF13C)in the gingival tissues of aggressive periodontitis was significantly higher than that in the healthy gingival tissues,suggesting that lnc RNA-TNFRSF13C may be involved in the disease process of periodontitis.lnc RNA-TNFRSF13C overlaps with the intron of the TNFRSF13C gene.The TNFRSF13C gene encodes a B cell activation factor receptor(BAFF-R),which is a specific receptor for B cell activation factor(BAFF).BAFF-R is the main receptor for BAFF mediating B cell survival,maturation and activation.This study aimed to detect the expressions of lnc RNA-TNFRSF13C,BAFF and BAFF-R in B cells of patients with different types of periodontitis;to analyze the effects of lnc RNA-TNFRSF13C on BAFF,BAFF-R and B cell function in vitro experiments;to preliminarily explore the role and possible regulatory mechanism of lnc RNA-TNFRSF13C in B cells of patients with periodontitis,so as to provide a new theoretical basis for the treatment of periodontitis.Methods:1.After obtaining informed consent from patients,blood samples were collected from healthy volunteers and patients with chronic periodontitis and aggressive periodontitis.Ficoll-Paque solution was used to isolate peripheral blood mononuclear cells(PBMCs)from human blood samples.CD19~+B cells were purified from PBMCs by MACS magnetic cell sorting.Total RNA was extracted from cells,and the expression levels of lnc RNA-TNFRSF13C,BAFF and BAFF-R in B cells of patients with different types of periodontitis were respectively detected by real-time polymerase chain reaction(RT-PCR).2.After obtaining informed consent from patients,blood samples from healthy volunteers were collected and CD19~+B cells were isolated according to the methods described above for this part and subsequent experiments.B cells were cultured in vitro,and then treated with 10μg/ml Porphyromonas gingivalis(P.g)lipopolysaccharide(LPS).And the expression levels of lnc RNA-TNFRSF13C,BAFF,and BAFF-R were detected by RT-PCR at 24h after stimulation.3.B cells cultured in vitro were transfected with lnc RNA-TNFRSF13C overexpression plasmid and lnc RNA-TNFRSF13C si RNA.Then,cell transfection efficiency was measured,and the expression changes of lnc RNA-TNFRSF13C,BAFF,and BAFF-R were detected after transfection.4.B cells were transfected with lnc RNA-TNFRSF13C overexpression plasmid and lnc RNA-TNFRSF13C si RNA,and cell transfection efficiency was measured.The cells were stimulated with 10μg/ml P.g LPS after transfection.Then,B cells proliferation and apoptosis were detected.Meanwhile,the cell supernatants were collected at 24h,48h,and 72h.And the secretion level changes in tumor necrosis factor-α(TNF-α),immunoglobulin A(Ig A)and immunoglobulin G(Ig G)were evaluated by enzyme-linked immunosorbent assay(ELISA).Results:1.The expressions of lnc RNA-TNFRSF13C,BAFF and BAFF-R in B cells of patients with chronic and aggressive periodontitis were significantly increased compared to healthy control groups(P<0.05).The expression level of lnc RNA-TNFRSF13C in patients with aggressive periodontitis was markedly higher than that in chronic periodontitis(P<0.05),while the expression levels of BAFF and BAFF-R in aggressive periodontitis were obviously lower than those in chronic periodontitis(P<0.05).2.Compared with the control group,the m RNA levels of lnc RNA-TNFRSF13C,BAFF and BAFF-R obviously increased after LPS stimulation of B cells(P<0.05).3.After lnc RNA-TNFRSF13C overexpression plasmid was transfected into B cells,the expression of lnc RNA-TNFRSF13C was significantly increased compared to the blank vector group(P<0.05),while the expressions of BAFF and BAFF-R were significantly reduced in the sno-TNFRSF13C group(P<0.05).After transfection B cells with lnc RNA-TNFRSF13C si RNA,compared with the negative control group,the expression of lnc RNA-TNFRSF13C was obviously reduced(P<0.05),while the expressions of BAFF and BAFF-R were obviously increased in the si-TNFRSF13C group(P<0.05).4.CCK8 assay was used to detect the proliferation of B cells.The results revealed that cell proliferation of the sno-TNFRSF13C group was evidently lower than that of the control group(P<0.05),while cell proliferation of the si-TNFRSF13C group was higher than that of the control group(P<0.05).Annexin V-FITC/PI double staining assay was used to analyze the apoptosis of B cells.Compared with the control group,the apoptosis of B cells in sno-TNFRSF13C group was higher(P<0.05),while that in si-TNFRSF13C group was inhibited(P<0.05).5.ELISA test results indicated that the TNF-αlevels in the sno-TNFRSF13C group at 24h,48h,and 72h were distinctly lower than those in the control group(P<0.05),while the TNF-αlevels in the si-TNFRSF13C group were evidently higher than those in the control group at 24h,48h,and 72h(P<0.05).There was no evident difference in Ig A and Ig G levels between groups at 24h and 48h(P>0.05).Compared with the control group,the levels of Ig A and Ig G in the sno-TNFRSF13C group were significantly reduced at 72h(P<0.05),while Ig A and Ig G levels in the si-TNFRSF13C group were significantly increased at 72h(P<0.05).Conclusion:1.lnc RNA-TNFRSF13C,BAFF and BAFF-R are highly expressed in B cells of patients with periodontitis,suggesting that lnc RNA-TNFRSF13C and related factors are involved in the occurrence and development of periodontitis.2.Overexpression of lnc RNA-TNFRSF13C can reduce the expression levels of BAFF and BAFF-R,and inhibit the secretion of cytokine TNF-α,antibodies Ig A and Ig G,indicating that lnc RNA-TNFRSF13C can inhibit the differentiation of B cells into plasma cells and hinder the defense function of B cells to the outside.3.Overexpression of lnc RNA-TNFRSF13C can inhibit the proliferation of B cells and promotes the apoptosis of B cells,thus further hindering the immune function of B cells.However,the regulatory mechanism still needs further research.4.lnc RNA-TNFRSF13C is highly expressed in aggressive periodontitis,and the function of B cells is affected,which may be one of the causes of aggressive periodontitis.