LncRNA OIP5-AS1 inhibits the lipopolysaccharide-induced inflammatory response and promotes osteogenic differentiation of human periodontal ligament cells by sponging miR-92a-3p

Abstract

Periodontitis is a chronic infectious disease that affects the oral health of adults. Long non-coding RNA OIP5 antisense RNA 1 (OIP5-AS1) has been reported to downregulated in the periodontal tissue of patients with periodontitis. Therefore, the study sought to look at the possible functions of OIP5-AS1 in periodontitis and the associated underlying mechanisms. In the present study, the expression level of OIP5-AS1 and microRNA-92a-3p were analyzed using reverse transcription-quantitative PCR. The levels of osteogenic proteins were determined using western blotting and inflammatory cytokines and oxidative stress were also examined. The proliferation of human periodontal ligament stem cells (hPDLSCs) was evaluated using MTT assays. Assay of osteogenic differentiation was undertaken by means of Alkaline phosphatase staining. The possible association between OIP5-AS1 and miR-92a-3p was determined applying dual-luciferase reporter assays and verified by RNA immunoprecipitation assay. We found that OIP5-AS1 was expressed at low levels in lipopolysaccharide (LPS)-stimulated hPDLSCs. OIP5-AS1 overexpression promoted proliferation and osteogenic differentiation ability and reduced LPS-induced inflammation in hPDLSCs. Furthermore, OIP5-AS1 directly targeted and reduced miR-92a-3p expression. The overexpression of miR-92a-3p partly abolished the effects of OIP5-AS1 on LPS-induced cell proliferation and osteogenic differentiation as well as inflammation in hPDLSCs. Collectively, the results indicated that OIP5-AS1 overexpression inhibited the LPS-induced inflammatory response and promoted the osteogenic differentiation of hPDLSCs by sponging miR-92a-3p. Thus, OIP5-AS1 is probably an essential objective for research during periodontitis treatment.

Keywords: Long non-coding RNA OIP5 antisense RNA 1; human periodontal ligament cells; inflammation; microRNA-92a-3p; osteogenic differentiation; periodontitis.

Graphical abstract

Figure 1.

OIP5-AS1 was poorly expressed in LPS-stimulated hPDLSCs. OIP5-AS1 expression in hPDLSCs was assayed by use of RT-qPCR. Error bars indicate the mean ± SD of three-independent replicate experiments. **P < 0.01, ***P < 0.001.

Figure 2.

OIP5-AS1 overexpression promoted the proliferation of hPDLSCs under LPS stimulation. (a) OIP5-AS1 expression in hPDLSCs was examined with the use of RT-qPCR. Error bars indicate the mean ± SD of three-independent replicate experiments. ***P < 0.001. (b) The proliferation of hPDLSCs was assessed with the application of MTT assay. Error bars indicate the mean ± SD of three-independent replicate experiments. ***P < 0.001 vs. Control. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. LPS.

Figure 3.

OIP5-AS1 overexpression promoted the osteogenic differentiation of hPDLSCs under LPS stimulation. (a) The detection of Alkaline phosphatase (ALP) activity was carried out employing ALP staining. Scale bar: 50 μm. (b) The mRNA levels of OCN, OPN, RUNX2 and BMP2 were quantified with RT-qPCR. (c) The protein expressions of OCN, OPN, RUNX2 and BMP2 were examined with the utilization of western blotting. Error bars indicate the mean ± SD of three-independent replicate experiments. **P < 0.01, ***P < 0.001.

Figure 4.

OIP5-AS1 overexpression inhibited the LPS-induced inflammation in hPDLSCs. (a) The levels of TNF-α, IL-6 and IL-1β were assayed with the utilization of ELISA kits. (b) The levels of MDA and LDH were measured by commercial assay kits. Error bars indicate the mean ± SD of three-independent replicate experiments. ***P < 0.001.

Figure 5.

OIP5-AS1 overexpression directly targeted miR-92a-3p and downregulated its expression. (a)The binding site of OIP5-AS1 and miR-92a-3p. (b) The miR-92a-3p mRNA level was quantified with RT-qPCR. (c) The association between OIP5-AS1 and miR-92a-3p was determined making use of luciferase activity assays. (d) OIP5-AS1 binding to miR-92a-3p detected by RIP assay. (e) The miR-92a-3p mRNA level was quantified with RT-qPCR. Error bars indicate the mean ± SD of three-independent replicate experiments. ***P < 0.001.

Figure 6.

miR-92a-3p upregulation reversed the effect of OIP5-AS1 on the proliferation and osteogenic differentiation of hPDLSCs. (a) The miR-92a-3p mRNA level was quantified with RT-qPCR. (b) The assessment of the proliferation in hPDLSCs was conducted employing MTT assay. (c) ALP activity was subjected to analysis with the aid of ALP staining. Scale bar: 50 μm. (d) The mRNA levels of OCN, OPN, RUNX2 and BMP2 were quantified with RT-qPCR. (e) The expressions of osteogenic differentiation markers OCN, OPN, RUNX2 and BMP2 were examined with the adoption of western blotting. Error bars indicate the mean ± SD of three-independent replicate experiments. ***P < 0.001.

Figure 7.

miR-92a-3p upregulation reversed the effect of OIP5-AS1 on LPS-induced inflammation in hPDLSCs. (a) The levels of TNF-α, IL-6 and IL-1β were assayed with the help of ELISA kits. (b) The levels of MDA and LDH were measured by commercial assay kits. Error bars indicate the mean ± SD of three-independent replicate experiments. ***P < 0.001.