Circ_0138960 knockdown alleviates lipopolysaccharide-induced inflammatory response and injury in human dental pulp cells by targeting miR-545-5p/MYD88 axis in pulpitis

Abstract

Background/purpose: Circular RNAs (circRNAs) have been shown to play important regulatory roles in many human diseases, yet their functions in pulpitis remain to be clarified. This study was designed to investigate the function of circ_0138960 in pulpitis progression and its underlying mechanism.

Material and methods: Cell viability and proliferation were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 5-Ethynyl-2'-deoxyuridine (EdU) assay. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) were conducted to analyze cell apoptosis rate and the release of inflammatory cytokines. The activity of superoxide dismutase (SOD) was analyzed using a SOD assay kit. Dual-luciferase reporter and RNA-pull down assays were performed to verify the interaction between microRNA-545-5p (miR-545-5p) and circ_0138960 or myeloid differentiation primary response gene 88 (MYD88).

Results: Lipopolysaccharide (LPS) treatment restrained the proliferation and promoted the apoptosis, inflammation, and oxidative stress of human dental pulp cells (hDPCs). LPS treatment dose-dependently up-regulated circ_0138960 expression in hDPCs. Circ_0138960 knockdown overturned LPS-induced inflammation and injury in hDPCs. Circ_0138960 could act as a molecular sponge for miR-545-5p, and circ_0138960 knockdown protected hDPCs from LPS-induced effects by up-regulating miR-545-5p. miR-545-5p directly interacted with the 3' untranslated region (3'UTR) of MYD88, and MYD88 overexpression reversed miR-545-5p-mediated effects in LPS-treated hDPCs. Circ_0138960 positively regulated MYD88 expression by sponging miR-545-5p in hDPCs. LPS could activate nuclear factor kappa-B (NF-κB) signaling by targeting circ_0138960/miR-545-5p/MYD88 axis in hDPCs.

Conclusion: Circ_0138960 knockdown attenuated LPS-induced inflammatory response and injury in hDPCs by targeting the miR-545-5p/MYD88/NF-κB axis.

Keywords: MYD88; NF-κB; Pulpitis; circ_0138960; miR-545-5p.

Fig. 1

LPS exposure dose-dependently suppresses the proliferation and induces the apoptosis, inflammation, and oxidative stress of hDPCs. (A–G) hDPCs were treated with LPS (0, 3, 6, or 9 μg/mL) for 24 h. (A) MTT assay was conducted to measure cell viability. (B) EdU assay was performed to analyze cell proliferation ability. (C) Flow cytometry was conducted to detect cell apoptosis rate. (D and E) Western blot assay was carried out to determine the expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 in hDPCs. (F) ELISA was conducted to analyze the concentrations of two inflammatory cytokines (IL-6 and TNF-α) in the culture supernatant of hDPCs. (G) The activity of SOD was analyzed using a SOD assay kit. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 2

Circ_0138960 knockdown reverses LPS-induced effects in hDPCs. (A) RT-qPCR was conducted to measure the expression of circ_0138960 in the pulp tissues of normal subjects (n = 17) and patients with pulpitis (n = 21). (B) hDPCs were treated with LPS (0, 3, 6, or 9 μg/mL) for 24 h, and the expression of circ_0138960 was examined by RT-qPCR. (C) RT-qPCR was performed to detect the levels of circ_0138960 and GAPDH in total RNA samples isolated from hDPCs after RNase R treatment. (D–J) hDPCs were divided into four groups: Control, LPS (6 μg/mL, 24 h), LPS + si-NC, and LPS + si-circ_0138960. (D) RT-qPCR was conducted to measure circ_0138960 expression in treated hDPCs. (E) Cell viability was analyzed by MTT assay. (F) EdU assay was conducted to assess cell proliferation ability. (G) The apoptosis of hDPCs was detected by flow cytometry. (H) The protein levels of Bax and Bcl-2 were determined by western blot assay. (I) The levels of IL-6 and TNF-α in the culture supernatant of hDPCs were examined by ELISA. (J) A SOD assay kit was used to analyze the activity of SOD. ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 3

miR-545-5p is a direct target of circ_0138960. (A) The binding sequence between circ_0138960 and miR-545-5p was predicted by bioinformatics tool circinteractome. (B) RT-qPCR was conducted to verify the overexpression efficiency of miR-545-5p mimics in hDPCs. (C and D) Dual-luciferase reporter and RNA-pull down assays were conducted to confirm the target relationship between circ_0138960 and miR-545-5p in hDPCs. (E) RT-qPCR was conducted to detect the expression of miR-545-5p in the pulp tissues of normal subjects (n = 17) and patients with pulpitis (n = 21). (F) The level of miR-545-5p was examined in hDPCs treated with LPS (0, 3, 6, or 9 μg/mL) for 24 h by RT-qPCR. ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 4

Circ_0138960 knockdown-mediated protective effects in LPS-induced hDPCs are reversed by the silence of miR-545-5p. (A–H) hDPCs were transfected with si-circ_0138960 alone or together with anti-miR-545-5p followed by LPS treatment (6 μg/mL, 24 h). (A) The expression of miR-545-5p was detected by RT-qPCR. (B) Cell viability was analyzed by MTT assay. (C and D) Cell proliferation was assessed by EdU assay. (E) Flow cytometry was conducted to analyze the apoptosis of hDPCs. (F) The protein levels of Bax and Bcl-2 were measured by western blot assay. (G) The release of two inflammatory cytokines (IL-6 and TNF-α) in the culture supernatant of hDPCs was analyzed by ELISA. (H) The activity of SOD was analyzed using a SOD assay kit. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 5

miR-545-5p directly targets the 3′UTR of MYD88. (A) The binding sites between miR-545-5p and MYD88 3′UTR were predicted by starbase tool. (B and C) Dual-luciferase reporter and RNA-pull down assays were conducted to confirm the interaction between miR-545-5p and MYD88 3′UTR. (D) The mRNA expression of MYD88 in the pulp tissues of normal subjects (n = 17) and patients with pulpitis (n = 21) was detected by RT-qPCR. (E) Expression association between miR-545-5p and circ_0138960 or MYD88 mRNA in the pulp tissues of pulpitis patients was performed using Pearson’s correlation coefficient. (F) Western blot assay was conducted to measure the protein expression of MYD88 in the pulp tissues of normal subjects and pulpitis patients. (G) hDPCs were exposed to LPS (0, 3, 6, or 9 μg/mL) for 24 h, and the protein expression of MYD88 was measured by western blot assay. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 6

miR-545-5p overexpression protects hDPCs against LPS-induced effects by down-regulating MYD88. (A-H) hDPCs were transfected with miR-545-5p mimics alone or together with MYD88 expressing plasmid followed by LPS treatment. (A) The protein level of MYD88 was detected by western blot assay. (B) Cell viability was analyzed by MTT assay. (C and D) EdU assay was carried out to assess the proliferation ability of hDPCs. (E) Flow cytometry was performed to analyze the apoptosis rate of hDPCs. (F) Western blot assay was performed to measure the protein levels of Bax and Bcl-2 in hDPCs. (G) ELISA was conducted to analyze the concentrations of IL-6 and TNF-α in the culture supernatant of hDPCs. (H) The activity of SOD was analyzed using a SOD assay kit. ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 7

Circ_0138960 knockdown suppresses LPS-induced activation of NF-κB signaling by targeting miR-545-5p/MYD88 axis. (A) hDPCs were transfected with si-circ_0138960 alone or together with anti-miR-545-5p followed by LPS exposure (6 μg/mL, 24 h). The protein level of MYD88 was detected by western blot assay. (B) hDPCs were transfected with si-circ_0138960 alone or together with anti-miR-545-5p/MYD88 expressing plasmid followed by LPS exposure (6 μg/mL, 24 h). The levels of p-P65, P65, p-IκBα, and IκBα were detected by western blot assay. ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. S1

Circ_0138960 overexpression aggravated LPS-induced effects in hDPCs. (A–F) hDPCs were divided into four groups: Control, LPS (6 μg/mL, 24 h), LPS + pCD5-ciR, and LPS + circ_0138960. (A) RT-qPCR was conducted to measure circ_0138960 expression in treated hDPCs. (B) Cell viability was measured by MTT assay. (C and D) EdU assay was performed to examine cell proliferation ability. (E) The apoptosis of hDPCs was analyzed by flow cytometry. (F) The levels of IL-6 and TNF-α in the culture supernatant of hDPCs were determined using ELISA. (G) A SOD assay kit was applied to assess the activity of SOD. ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.