Long Non-Coding RNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1)Relieves Sepsis-Induced Kidney Injury and Lipopolysaccharide (LPS)-Induced Inflammation in HK-2 Cells

Abstract

BACKGROUND Long non-coding RNAs (lncRNAs) play key roles in the development and progression of diseases, including sepsis. Therefore, this study aimed to clarify the role and underlying molecular mechanisms of lncRNA NEAT1 in sepsis. MATERIAL AND METHODS We used real-time quantitative polymerase chain reaction (RT-qPCR) to analyze the expression of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1), let-7b-5p, and tumor necrosis factor receptor-associated factor 6 (TRAF6). Western blot assay was used to measure the protein expression levels. After treatment with lipopolysaccharide (LPS), the biological behaviors of human renal tubular epithelial cells (HK-2), such as proliferation and apoptosis, were determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT) and flow cytometry assays, respectively. The interaction relationship among NEAT1, TRAF6, and let-7b-5p was analyzed by the bioinformatics starBase database and dual-luciferase reporter assay. RESULTS lncRNA NEAT1 was expressed at higher levels in kidney tissues from sepsis patients than in healthy kidney tissues. Interestingly, LPS induced high expression of lncRNA NEAT1 in HK-2 cells in a time- and dose-dependent manner. Furthermore, silencing of NEAT1 weakened LPS-induced apoptosis, inflammation, and inhibition of proliferation, which was overturned by silencing of let-7b-5p. In addition, overexpression of TRAF6 abolished the overexpression of let-7b-5p-induced effects on apoptosis, inflammation, and growth of HK-2 cells exposed to LPS. In summary, NEAT1 regulated TRAF6 expression by sponging let-7b-5p in HK-2 cells, which promotes LPS-induced injury and inflammation in HK-2 cells. CONCLUSIONS Our data show that the lower expression of NEAT1 impeded sepsis development and LPS-induced injury inflammation by targeting let-7b-5p/TRAF6 axis, and NEAT1 may be a target for treatment of sepsis patients.

Figure 1

The expression level of NEAT1 in sepsis patients and healthy volunteers. (A) The relative expression level of NEAT1 in kidney tissues from sepsis patients and healthy kidney tissues was assessed with RT-qPCR assay. (B, C) BUN and serum creatinine were analyzed in kidney tissues from sepsis patients. (D–F) Western blot analysis was used to detect TNF-α, IL-6, and IL-1β levels in sepsis patients and healthy kidney tissues. * P<0.05.

Figure 2

Effects of NEAT1 silencing on proliferation, apoptosis, and inflammation of LPS-treated HK-2 cells. (A) NEAT1 level was assessed in HK-2 cells treated with different concentrations LPS (0, 0.1, 1.0, 10, and 20 mg/L) for 24 h by RT-qPCR assay. (B) NEAT1 level was detected in HK-2 cells stimulated with 1.0 mg/L LPS at various time points (0, 6, 12, 18, and 24 h) by RT-qPCR assay. (C–F) HK-2 cells were separated into 4 groups: control, LPS, LPS+si-NC, and LPS+si-NEAT1 groups. (C) The relative expression level of NEAT1 was quantified with RT-qPCR analysis in HK-2 cells. (D) The growth curves of HK-2 cells were drawn using MTT assay. (E) Flow cytometry was used to measure the apoptosis rate of HK-2 cells. (F) The protein expression levels of TNF-α, IL-6, and IL-1β were assessed with Western blot analysis. * P<0.05.

Figure 3

Let-7b-5p was a direct target of NEAT1. (A) The complementary sequence between let-7b-5p and NEAT1 along with mutant sites were shown. (B) The luciferase activity was analyzed by dual-luciferase reporter assay. (C) RT-qPCR assay was used to test the expression level of let-7b-5p in kidney tissues in sepsis patients and healthy kidney tissues. (D, E) Let-7b-5p expression was analyzed by RT-qPCR assay in HK-2 cells. (F) RT-qPCR analysis was used to analyze let-7b-5p expression in HK-2 cells treated with LPS+si-NC, LPS+si-NEAT1, LPS+pcDNA, or LPS+NEAT1. * P<0.05.

Figure 4

Knockdown of NEAT1-mediated effects on proliferation, apoptosis, and inflammation in LPS-induced HK-2 cells was abolished by silencing of let-7b-5p. (A) Relationship between the expression levels of NEAT1 and let-7b-5p was analyzed by Pearson’s correlation analysis. (B–E) HK-2 cells were exposed to LPS+si-NC, LPS+si-NEAT1, LPS+si-NEAT1+anti-let-NC, or LPS+si-NEAT1+anti-let-7b-5p. (B) RT-qPCR was enforced to measure expression level of let-7b-5p in HK-2 cells. (C) MTT assay was performed for examining the cell viability of HK-2 cells. (D) The apoptosis rate of HK-2 cells was assessed by performing flow cytometry assay. (E) The protein expressions of TNF-α, IL-6, and IL-1β in HK-2 cells were examined by Western blot assay. * P<0.05.

Figure 5

Let-7b-5p negatively regulated TRAF6 expression. (A) Binding region between let-7b-5p and TRAF6 as well as mutated nucleotides ofTRAF6 3′UTR were shown. (B) Dual-luciferase reporter assay was used to show the luciferase activity of TRAF6 3′UTR-WT and TRAF6 3′UTR-MUT in HK-2 cells. (C, D) The expression levels of TRAF6 in kidney tissues from sepsis patients and healthy kidney tissues were evaluated by RT-qPCR and Western blot assay, respectively. (E–H) LPS elevated TRAF6 expression in HK-2 cells. (I, J) Transfection efficiency of let-7b-5p and anti-let-7b-5p was checked by RT-qPCR assay, with let-NC and anti-let-NC as controls. (K, L) The expression level of TRAF6 was analyzed with RT-qPCR and Western blot assay in HK-2 cells treated with LPS+let-NC, LPS+let-7b-5p, LPS+anti-let-NC, or LPS+anti-let-7b-5p. * P<0.05.

Figure 6

Overexpression of TRAF6 reversed let-7b-5p-induced the effect on proliferation, apoptosis, and inflammation in LPS-induced HK-2 cells. (A–F) HK-2 cells were treated with LPS+let-NC, LPS+let-7b-5p, LPS+let-7b-5p+pcDNA, or LPS+let-7b-5p+TRAF6. (A, B) The level of TRAF6 in HK-2 cells was measured by using RT-qPCR and Western blot assays. (C) Pearson’s correlation analysis revealed a negative correlation between the levels of TRAF6 mRNA and let-7b-5p. (D) Cell proliferation capability was examined in HK-2 with MTT analysis. (E) The apoptosis of HK-2 cells was performed using flow cytometry assay. (F) The expression levels of TNF-α, IL-6, and IL-1β were shown in HK-2 cells with Western blot analysis. * P<0.05.

Figure 7

NEAT1 regulated let-7b-5p/TRAF6 axis inHK-2 cells. (A) The relationship between NEAT1 and TRAF6 was shown by Pearson’s correlation analysis. (B, C) The expression level ofTRAF6 was estimated with RT-qPCR and Western blot assays in HK-2 cells treated with LPS+si-NC, LPS+si-NEAT1, LPS+si-NEAT1+anti-let-NC, or LPS+si-NEAT1+anti-let-7b-5p. * P<0.05.