Long noncoding RNA X-inactive specific transcript regulates NLR family pyrin domain containing 3/caspase-1-mediated pyroptosis in diabetic nephropathy

Abstract

Background: NLRP3-mediated pyroptosis is recognized as an essential modulator of renal disease pathology. Long noncoding RNAs (lncRNAs) are active participators of diabetic nephropathy (DN). X inactive specific transcript (XIST) expression has been reported to be elevated in the serum of DN patients.

Aim: To evaluate the mechanism of lncRNA XIST in renal tubular epithelial cell (RTEC) pyroptosis in DN.

Methods: A DN rat model was established through streptozotocin injection, and XIST was knocked down by tail vein injection of the lentivirus LV sh-XIST. Renal metabolic and biochemical indices were detected, and pathological changes in the renal tissue were assessed. The expression of indicators related to inflammation and pyroptosis was also detected. High glucose (HG) was used to treat HK2 cells, and cell viability and lactate dehydrogenase (LDH) activity were detected after silencing XIST. The subcellular localization and downstream mechanism of XIST were investigated. Finally, a rescue experiment was carried out to verify that XIST regulates NLR family pyrin domain containing 3 (NLRP3)/caspase-1-mediated RTEC pyroptosis through the microRNA-15-5p (miR-15b-5p)/Toll-like receptor 4 (TLR4) axis.

Results: XIST was highly expressed in the DN models. XIST silencing improved renal metabolism and biochemical indices and mitigated renal injury. The expression of inflammation and pyroptosis indicators was significantly increased in DN rats and HG-treated HK2 cells; cell viability was decreased and LDH activity was increased after HG treatment. Silencing XIST inhibited RTEC pyroptosis by inhibiting NLRP3/caspase-1. Mechanistically, XIST sponged miR-15b-5p to regulate TLR4. Silencing XIST inhibited TLR4 by promoting miR-15b-5p. miR-15b-5p inhibition or TLR4 overexpression averted the inhibitory effect of silencing XIST on HG-induced RTEC pyroptosis.

Conclusion: Silencing XIST inhibits TLR4 by upregulating miR-15b-5p and ultimately inhibits renal injury in DN by inhibiting NLRP3/caspase-1-mediated RTEC pyroptosis.

Keywords: Diabetic nephropathy; Long noncoding RNA X-inactive specific transcript; NLR family pyrin domain containing 3/caspase-1 pathway; Pyroptosis; Renal tubular epithelial cell; Toll-like receptor 4; microRNA-15b-5p.

Figure 1

Silencing lncRNA X inactive specific transcript can inhibit renal injury in DN rats. After establishing DN model rats, lentivirus-LV sh- X inactive specific transcript (XIST) was injected into the tail vein to knock down XIST expression in vivo. A: XIST expression detected by qRT-PCR; B: Kidney weight/body weight (KW/BW); C: Fasting blood glucose (FBG); D: Blood urea nitrogen (BUN); E: Serum creatinine (Cr); F: Urine protein for 24 h (UP 24 h); G: Histological changes of renal tissue estimated by HE staining, PAS staining and Masson staining; scale bar: 25 μm; arrows indicate inflammatory cell infiltration (black arrows) and fibrosis (white arrows). (g) glomerulus, (t) tubules; n = 8/group. The data were described as mean ± SD and analyzed by one-way ANOVA and Tukey's multiple comparisons test; ^aP < 0.05. DN: Diabetic nephropathy; LV: Lentivirus; XIST: X inactive specific transcript; sh: Short hairpin RNA; HE: Hematoxylin-eosin staining; PAS: Periodic Acid-Schiff stain.

Figure 2

Silencing lncRNA X inactive specific transcript can inhibit renal tubular epithelial cell pyroptosis in diabetic nephropathy rats. After establishing diabetic nephropathy model rats, lentivirus-LV sh- X inactive specific transcript was injected into the tail vein. A: Enzyme-linked immunosorbent assay detected IL-1β and IL-18 expressions; B: Western blot tested the levels of NLR family pyrin domain containing 3, ASC, Cleaved Caspase-1, Caspase-1, GSDMD, and GSDMD-N; C: Immunofluorescence tested the expression of Caspase-1; scale bar: 25 μm; N = 8/group. The data were described as mean ± SD and analyzed by one-way ANOVA and Tukey's multiple comparisons test; ^aP < 0.05. DN: Diabetic nephropathy; LV: Lentivirus; XIST: X inactive specific transcript; sh: Short hairpin RNA; NLRP3: NLR family pyrin domain containing 3; ASC: Apoptosis speck-like protein; GSDMD: Gasdermin D; DAPI: 4',6-diamidino-2-phenylindole.

Figure 3

Silencing X inactive specific transcript in diabetic nephropathy can inhibit renal tubular epithelial cell pyroptosis in high glucose-induced HK2 cells. si-NC or si- X inactive specific transcript (XIST) was delivered into HK2 cells treated with high glucose. A: The XIST expression detected by qRT-PCR; B: Enzyme-linked immunosorbent assay detected the expression of IL-1β and IL-18; C: Western blot tested the levels of NLR family pyrin domain containing 3, ASC, Cleaved Caspase-1, Caspase-1, GSDMD, and GSDMD-N; D: Cell viability; E: Lactate dehydrogenase activity. The cell experiment was performed in triplicate. The data were described as mean ± SD and analyzed by one-way ANOVA (A/D-E) or two-way ANOVA (B/C) and Tukey's multiple comparisons test; ^aP < 0.05. NG: Normal glucose; HG: High glucose; si: Small interfering RNA; XIST: X inactive specific transcript; LDH: Lactate dehydrogenase; NLRP3: NLR family pyrin domain containing 3; SC: Apoptosis speck-like protein; GSDMD: Gasdermin D.

Figure 4

X inactive specific transcript can adsorb and bind to miR-15b-5p to reduce miR-15b-5p expression in diabetic nephropathy. A: Fluorescence in situ hybridization assay (scale bar: 25 μm); B: Nuclear/cytosol fractionation assay verified the subcellular localization of X inactive specific transcript (XIST) in HK2 cells; C: Starbase website (starbase.sysu.edu.cn/index.php) predicted the binding sites of XIST and miR-15b-5p; D: Dual-luciferase experiment and E: RNA immunoprecipitation experiment verified the binding relation of XIST and miR-15b-5p; F: qRT-PCR detected miR-15b-5p and pre-miR-15b expression in renal tissue; G: qRT-PCR detected miR-15b-5p and pre-miR-15b expression in HK2 cells. The cell experiment was performed in triplicate. The data were described as mean ± SD and analyzed by one-way ANOVA (F/G) or two-way ANOVA (D/E) and Tukey's multiple comparisons test; ^aP < 0.05. XIST: X inactive specific transcript; DN: Diabetic nephropathy; DAPI: 4',6-diamidino-2-phenylindole; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; U6: U6 small nuclear RNA; WT: Wild type; MUT: Mutant.

Figure 5

Inhibition of miR-15b-5p in X inactive specific transcript -silenced HK2 cells activates renal tubular epithelial cell pyroptosis. In HG-treated HK2 cells, both X inactive specific transcript (XIST) and miR-15b-5p were silenced. A: qRT-PCR verified the transfection efficiency of miR-15b-5p inhibitor; B: Enzyme-linked immunosorbent assay detected the expression of IL-1β and IL-18; C: Western blot tested the levels of NLR family pyrin domain containing 3, ASC, Cleaved Caspase-1, Caspase-1, GSDMD, and GSDMD-N; D: Cell viability; E: Lactate dehydrogenase activity. The cell experiment was performed in triplicate. The data were described as mean ± SD and analyzed by one-way ANOVA (A/D-E) or two-way ANOVA (B/C) and Tukey's multiple comparisons test; ^aP < 0.05. LDH: Lactate dehydrogenase; XIST: X inactive specific transcript; si: Small interfering RNA; NLRP3: NLR family pyrin domain containing 3; ASC: Apoptosis speck-like protein; GSDMD: Gasdermin D.

Figure 6

Toll like receptor 4 expression is recovered by using miR-15b-5p inhibitor. A: Starbase website (starbase.sysu.edu.cn/index.php) predicted the binding sites of miR-15b-5p and toll like receptor 4 (TLR4); B: Dual-luciferase experiment and C: RNA immunoprecipitation experiment verified the binding relation of miR-15b-5p and TLR4; D: qRT-PCR detected TLR4 expression in renal tissues; E: Western blot detected TLR4 level in renal tissues; F: qRT-PCR detected TLR4 expression in HK2 cells; G: Western blot detected TLR4 level in HK2 cells. The cell experiment was performed in triplicate. The data were described as mean ± SD and analyzed by one-way ANOVA (D-G) or two-way ANOVA (B/C) and Tukey's multiple comparisons test; ^aP < 0.05. WT: Wild type; MUT: Mutant; TLR4: Toll like receptor 4; DN: Diabetic nephropathy; LV: Lentivirus; XIST: X inactive specific transcript; NG: Normal glucose; HG: High glucose; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 7

Toll like receptor 4 overexpression in X inactive specific transcript-silenced cells causes increased pyroptosis. In HG-treated HK2 cells, X inactive specific transcript (XIST) was silenced and toll like receptor 4 (TLR4) was overexpressed. A and B: The expression of TLR4 levels detected by qRT-PCR and Western blot; C: Enzyme-linked immunosorbent assay detected the expression of IL-1β and IL-18; D: Western blot tested the levels of NLR family pyrin domain containing 3, ASC, Cleaved Caspase-1, Caspase-1, GSDMD, and GSDMD-N; E: Cell viability; F: LDH activity. The cell experiment was performed in triplicate. The data were described as mean ± SD and analyzed by one-way ANOVA (A/B/E/F) or two-way ANOVA (C/D) and Tukey's multiple comparisons test; ^aP < 0.05. XIST: X inactive specific transcript; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; LDH: Lactate dehydrogenase; NLRP3: NLR family pyrin domain containing 3; ASC: Apoptosis speck-like protein; GSDMD: Gasdermin D.

Figure 8

Mechanism diagram. STZ and HG treatment induced high expression of X inactive specific transcript (XIST); silencing XIST promoted the targeted inhibition of miR-15b-5p on toll like receptor 4 (TLR4) by upregulating miR-15b-5p, thereby inhibiting TLR4, and ultimately alleviating the renal injury of diabetic nephropathy by inhibiting NLRP3/Caspase-1-mediated RTEC pyroptosis. mRNA: messenger RNA; DN: Diabetic nephropathy; NLRP3: NLR family pyrin domain containing 3; ASC: Apoptosis speck-like protein; GSDMD: Gasdermin D.