lncRNA NEAT1 mediates LPS-induced pyroptosis of BEAS-2B cells via targeting miR-26a-5p/ROCK1 axis

Abstract

Acute lung injury (ALI) is an adverse disease of the respiratory system, and one of its prevalent causes is sepsis induction. Cell pyroptosis facilitates the progression of ALI and lncRNAs play critical roles in ALI. Thus, this research seeks to investigate the specific mechanism of NEAT1 in sepsis-ALI.BEAS-2B cells were exposed to lipopolysaccharide (LPS) to construct a cell model of sepsis-induced ALI. The gene and protein expression were assessed using qRT-PCR and western blot. Cell viability was identified by CCK-8. Cell death was discovered using PI staining. The secretion of IL-1β and IL-18 was examined using ELISA. The interconnections among NEAT1, miR-26a-5p, and ROCK1 were confirmed using starbase, luciferase assay, and RIP.LPS treatment augmented NEAT1 and ROCK1 levels while mitigating miR-26a-5p level in BEAS-2B cells. Additionally, LPS treatment facilitated cell death and cell pyroptosis, whereas NEAT1 silencing could reverse these effects in BEAS-2B cells. Mechanistically, NEAT1 positively mediated ROCK1 expression by targeting miR-26a-5p. Furthermore, miR-26a-5p inhibitor offset NEAT1 depletion-mediated suppressive effects on cell death and cell pyroptosis. ROCK1 upregulation decreased the inhibitory impacts produced by miR-26a-5p overexpression on cell death and cell pyroptosis. Our outcomes demonstrated NEAT1 could reinforce LPS-induced cell death and cell pyroptosis by repressing the miR-26a-5p/ROCK1 axis, thereby worsening ALI caused by sepsis. Our data indicated NEAT1, miR-26a-5p, and ROCK1 might be biomarkers and target genes for relieving sepsis-induced ALI.

Keywords: ROCK1; acute lung injury; lncRNA NEAT1; miR-26a-5p; sepsis.

FIGURE 1

NEAT1 knockdown attenuated LPS‐induced pyroptosis in BEAS‐2B cells. (A) NEAT1 was analyzed using qRT‐PCR in BEAS‐2B cells with/without LPS treatment. BEAS‐2B cells suffered si‐NEAT1 or si‐NC transfection upon LPS administration. (B) NEAT1 was evaluated using qRT‐PCR. (C) Cell viability was analyzed using CCK8. (D) Cell death was determined by PI staining. (E) The protein levels of pyroptosis‐related proteins were investigated using a western blot. (F) The levels of IL‐1β and IL‐18 were determined using ELISA. All data collected from at least three replicate experiments were indicated as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

NEAT1 restricted miR‐26a‐5p expression. (A) MiR‐26a‐5p was calculated using qRT‐PCR in BEAS‐2B cells with/without LPS treatment. (B–D) The intercorrelation of NEAT1 and miR‐26a‐5p was forecasted and validated using Starbase, dual luciferase assay, and RIP. (E) miR‐26a‐5p was identified using qRT‐PCR in BEAS‐2B cells with si‐NEAT1 or si‐NC transfection upon LPS treatment. All data collected from at least three replicate experiments were expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

miR‐26a‐5p inhibition abolished the suppression of NEAT1 knockdown on cell pyroptosis. si‐NEAT1 or in conjunction with miR‐26a‐5p inhibitor was transferred into BEAS‐2B cells and followed LPS treatment. (A) NEAT1 and miR‐26a‐5p were evaluated using qRT‐PCR. (B) Cell viability was calculated using CCK8. (C) Cell death was assessed by PI staining. (D) The protein levels of pyroptosis‐related proteins including NLRP3, ASC, Cleaved caspase1, and Cleaved Gasdermin‐D were identified using western blot. (E) The levels of IL‐1β and IL‐18 were tested using ELISA. All data collected from at least three replicate experiments were denoted as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

miR‐26a‐5p targeted ROCK1 and mediated ROCK1 expression. (A,B) ROCK1 was evaluated using qRT‐PCR and western blot in BEAS‐2B cells with/without LPS treatment. (C,D) the connection between ROCK1 and miR‐26a‐5p was forecasted and validated using Starbase and dual luciferase assay. (E,F) ROCK1 was analyzed using qRT‐PCR and western blot in BEAS‐2B cells with miR‐26a‐5p mimic or miR‐NC transfection upon LPS administration. All data procured from at least three replicate experiments were denoted as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

ROCK1 upregulation compromised the suppression of miR‐26a‐5p mimic on pyroptosis. miR‐26a‐5p mimic or in conjunction with pcDNA3.1‐ROCK1 was transfected into BEAS‐2B cells and followed LPS treatment. (A) ROCK1 and miR‐26a‐5p were evaluated using qRT‐PCR. (B) ROCK1 was determined using a western blot. (C) cell viability was identified using CCK8. (D) cell death was determined with PI staining. (E) protein levels of pyroptosis‐related proteins including NLRP3, ASC, Cleaved caspase1, and Cleaved Gasdermin‐D were assessed by western blot. (F) The levels of IL‐1β and IL‐18 were tested using ELISA. All data procured from at least three replicate experiments were implied as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.