MALAT1 promotes colonic epithelial cell apoptosis and pyroptosis by sponging miR-22-3p to enhance NLRP3 expression

Abstract

Background: Colonic epithelial cell apoptosis and pyroptosis had a close relationship with the pathological progression of ulcerative colitis (UC). LncRNA play a crucial role in the progression of UC. However, the role of the lncRNA MALAT1 in colonic epithelial cell apoptosis and pyroptosis remains unclear.

Methods: UC colitis cell model was established through lipopolysaccharide (LPS) treatment. MiR-22-3p and MALAT1 expression in fetal human colon (FHC) cells were analyzed by qRT-PCR. Proliferation and apoptosis of FHCs were measured using CCK-8 assay and flow cytometry, respectively. Pyroptosis indicators including interleukin (IL)-1β, IL-18, tumor necrosis factor-α (TNF-α), NLR family pyrin domain containing 3 (NLRP3), caspase-1, and N-gasdermin D (N-GSDMD) in FHCs were detected using ELISA, qRT-PCR, western blotting, and immunofluorescence.

Results: In this study, apoptosis was facilitated, IL-1β, IL-18, and TNF-α levels were enhanced, NLRP3, caspase-1, N-GSDMD protein were increased, and MALAT1 expression was markedly increased in LPS-treated FHCs (LTFs). MALAT1 knockdown remarkably facilitated proliferation and suppressed apoptosis, reduced IL-1β, IL-18, and TNF-α levels, and decreased the protein of NLRP3, caspase-1, N-GSDMD. Furthermore, NLRP3 overexpression remarkably reversed the effect of MALAT1-downexpression in LTFs. In addition, miR-22-3p could bind with MALAT1 and NLRP3 3' UTR. Furthermore, miR-22-3p inhibition remarkably reversed the effect of MALAT1 overexpression in LTFs.

Conclusions: These findings suggest that MALAT1 represents a promising therapeutic target for the treatment of UC by modulating the miR-22-3p/NLRP3 pathway, potentially leading to novel strategies for reducing inflammation and cell death in the colon.

Keywords: Fetal human colon cell; Inflammasome; Long non-coding RNAs; Proliferation.

Figure 1. MALAT1 expression affected proliferation and apoptosis in LPS treated FHCs (LTFs).

(A) The MALAT1 expression in FHC cells and after transfection with si-MALAT1 were measured through qRT-PCR. (B) The MALAT1 expression in LTFs alone and after transfection with ov-MALAT1 or si-MALAT1 were measured through qRT-PCR. (C) Cell proliferation of LTFs and LTFs transfected with ov-MALAT1 and si-MALAT1 were studied using CCK-8 assay. (D) Cell apoptosis of LTFs and LTFs transfected with ov-MALAT1 and si-MALAT1 were evaluated through flow cytometry (*P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 2. The interference of MALAT1 expression inhibited the pyroptosis of LPS treated FHCs (LTFs).

(A) The secretion levels of inflammatory cytokine were examined through ELISA in LTFs. (B) The NLRP3 mRNA in LTFs were measured by qRT-PCR (C) the pyroptosis biomarker NLRP3, caspase-1, and N-GSDMD levels in LTFs were measured by western blot analysis. (D and E) Immunofluorescence was used to measure N-GSDMD protein in LTFs (***P < 0.001).

Figure 3. NLRP3 overexpression could block proliferation while promote apoptosis and pyroptosis of LPS treated FHCs (LTFs) in the presence of MALAT1 interference.

(A) The NLRP3, cl-caspase-1, and N-GSDMD expression levels in LTFs transfected with ov-NLRP3 +si-MALAT1 were assessed by western blot. (B) N-GSDMD protein in LTFs was analyzed through Immunofluorescence after transfected with ov-NLRP3+si-MALAT1. (C and D) The proliferation and apoptosis of LTFs transfected with ov-NLRP3 plus si-MALAT1 were analyzed. (E) ELISA was used to examine the secretion of inflammatory cytokine in LTFs transfected with ov-NLRP3 plus si-MALAT1 (*P < 0.05 and ***P < 0.001).

Figure 4. MALAT1 and NLRP3 3′- UTR combine miR-22-3p through competing endogenous.

(A) The miRNAs combined with MALAT1 were analyzed using starbase and RNAInter while it combined with NLRP3 3′-UTR were analyzed using targetscan and RNAInter, then take the intersection of the four sets of results. (B and C) Wild type and mutant sequences bind to miR-22-3p. (D) Double Luciferase experiment confirmed that both MALAT1 and NLRP3 3′- UTR could bind to miR-22-3p. (E) miR-22-3p expression was measured by qRT-PCR in LPS treated FHCs (LTFs) after MALAT1 overexpression and downexpression (***P < 0.001).

Figure 5. miR-22-3p interference could block proliferation while promote apoptosis of LPS treated FHCs (LTFs) in the presence of MALAT1 interference.

(A) miR-22-3p expression in LTFs was assessed by qRT-PCR after co-transfected with si-MALAT1+miR-inhibitor. (B and C) The proliferation and apoptosis of LTFs transfected with si-MALAT1+miR-22-3p inhibitor were analyzed after co-transfected with si-MALAT1+miR-inhibitor (**P < 0.01 and ***P < 0.001).

Figure 6. miR-22-3p interference could promote pyroptosis of LPS treated FHCs (LTFs) even in the presence of MALAT1 interference.

(A) ELISA was used to examine the secretion levels of inflammatory cytokine in LTFs co-transfected with si-MALAT1+miR-22-3pinhibitor. (B) Proteins in LTFs co-transfected with si-MALAT1+miR-22-3p inhibitor were assessed by western blot. (C) N-GSDMD protein in LTFs was analyzed through immunofluorescence (***P < 0.001).