LncRNA SNHG16 promotes LPS-induced human bronchial epithelial cell pyroptosis through miR-339-5p/NLRP1 axis mediation

Abstract

Introduction: Pyroptosis can aggravate lung injury in sepsis. It has been reported that lncRNA SNHG16 can regulate the inflammatory response. However, the role and underlying mechanism of SNHG16 in sepsis-induced pyroptosis and lung injury remain unclear.

Material and methods: To mimic septic lung injury in vitro, cells were treated with 1 µg/ml LPS. The Cell Counting Kit-8 (CCK-8) assay was performed to test cell viability. The lactate dehydrogenase (LDH) level was detected using a commercial kit. Interleukin (IL)-18 and IL-1 β secretion was tested using ELISA. Pyroptosis was investigated via flow cytometry. The relationship among SNHG16, miR-339-5p, and NLR family pyrin domain containing 1 (NLRP1) was explored using the dual luciferase assay.

Results: LPS significantly upregulated the levels of SNHG16 and NLRP1 in BEAS-2B cells. In addition, LPS significantly induced pyroptosis in BEAS2B cells, while this phenomenon was reversed by SNHG16 silencing. SNHG16 could bind with miR-339-5p, and NLRP1 was found to be the downstream mRNA of miR-339-5p. SNHG16 silencing significantly abolished the LPS-induced upregulation of NLRP1 through miR-339-5p downregulation. The upregulation of miR-339-5p inhibited the pro-apoptotic effect of LPS on BEAS-2B cells, which was abolished by NLRP1 overexpression. Furthermore, the anti-pyroptotic effect of SNHG16 siRNA was abolished by NLRP1 upregulation.

Conclusions: SNHG16 silencing reversed LPS-induced pyroptosis in BEAS-2B cells via miR-339-5p/NLRP1 axis mediation. Our study might shed new light on exploring therapeutic strategies for the treatment of septic lung injury.

Keywords: NLRP1; SNHG16; miR-339-5p; pyroptosis.

Fig. 1

SNHG16 downregulation relieved LPS-induced pyroptosis. BEAS-2B cells were treated with LPS (1 μg/ml) for 24 h. A) The level of NLRP1 in BEAS-2B cells was detected by RT-qPCR. B) NLRP1 expression in BEAS-2B cells was analyzed via western blotting. C) Cells were transfected with sh-NC or sh-SNHG16. SNHG16 levels in BEAS-2B cells were determined by RT-qPCR. BEAS-2B cells were treated with LPS, LPS + si-NC, or LPS + si-SNHG16. D) SNHG16 levels in BEAS-2B cells were evaluated via RT-qPCR. E) BEAS-2B cell viability was tested through CCK-8 assay. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001. F) LDH release was examined via the LDH kit. G) IL-18 and IL-1β levels in supernatants of BEAS-2B cells were examined via ELISA. H) The mRNA level of NLRP1 was determined by RT-qPCR. I) Protein levels of NLRP1, ASC, cleaved caspase-1, and cleaved GSDMD in BEAS-2B cells were analyzed by western blotting. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001. J) BEAS-2B cell pyroptosis was tested by flow cytometry. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

SNHG16 positively regulated NLRP1 expression through miR-339-5p sponging. A) The starBase and miRWalk databases were used to explore miRNAs that shared common binding sites with SNHG16 and NLRP1. B) The expression of miR-339-5p in BEAS-2B cells was investigated by RT-qPCR after si-NC or si-SNHG16 transfection. BEAS-2B cells were transfected with NC mimics, miR-339-5p mimics, inhibitor NC, or miR-339-5p inhibitor. C) The expression of miR-339-5p in BEAS- 2B cells was examined via RT-qPCR. D) The binding sites among SNHG16 and miR-339-5p were tested by the starBase database. E) The dual luciferase assay was performed to investigate luciferase activity. F) NLRP1 expression in BEAS-2B cells was examined via RT-qPCR and western blotting. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001 G) NLRP1 expression in BEAS-2B cells was examined via RT-qPCR and western blotting. H) The binding sites between NLRP1 and miR-339-5p were predicted by the starBase database. I) The dual luciferase assay was performed to measure luciferase activity. Cells were treated with inhibitor NC + si-NC, si-SNHG16 + NC inhibitor, or si-SNHG16 + miR-339-5p inhibitor. J, K) Levels of miR-339-5p and NLRP1 were examined via RT-qPCR. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001. L) NLRP1 expression in BEAS-2B cells was tested by western blotting. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

MiR-339-5p downregulation reversed the inhibitory effect of SNHG16 silencing on the NLRP1 level in LPS-treated BEAS-2B cells. A) BEAS-2B cells were treated with LPS, LPS + si-NC, or LPS + si-SNHG16. The level of miR-339-5p in BEAS-2B cells was determined via RT-qPCR. BEAS-2B cells were treated with LPS, LPS + mimics NC, or LPS + miR-339-5p mimics. B, C) Levels of miR-339-5p and NLRP1 were determined via RT-qPCR. D) The protein level of NLRP1 was tested by western blotting. BEAS-2B cells were treated with LPS, LPS + inhibitor NC + si-NC, LPS + si-SNHG16 + inhibitor NC, or LPS + si-SNHG16 + miR-339-5p inhibitor. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001. E, F) Levels of miR-339-5p and NLRP1 in BEAS-2B cells were examined via RT-qPCR. G) NLRP1 expression in BEAS-2B cells was tested by western blotting. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

NLRP1 reversed miR-339-5p overexpression-mediated pyroptosis inhibition in LPS-induced BEAS-2B cells. A, B) The overexpression effect of NLRP1 was tested by RT-qPCR and western blotting after transfection with oe-NLRP1. BEAS-2B cells were exposed to LPS, LPS + mimics NC + pcDNA3.1, LPS + miR-339-5p mimics + pcDNA3.1, or LPS + miR-339-5p mimics + pcDNA3.1-NLRP1. C, D) NLRP1 levels in BEAS-2B cells were tested by RT-qPCR and western blotting. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001. E) BEAS-2B cell viability was assessed by the CCK-8 assay. F) LDH release was tested using the LDH kit. G) IL-18 and IL-1β levels in cell supernatants were tested by ELISA. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001. H) Protein levels of ASC, cleaved caspase-1, and cleaved GSDMD in BEAS-2B cells were detected by western blotting. I) BEAS-2B cell pyroptosis was determined by flow cytometry. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001. I) BEAS-2B cell pyroptosis was determined by flow cytometry. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

NLRP1 upregulation abolished SNHG16 knockdown-reduced pyroptosis. BEAS2B cells were transfected with LPS, si-NC + LPS + pcDNA3.1, LPS + si-SNHG16 + pcDNA3.1, or LPS + si-SNHG16 + pcDNA3.1-NLRP1. A, B) NLRP1 levels in BEAS-2B cells were tested by RT-qPCR and western blotting. C) BEAS-2B cell viability was assessed by the CCK-8 assay. D) The LDH kit was used to test for LDH release. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001. E) IL-1β and IL-18 levels in supernatants of BEAS-2B cells were tested by ELISA. F) Protein levels of ASC, cleaved caspase-1, and cleaved GSDMD in BEAS-2B cells were detected by western blotting. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001. G) BEAS-2B cell pyroptosis was determined by flow cytometry. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001 G) BEAS-2B cell pyroptosis was determined by flow cytometry. n = 3 per group. * p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

The mechanism underlying the function of SNHG16 in septic ALI. SNHG16 sponged miR-339-5p to indirectly upregulate NLRP1 in LPS-treated bronchial epithelial cells BEAS-2B. Then, the upregulated NLRP1 promoted activation of caspase-1 and secretion of pyroptosis-related factors (IL-1β and IL-18), thus aggravating BEAS-2B pyroptosis