Long Noncoding RNA MIAT Regulates Hyperosmotic Stress-Induced Corneal Epithelial Cell Injury via Inhibiting the Caspase-1-Dependent Pyroptosis and Apoptosis in Dry Eye Disease

Abstract

Purpose: The biological role and mechanism of long noncoding RNA (lncRNA) myocardial infarction-associated transcript (MIAT) in dry eye remain to be illustrated. Pyroptosis is a noticeable form of inflammatory activation, which is characteristic of gasdermin D (GSDMD)-driven cell death. The present study was designed to explore the role of MIAT in pyroptosis and apoptosis induced by hyperosmolarity stress (HS) in human corneal epithelial cells (HCECs).

Methods: HCECs were cultured in 70-120 mM hyperosmotic medium for 24 h to create a dry eye model in vitro. The level of the pyroptosis marker GSDMD was measured, and the cell inflammatory response was evaluated by detecting IL-1β and IL-18 levels. Exogenous caspase-1 inhibitor Ac-YVAD-CHO was used. The pyroptosis in HCECs was examined by caspase-1 activity, immunofluorescent staining, and Western blotting. Flow cytometry was performed to test the apoptosis rate of HCECs. Cell migration and proliferation were detected. The expression of the lncRNA MIAT in HCECs was detected by quantitative real-time PCR. MIAT was knocked down by small interfering RNA (siRNA) transfection. The effects of caspase-1 inhibition on pyroptosis, apoptosis, migration, and proliferation were observed.

Results: HS promoted pyroptosis in HCECs by elevating caspase-1, GSDMD, and the active cleavage of GSDMD (N-terminal domain, N-GSDMD), and increased the release of IL-1β, IL-18, LDH and the rate of apoptosis, with reduced cell migration. These changes were prevented by the inhibition of caspase-1. The expression of MIAT was significantly increased in HCECs exposed to a hyperosmotic medium. Silencing MIAT increased the expression of GSDMD, caspase-1, and inflammatory chemokines IL-1β and IL-18, and promoted apoptosis while inhibiting migration and proliferation in HCECs.

Conclusion: The lncRNA MIAT is involved in HS-induced pyroptosis and apoptosis and the inflammatory response of HCECs and provides a new understanding of the pathogenesis of dry eye.

Keywords: apoptosis; dry eye; myocardial infarction-associated transcript; pyroptosis.

Figure 1

Hyperosmolarity induced GSDMD-dependent pyroptosis in HCECs. (A) The mRNA level of pyroptosis marker GSDMD was detected by RT-PCR. (B–D) The protein levels of pyroptosis markers (GSDMD and N-GSDMD) were detected by Western blotting. (E) Cell viability of HCECs treated with hyperosmolarity medium (90 mM) after 3 days. (F) The protein level of caspase-1 was detected by caspase-1 activity assay kit. (G) The release of lactate dehydrogenase (LDH) increased in hyperosmolarity stress (HS)-stimulated cells. (H and I) IL-1β and IL-18 levels in the cell were detected by qRT-PCR. (J) Showing the distributions of GSDMD puncta in HCECs exposed to hyperosmotic medium (90mM). Values are shown as mean ± SD. *P<0.05, **P<0.01, ****P <0.0001.

Figure 2

Hyperosmolarity reduced the migration of HCECs and promoted apoptosis. (A and B) Cell ability of migration was measured by transwell assay after HCECs treated with various concentrations of hyperosmolarity medium. The data are presented as the mean ± SD, *p<0.05 compared with iso-osmotic as the control. (C) The mRNA level of apoptosis marker caspase-3 was detected by qRT-PCR. (D and E) The ratio of protein level of apoptosis marker (Bcl-2/Bax) was detected by Western blotting. (F and G) Cell apoptosis rate was evaluated in flow cytometry analysis. Data obtained from more than three repeated experiments were shown as mean ± SD. *P<0.05, **P<0.01, ****P <0.0001.

Figure 3

Hyperosmolarity induced MIAT upregulation and knocking-down MIAT promoted pyroptosis but inhibited proliferation in HCECs. (A) qRT-PCR assay revealed MIAT expression. (B) qRT-PCR assay revealed MIAT expression after being treated with transfected with MIAT-specific siRNA or a negative control siNC. (C and D) The mRNA levels of GSDMD and caspase-1 were detected by qRT-PCR versus the control group. (E–H) The protein levels of GSDMD and caspase-1 were detected by Western blotting and caspase-1 activity assay kit. (I and J) The mRNA levels of IL-1β and IL-18 were detected by qRT-PCR. (K and L) The contents of IL-1β and IL-18 in supernatants were examined by ELISA assay. (M) The release of lactate dehydrogenase (LDH) increased in siMIAT. (N) Cell viability of HCECs was decreased after knocking down MIAT in 3 days. (O) Showing the distributions of GSDMD puncta in HCECs after MIAT knockdown treatment. Data obtained from more than three repeated experiments were shown as mean ± SD. *P < 0.05, **P < 0.01.

Figure 4

Knocking down MIAT inhibited the migration and promoted apoptosis of HCECs. (A and B) Cell ability of migration was measured by transwell assay after HCECs treated with transfection siRNA. (C–E) The mRNA levels of caspase-3, caspase-8, and the ratio of Bcl2/Bax were detected by qRT-PCR. **P <0.01, ***P <0.001 compared with the control. (F and G) The protein levels of caspase-3 and caspase-8 were detected by Western blotting. *P<0.05 compared with the control. (H and I) Cell apoptosis rate was evaluated in flow cytometry analysis. **P <0.01 compared with siNC as the control.

Figure 5

Caspase-1 inhibitor attenuates pyroptosis induced by hyperosmolarity stress (HS) and MIAT knockdown and regulates apoptosis in HCECs. (A) Cell viability of HCECs treated with various concentrations of Ac-YVAD-CHO (1, 5, 10 μM). (B) Ac-YVAD-CHO treatment (10 μM) suppressed caspase-1, GSDMD, IL-1β, and IL-18 in mRNA levels in HS-stimulated cells. (C) Ac-YVAD-CHO treatment (10 μM) suppressed caspase-1, GSDMD, IL-1β, and IL-18 in mRNA level in siRNA cells. (D–I) Ac-YVAD-CHO treatment (10 μM) suppressed GSDMD and N-GSDMD in protein level in both HS-stimulated cells and siMIAT cells, values are shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001.

Figure 6

Caspase-1 inhibitor reversed migration and apoptosis in HCECs induced by hyperosmolarity stress (HS) and MIAT knockdown. (A and B) Cell ability of migration was measured by transwell assay after HCECs were treated with 90mM concentrations of hyperosmolarity medium and Ac-YVAD-CHO treatment (10 μM). (C and D) Cell ability of migration was measured by transwell assay treated with transfection siRNA and Ac-YVAD-CHO treatment (10 μM). (E and F) Ac-YVAD-CHO treatment (10 μM) reduced the proportion of apoptotic HS-stimulated cells, shown as ratio of Bcl-2/Bax by Western blotting. (G and H) Cell apoptosis rate decreased in flow cytometry analysis after Ac-YVAD-CHO treatment (10 μM) in the DE group. (I and J) Cell apoptosis rate decreased in flow cytometry analysis after Ac-YVAD-CHO treatment (10 μM) in knockdown MIAT group. The ratio of values are shown as mean ± SD. *P<0.05, **P<0.01.