| Purpose: To investigate the regulatory effect of long noncoding RNA(lncRNA)myocardial infarction-associated transcripts(MIAT)on pyroptosis and apoptosis in dry eye model of human corneal epithelial cells(HCECs)induced by hyperosmolarity stress.Methods: 1.Human corneal epithelial cells were cultured in hyperosmotic medium with Na Cl concentrations of 70,90,100,110 and 120 m M for 24 h,and a dry eye model was established.The m RNA and protein expression of MIAT and gasdermin D(GSDMD)was detected by q RT-PCR and western blotting after stimulating HCECs with different concentrations of hyperosmolarity medium,and the optimal hyperosmotic concentration was selected for following experiments.2.Experimental grouping,divided into(1)normal control group(NC): 0m M isotonic solution cultured for 24h;(2)hyperosmotic group: 90 m M hyperosmotic solution cultured for 24h;(3)si NC group: small interfering RNA si NC transfected to cells,cultured for 24h;(4)si MIAT group: small interfering RNA si MIAT transfected to cells,cultured for 24 h.q RT-PCR and ELISA methods measured the levels of interleukin-1β(IL-1β)and interleukin-18(IL-18)in each group of the cells to assess the cellular inflammatory response.The LDH kit detected the level of LDH release after cell lysis in the cell supernatant.Pyroptosis phenotype of the cells in each group was detected by the activity of caspase-1,immunofluorescence staining,and western blotting.The expression of apoptotic markers of the cells in each group was detected by q RT-PCR and western blotting.Annexin V-FITC/PI double-labeled flow cytometry was used to detect the apoptosis rate of each group of cells.CCK8 method detected the proliferation viability of the cells in each group.Transwell method detected the migration capacity of the cells in each group.3.After pretreating HCECs with 1,5and 10 μM exogenous caspase-1 inhibitor Ac-YVAD-CHO,the cells were stimulated with hyperosmotic medium or transfected si MIAT,and a dimethyl sulfoxide(DMSO)control group was set up,and the optimal inhibitor concentration was selected by CCK8 experiments,and the changes of pyroptosis,apoptosis and migration in HCECs were detected respectively.Results: Compared with the NC group,the expression of MIAT in the 90,110,and120 m M hyperosmotic groups was significantly increased.At the same time,the expression of m RNA and protein of GSDMD was also significantly increased in the 90 m M hyperosmotic group relative to the NC group,and the protein expression of the active cleavage GSDMD(N-terminal domain,N-GSDMD)was consistent with GSDMD,so 90 m M(500 m Osm/L)hyperosmotic medium was chosen to build an in vitro dry eye model.2.In the HCECs hyperosmotic treatment group,the m RNA and protein expressions of caspase-1 and inflammatory factors IL-1β and IL-18 were significantly increased.The release of LDH was significantly elevated.Meanwhile,the expression of the apoptotic marker caspase-3 in the hyperosmotic group increased,and the ratio of Bcl-2/Bax significantly decreased.The results of flow cytometry showed that the apoptosis rate of cells in the hyperosmotic group was significantly greater than that in the control group.After hyperosmotic media treatment for 24 h,the proliferation and migration ability of cells reduced significantly.3.The expression of m RNA and protein in GSDMD and caspase-1 in the si MIAT group was significantly increased,and the protein expression of the N-GSDMD fragment was also significantly increased;similarly,the expression of inflammatory factors IL-1β and IL-18 m RNA and protein was higher than that in the si NC group.LDH detection experiments showed that the LDH release in the si MIAT group was significantly higher than that in the si NC group.The expression of caspase-3 and caspase-8 in the si MIAT group was significantly increased,while the ratio of Bcl/Bax decreased significantly,indicating that after knocking down MIAT,the apoptosis of HCECs was promoted,and the experimental results of flow cytometry also showed a higher apoptosis rate in the si MIAT group.Moreover,the cell proliferation and migration ability of the si MIAT group decreased significantly.4.Compared with the simple hyperosmotic group,the expression of pyroptosis and apoptosis markers and inflammatory factors in the Ac-YVAD-CHO combined with hyperosmotic group was significantly reversed.The proliferation and migration capacity of HCECs in the AcYVAD-CHO combined with hyperosmotic group was increased compared with that in the simple hyperosmotic group.The pyroptosis and apoptosis phenotypes in the AcYVAD-CHO combined si MIAT group were also significantly lower than those in the AcYVAD-CHO combined si NC group,and the proliferation and apoptosis ability of cells were also significantly increased.Conclusion: 1.Hyperosmotic stress can increase the expression of GSDMD,caspase-1,caspase-3,and inflammatory chemokines IL-1β and IL-18 in HCECs,promoting pyroptosis and apoptosis,and inhibiting migration and proliferation.2.MIAT is elevated in HCECs cultured with hyperosmolarity,and participates in hyperosmotic stress induced pyroptosis,apoptosis and inflammatory responses in HCECs.3.MIAT inhibits the pyroptosis and apoptosis of HCECs induced by hyperosmotic stress through caspase-1,and promotes cell proliferation and migration. |
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