Impact and related mechanism on the improvement of hyperglycemia?induced pyroptosis in H9c2 cells by mircoRNA?214
10.3760/cma.j.issn.0253?3758.2019.10.009
- VernacularTitle:微小RNA?214改善高糖诱导的H9c2细胞焦亡的机制探讨
- Author:
Yan WANG
1
;
Ranzun ZHAO
;
Panke CHEN
;
Guanxue XU
;
Zhijiang LIU
;
Xianping LONG
;
Zhimei QIU
;
Bei SHI
Author Information
1. 遵义医科大学附属医院心内科563000
- Keywords:
Diabetic cardiomyopathies;
Myocytes,cardiac;
Pyrotosis;
MicroRNA?214;
Caspase?1
- From:
Chinese Journal of Cardiology
2019;47(10):820-828
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate whether microRNA(miR)?214 can improve hyperglycemia induced pyroptosis in H9c2 cells through targeting caspase?1. Methods H9c2 cells of rats those in good growth condition were selected and incubated into the T25 culture bottle after digestion and passage. Cells were cultured in an incubator at 37℃with 5%CO2, repeat passage was made after cell density reached about 80%, The 5th to 8th generations of cells were selected for the subsequent experiments. To observe the effect of overexpression of miR?214 on pyroptosis and caspase?1 expression in H9c2 cells induced by hyperglycemia, the cells were divided into 4 groups: Control group(H9c2 cells cultured normally), Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR?214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR?214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR?214 mimic?negative control+hyperglycaemic group(MNC+HG group, H9c2 cells were transfected with miR?214 mimic?negative control for 24 hours and then treated with 50 mmol/L hyperglycaemic for 24 hours). In order to further verify the anti?pyroptosis effect of miR?214 was mediated by targeted inhibition on caspase?1, cells overexpressing caspase?1 were used in the rescue experiment. The cells overexpressing caspase?1 were divided into 4 groups: Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR?214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR?214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR?214 mimics+hyperglycosis+recombinant adenovirus (Ad?caspase?1?EGFP) group with caspase?1 gene and EGFP green fluorescent protein expression (mimics+HG + Ad?caspase?1?EGFP group, H9c2 cells were transfected with caspase?1?green fluorescent protein?carrying adenovirus for 48 hours, followed by transfection of miR?214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycaemia for 24 hours), miR?214 mimics+HG+Ad?EGFP empty virus group (mimics+HG+Ad?EGFP group, H9c2 cells were transfected with empty adenovirus containing green fluorescent protein for 48 hours, followed by transfection with miR?214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycosis for 24 hours). The mRNA expression levels of miRNA?214 and caspase?1 in cells were detected by real?time quantitative PCR. The expression and localization of caspase?1 protein were detected by immunofluorescence assay. Western blot was used to detect protein expression levels of procaspase?1, cleaved caspase?1, NLRP3 and ACS with β?actin as internal reference. The secretion of IL?1β and IL?18 in cell culture medium was detected by ELISA. The correlation between miR?214 and caspase?1 was detected by double luciferase reporter gene. Results (1) The mRNA expression levels of miR?214 and caspase?1 in each group: the mRNA expressions of miR?214 in HG group and MNC+HG group were significantly lower than that in control group(P<0.05). The mRNA expression of miR?214 in mimics+HG group was significantly higher than that in control group (P<0.05). The mRNA expression levels of caspase?1 in HG group and MNC+HG group were significantly higher than that in control group(P<0.05). The mRNA expression level of caspase?1 in mimics+HG group was lower than that in control group(P<0.05). (2) The expression of caspase?1 in each group: the green fluorescence intensity in the control group was weak, which was strong in the HG group and MNC+HG group. The green fluorescence expression was weaker in mimics+HG group than in HG group. (3) ASC and NLRP3 protein expression levels in each group: ASC and NLRP3 protein expression levels in HG group and MNC+HG group were higher than those in control group(P<0.05). ASC and NLRP3 protein expression levels were significantly lower in mimics+HG group than in mimics+HG group (P<0.05). (4) The secretion of IL?1β and IL?18 in the cell culture medium of each group: the content of IL?1β and IL?18 in the cell culture medium of HG group and MNC+HG group was significantly higher than that of control group (P<0.05). The content of IL?1β and IL?18 in the cell culture medium of mimics+HG group was significantly lower than that of the HG group (P<0.05). (5) Correlation between miR?214 and caspase?1: miR?214 specifically binds to caspase?1 3 ＇UTR. Meanwhile, Western blot results showed that cleaved caspase?1 protein expression levels were significantly higher in both HG group and MNC+HG group than in control group (P<0.05). The levels of cleaved caspase?1 were significantly lower in mimics+HG group than in HG group (P<0.05). There was no significant difference in procaspase?1 expression among groups (P>0.05). (6) The expression levels of procaspase?1, cleaved caspase?1, ASC and NLRP3 in each group in rescue experiment: there was no significant difference in the expression of procaspase?1 in each group (P>0.05). Cleaved caspase?1 , ASC and NLRP3 protein expressions were significantly lower in mimics+HG group than in HG group (P<0.05). However, cleaved caspase?1, ASC and NLRP3 protein expressions were significantly higher in mimics+HG+Ad?caspase?1?EGFP group than in mimics+HG group (P<0.05). (7) The expression of IL?1β and IL?18 in rescue experiment: the secretions of IL?1β and IL?18 in the cell culture medium of the mimics+HG group were significantly lower than that of HG group (P<0.05), which were significantly higher in mimics+HG+Ad?caspase?1?EGFP group than in mimics+HG group (P<0.05). Conclusion miR?214 can improve the hyperglycemia induced pyroptosis in H9c2 cells by targeted inhibition of the caspase?1.
