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Title: [Impact and related mechanism on the improvement of hyperglycemia-induced pyroptosis in H9c2 cells by mircoRNA-214].
Author: Wang Y, Zhao RZ, Chen PK, Xu GX, Liu ZJ, Long XP, Qiu ZM, Shi B.
Journal: Zhonghua Xin Xue Guan Bing Za Zhi; 2019 Oct 24; 47(10):820-828. PubMed ID: 31648465.
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%CO(2), repeat passage was made after cell density reached about 80%, The 5(th) to 8(th) 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. 目的： 探讨微小RNA（miR）-214是否通过靶向抑制天冬氨酸蛋白水解酶-1（caspase-1）改善高糖诱导的H9c2细胞焦亡。 方法： 选取生长状态良好的融合约80%以上的大鼠心肌细胞系H9c2细胞，经消化传代后接种于新的T25培养瓶中，于37 ℃ 5%CO(2)培养箱中培养，待细胞密度达到约80%时重复传代操作，取第5~8代细胞用于后续实验。为了观察过表达miR-214对高糖所致的H9c2细胞焦亡及caspase-1表达的影响，将细胞分为4组。对照组：正常培养的H9c2细胞。高糖处理组（HG组）：50 mmol/L葡萄糖干预H9c2细胞24 h。miR-214模拟物+高糖处理组（mimics+HG组）：H9c2细胞转染miR-214模拟物24 h后予以50 mmol/L高糖处理24 h。miR-214模拟物阴性对照+高糖处理组（MNC+HG组）：H9c2细胞转染miR-214模拟物阴性对照24 h后予以50 mmol/L高糖处理24 h。为进一步验证miR-214通过靶向抑制caspase-1发挥抗高糖诱导的细胞焦亡效应，以细胞过表达caspase-1作为回复实验，将细胞分为4组。高糖处理组（HG组）：50 mmol/L葡萄糖干预H9c2细胞24 h。miR-214模拟物+高糖处理组（mimics+HG组）：H9c2细胞转染miR-214模拟物24 h后予以50 mmol/L高糖处理24 h。miR-214模拟物+高糖+携带caspase-1基因并表达EGFP绿色荧光蛋白的重组腺病毒（Ad-caspase-1-EGFP）组（mimics+HG+Ad-caspase-1-EGFP组）：采用携带绿色荧光蛋白的腺病毒转染caspase-1至H9c2细胞48 h，随后转染miR-214模拟物24 h，然后予以50 mmol/L高糖处理24 h。miR-214模拟物+高糖+表达EGFP的空病毒组（mimics+HG+Ad-EGFP组）：采用携带绿色荧光蛋白的空载腺病毒转染H9c2细胞48 h，随后转染miR-214模拟物24 h，然后给予50 mmol/L高糖处理24 h。实时荧光定量PCR检测细胞中miR-214及caspase-1 mRNA表达水平。免疫荧光法检测细胞中caspase-1蛋白表达定位。Western blot法检测细胞中天冬氨酸蛋白水解酶前体-1（procaspase-1）、天冬氨酸蛋白水解酶剪切体-1（cleaved-caspase-1）、炎症复合体（NLRP3）、凋亡相关斑点样蛋白（ACS）的表达水平。ELISA法检测细胞培养液中白细胞介素（IL）-1β和IL-18的分泌量。双荧光素酶报告基因检测miR-214与caspase-1的相关性。 结果： （1）各组细胞miR-214及caspase-1 mRNA的表达水平：HG组和MNC+HG组细胞中miR-214表达均明显低于对照组（P均<0.05），而mimics+HG组细胞中miR-214表达则明显高于对照组（P<0.05）。HG组及MNC+HG组细胞中caspase-1 mRNA表达水平均明显高于对照组（P均<0.05），mimics+HG组细胞中caspase-1 mRNA表达水平则低于对照组（P<0.05）。（2）各组细胞中caspase-1的表达：对照组细胞中绿色荧光强度表达较弱，而HG组及MNC+HG组绿色荧光表达较强。mimics+HG组细胞中绿色荧光表达较HG组弱。（3）各组细胞中ASC和NLRP3蛋白的表达水平：HG组和MNC+HG组细胞中ASC和NLRP3蛋白表达水平均高于对照组（P均<0.05）。mimics+HG组细胞中ASC和NLRP3蛋白表达水平均明显低于HG组（P均<0.05）。（4）各组细胞培养液中IL-1β和IL-18的分泌量：HG组和MNC+HG组细胞培养液中IL-1β、IL-18含量均明显高于对照组（P均<0.05）。mimics+HG组细胞培养液中IL-1β、IL-18含量均明显低于HG组（P均<0.05）。（5）miR-214与caspase-1的相关性：miR-214特异性结合caspase-1 3′UTR。同时Western blot结果显示，HG组和MNC+HG组细胞中cleaved-caspase-1蛋白表达水平均明显高于对照组（P均<0.05），mimics+HG组细胞中cleaved-caspase-1蛋白表达水平则明显低于HG组（P<0.05）。而各组细胞中procaspase-1蛋白表达水平差异无统计学意义（P>0.05）。（6）回复实验中各组细胞中procaspase-1、cleaved-caspase-1以及ASC、NLRP3蛋白的表达水平：各组细胞中procaspase-1表达差异无统计学意义（P>0.05）。mimics+HG组细胞中cleaved-caspase-1表达明显低于HG组（P<0.05），ASC和NLRP3蛋白表达亦明显低于HG组（P均<0.05）。而mimics+HG+Ad-caspase-1-EGFP组细胞中cleaved-caspase-1表达则明显高于mimics+HG组，ASC和NLRP3蛋白表达亦明显高于mimics+HG组（P均<0.05）。（7）回复实验中各组细胞培养液中IL-1β和IL-18的分泌量：mimics+HG组细胞培养液中IL-1β和IL-8含量均明显低于HG组（P均<0.05）。而mimics+HG+Ad-caspase-1-EGFP组细胞培养液中IL-1β和IL-18含量均明显高于mimics+HG组（P均<0.05）。 结论： miR-214可通过靶向抑制caspase-1改善高糖诱导的H9c2细胞焦亡。.

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