Impact of Pax-8 gene interference on mitochondrial function and cardiomyocyte apoptosis
10.3760/cma.j.issn.0253-3758.2013.01.012
- VernacularTitle:转录因子Pax-8基因干扰后线粒体功能对心肌细胞凋亡的影响
- Author:
Xiao-Chun DAI
1
;
Xi ZHOU
;
Xiao-Yan HUANG
;
Liang-Guo WANG
;
Su LIN
;
De-Ye YANG
Author Information
1. 325000,温州医学院附属第一医院心内科温州医学院心血管生物和基因研究所
- Keywords:
Myocytes,cardiac;
Apoptosis;
Mitochondria;
Transcription factors
- From:
Chinese Journal of Cardiology
2013;41(1):54-59
- CountryChina
- Language:Chinese
-
Abstract:
Objective To observe the effects of paired box gene 8 (Pax-8) silencing by RNA interference on mitochondrial function and cardiomyocytes apoptosis.Methods The cultured H9C2 (2-1) myocytes were divided into 3 groups:short interference RNA targeting Pax-8 (Pax-8 siRNA) group,nonspecific siRNA group as the negative control (NC siRNA),and blank control group (BC siRNA).Fluo rescence spectrophotometry was used to detect the activity of caspase-3.RT-PCR was performed to detect mRNA expression of Bcl2 and Bax.The protein expression of Bcl2、Bax and cytoplasm of Cytothrome was examined by Western blot.Changes of △ψm were detected by flow cytometry.△ψm with JC-1 monomer/polymer ratio was calculated for measuring mitochondrial depolarization proportion.Results Compared to NC siRNA and BC siRNA group(0.075 ±0.021,0.072 ±0.019),the activity of caspase-3 in Pax-8 siRNA group(0.167 ±0.012) was significantly increased (P ＜0.05) ； Bcl2 mRNA and protein expression in Pax-8siRNA group (0.61 ±0.06,0.94 ±0.11) were significantly downregulated compared with NC siRNA group (0.90±0.070,1.39±0.15) and BC siRNA group (0.94 ±0.087,1.49±0.20) (P＜0.05)； Bax mRNA and protein expression in Pax-8 siRNA group (1.05 ± 0.10,1.25 ± 0.12) were markedly upregulated compared with NC siRNA group (0.72 ± 0.03,0.99 ± 0.12) and BC siRNA group (0.64 ±0.03,0.92 ± 0.06),P ＜ 0.05 ； cytosolic cytochrome expression in Pax-8 siRN A group (0.75 ± 0.14) was significantly upregulated compared with NC siRNA group (0.51 ± 0.06) and BC siRNA group (0.48 ±0.07) (P ＜ 0.05) ； JC-1 monomer/polymer ratio in Pax-8 siRNA group (0.163 ± 0.011) was significantly increased compared with NC siRNA group(0.092 ±0.015) and BC siRNA group (0.072 ± 0.025) (P ＜0.05) indicating mitochondrial membrane potential was significantly reduced in Pax-8 siRNA group.Above parameters were similar between NC siRNA group and BC siRNA group (P ＞ 0.05).Conclusion Inhibiting Pax-8 results in enhanced cardiomyocytes apoptosis through the mitochondrial pathway.
