Down-regulation of STAT3 expression using vector-based RNA interference promotes apoptosis in Hepatocarcinoma cells
Down-regulation of STAT3 expression using vector-based RNA interference promotes apoptosis in Hepatocarcinoma cells
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DOI：
10.3109/21691401.2015.1029628
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发表时间：
2016-07
期刊：
影响因子：
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通讯作者：
Junwei Zhang;Jiajun Du;Qi Liu;Yi Zhang
中科院分区：
文献类型：
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作者：
Junwei Zhang;Jiajun Du;Qi Liu;Yi Zhang
在本研究中，我们采用基于DNA载体的RNAi方法沉默Bel-7402细胞中信号转导和转录激活因子3(STAT3)的表达，以探讨Janus激酶(JAK)/STAT3信号通路如何影响肝癌细胞的凋亡。根据GenBank的STAT3基因，构建并合成了STAT3小干扰RNA(SiRNA)表达载体pGCsi.U6/new RFP STAT3，用脂质体2000将其导入Bel-7402细胞。将携带或不携带siRNA基因的细胞分别置于水井中处理。流式细胞仪(FCM)和Annexin V/PI细胞凋亡检测试剂盒染色检测细胞凋亡率。同时用JC-1荧光染色观察线粒体膜电位(ΔΨm)，并用倒置荧光显微镜观察。用Western blotting分析caspase-3蛋白的表达。结果表明，STAT3 siRNA处理Bel-7402细胞后，细胞凋亡率显著增加(P<；0.05)。JC-1荧光染色观察到STAT3 siRNA组线粒体膜电位显著降低。经STAT3 siRNA处理后，活化的caspase-3蛋白表达增加，与对照组相比差异有统计学意义(P<；0.05)。STAT3基因沉默显著提高了对Bel-7402细胞的凋亡效应。
In this study, we followed a DNA vector-based RNAi approach to silence the signal transducer and activator of transcription 3 (STAT3) expression in Bel-7402 cells, to explore how the Janus kinase (JAK)/STAT3 signaling pathway influences the apoptosis of hepatocarcinoma cells. According to GenBank's STAT3 cDNA, the plasmid pGCsi.U6/neoRFP STAT3, which was designed for expression of STAT3 small interfering RNA (siRNA), was constructed and synthesized, and then transfected into Bel-7402 cells using Lipofectamine 2000. Cells with or without siRNA transfection were treated in wells. The apoptotic rate was detected by flow cytometry (FCM) and by staining with the Annexin V/propidium iodide (PI) apoptosis detection kit. Simultaneously, the mitochondrial membrane potential (ΔΨm) was visualized by JC-1 fluorescence staining and observed using the inverted fluorescence microscope. Furthermore, the expression of caspase-3 protein was analyzed by Western blotting. The results showed that treatment with STAT3 siRNA displayed effects in the Bel-7402 cells, causing a significantly increased apoptotic ratio (P < 0.05). The mitochondrial membrane potential of the STAT3 siRNA group, observed by the JC-1 fluorescence staining, decreased significantly. The protein expression of active caspase-3 increased with STAT3 siRNA treatment, and was significantly higher than that of the control group (P < 0.05). STAT3 gene-silencing significantly improves the apoptotic effect against Bel-7402 cells.