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丙戊酸钠下调SH-SY5Y细胞中Bcl-2表达的作用及机制分析
Sodium valprovate upregulates miR-34a to inhibit Bcl-2 expression in human neuroblastoma SH-SY5Y cells
in vitro - 第三军医大学学报 2018年40卷第12期 页码：1060-1065
- 作者机构：
1.400047 重庆，重庆师范大学教育科学学院:重庆市特殊儿童心理诊断与教育技术重点实验室
2.00047 重庆，重庆师范大学教育科学学院:特殊教育系
3.400038 重庆，陆军军医大学(第三军医大学)：第一附属医院输血科
4.400038 重庆，陆军军医大学(第三军医大学)：基础医学院生物化学与分子生物学教研室
- 作者简介：
[ 戴旭芳，E-mail：xufangd@aliyun.com DAI Xufang, E-mail:xufangd@aliyun.com
- 基金信息：国家自然科学基金青年科学基金(31201068)
DOI：10.16016/j.1000-5404.201712088
中图分类号： R394.6; R749.94; R971.6收稿：2017-12-11，
修回：2018-02-12，
纸质出版：2018-01
- 稿件说明：
移动端阅览
戴旭芳, 秦利燕, 连继勤. 丙戊酸钠下调SH-SY5Y细胞中Bcl-2表达的作用及机制分析[J]. 第三军医大学学报, 2018,40(12):1060-1065.
Xufang DAI, Liyan QIN, Jiqin LIAN. Sodium valprovate upregulates miR-34a to inhibit Bcl-2 expression in human neuroblastoma SH-SY5Y cells
in vitro [J]. Journal of Third Military Medical University, 2018, 40(12): 1060-1065.戴旭芳, 秦利燕, 连继勤. 丙戊酸钠下调SH-SY5Y细胞中Bcl-2表达的作用及机制分析[J]. 第三军医大学学报, 2018,40(12):1060-1065. DOI： 10.16016/j.1000-5404.201712088.
Xufang DAI, Liyan QIN, Jiqin LIAN. Sodium valprovate upregulates miR-34a to inhibit Bcl-2 expression in human neuroblastoma SH-SY5Y cells
in vitro [J]. Journal of Third Military Medical University, 2018, 40(12): 1060-1065. DOI： 10.16016/j.1000-5404.201712088.
摘要
目的
2
探讨丙戊酸钠(sodium valproate，VPA)在神经细胞中下调Bcl-2表达的作用及可能机制。
方法
2
用不同浓度的VPA处理SH-SY5Y细胞，定量PCR检测Bcl-2 mRNA与miR-34a水平变化，Western blot检测Bcl-2蛋白水平变化，报告基因系统检测Bcl-2启动子活性变化，联合转录抑制剂处理细胞，分析Bcl-2 mRNA稳定性变化，联合miR-34a模拟物及抑制剂处理细胞，进一步分析miR-34a在VPA下调Bcl-2表达中的作用。Annexin V/PI双染结合流式分析细胞凋亡情况。
结果
2
VPA可浓度依赖性下调SH-SY5Y细胞中Bcl-2的mRNA与蛋白水平(
P
<
0.05)。VPA暴露不改变Bcl-2的启动子活性(
P
>
0.05)，但显著降低了其mRNA的稳定性(
P
<
0.05)。同时，VPA可上调SH-SY5Y细胞中miR-34a的表达(
P
<
0.05)，联合使用miR-34a的抑制剂可逆转VPA对Bcl-2表达的下调作用，且单独的miR-34a模拟物也可以下调细胞中Bcl-2的表达。VPA暴露还可引起SH-SY5Y细胞的凋亡增加(
P
<
0.05)。
结论
2
VPA可通过上调SH-SY5Y细胞中miR-34a水平而抑制Bcl-2的表达。
Abstract
Objective
2
To investigate the inhibitory effect of sodium valproate (VPA) exposure on Bcl-2 expression and explore the possible mechanism in human neuroblastoma SH-SY5Y cells
in vitro
.
Methods
2
SH-SY5Y cells exposed to different concentrations of VPA were examined for expression levels of bcl-2 mRNA and miR-34a using qRT-PCR and for Bcl-2 protein expression using Western blotting. The changes in the promoter activity of bcl-2 gene in VPA-treated cells were analyzed using a reporter gene assay
and the stability of bcl-2 mRNA was evaluated after transcription inhibition. A miR-34a mimic and a miR-34a antagonist were used to analyze the role of miR-34a in VPA-induced Bcl-2 down-regulation in the cells. The apoptosis of SH-SY5Y cells following VPA exposures was assessed using flow cytometry with Annexin V and PI staining.
Results
2
VPA exposure dose-dependently down-regulated the expression of Bcl-2 at both the mRNA and protein levels in SH-SY5Y cells (
P
<
0.05). VPA treatment significantly decreased the stability of bcl-2 mRNA (
P
<
0.05) without affecting its transcriptional activity (
P
>
0.05)
and obviously up-regulated the expression of miR-34a in SH-SY5Y cells (
P
<
0.05). Treatment of the cells with the miR-34a mimic alone inhibited the expression of Bcl-2
and application of the miR-34a antagonist obviously reversed the inhibitory effect of VPA on Bcl-2 expression in SH-SY5Y cells. VPA exposure also significantly increased the apoptosis of SH-SY5Y cells (
P
<
0.05).
Conclusion
2
VPA exposure down-regulates the expression of Bcl-2 possibly by up-regulating miR-34a level in SH-SY5Y cells.
关键词
Keywords
references
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