LncRNA JHDM1D-AS1 对MPP+ 诱导的帕金森细胞模型线粒体功能的机制研究

目的 探讨长链非编码RNA (long non-coding RNA, LncRNA) JHDM1D 反义RNA 1( JHDM1D antisense RNA1，JHDM1D-AS1) 对帕金森细胞模型线粒体功能的影响及分子机制。方法 采用1- 甲基-4- 苯基吡啶离子（MPP+）处理 SH-SY5Y 细胞构建帕金森细胞模型，记为MPP+ 组，正常细胞作为对照组（control 组）。将JHDM1D-AS1 mimic和JHDM1D-AS1 siRNA 分别转染至MPP+ 诱导的SH-SY5Y 细胞中，利用RT-PCR 检测JHDM1D-AS1 表达。流式细胞技术分析JHDM1D-AS1 表达对SH-SY5Y 细胞凋亡、线粒体膜电位及活性氧（reactive oxygen species，ROS）含量的影响。Western blot 实验分析JHDM1D-AS1 表达对沉默调节蛋白1（SIRT1）表达的影响，并利用双荧光素酶报告验证两者的靶向关系。使用SIRT1 激活剂 Resveratrol 对转染JHDM1D-AS1 mimic 的细胞进一步处理，证实JHDM1D-AS1 调控SIRT1 对帕金森细胞线粒体的影响。结果 control 组JHDM1D-AS1 相对表达为0.85±0.21，MPP+ 模型组JHDM1DAS1相对表达为1.25±0.33，较control 组增加，差异有统计学意义（t=62.017，P ＜ 0.05）。转染JHDM1D-AS1 mimic和JHDM1D-AS1 siRNA 序列后，JHDM1D-AS1 相对表达分别为1.63±0.38 和0.72±0.17，与MPP+ 组比较差异有统计学意义（F=112.035，P ＜ 0.05）。MPP+ 组细胞凋亡率为17.64%，JHDM1D-AS1 mimic 和JHDM1D-AS1 siRNA 组细胞凋亡率分别为25.92% 和10.74%，差异有统计学意义（F=49.052，P ＜ 0.05）。MPP+ 组绿色荧光强度为22.20%，JHDM1D-AS1-mimic 和JHDM1D-AS1siRNA 组绿色荧光强度分别为43.97% 和10.65%，差异有统计学意义（F=57.390，P ＜ 0.05）。流式结果显示，MPP+ 组相对荧光强度为27.58%±4.25%，JHDM1D-AS1 mimic 和JHDM1D-AS1 siRNA组相对荧光强度分别为45.10%±6.05% 和14.82%±3.70%，差异有统计学意义（F=25.794，P ＜ 0.05）。Control 组SIRTI 蛋白表达为1.00±0.23，MPP+ 模型组SIRT1 表达下调为0.70±0.27，差异有统计学意义（t=35.740，P ＜ 0.05）。转染JHDM1D-AS1 mimic 和JHDM1D-AS1 siRNA 后，SIRT1 表达分别为0.44±0.16 和1.34±0.22，与MPP+ 组比较差异有统计学意义（F=29.508，P ＜ 0.05）。Target Scan 软件预测，JHDM1D-AS1 与SIRT1 序列存在结合位点，双荧光素酶实验显示，JHDM1D-AS1 过表达可降低WT- SIRT1 荧光素酶活性。JHDM1D-AS1 过表达时线粒体膜电位降低，SIRT1 激活过表达时线粒体膜电位升高，转染 JHDM1D-AS1 mimic 并激活 SIRT1 后线粒体膜电位水平回升。结论 沉默JHDM1D-AS1 表达可抑制MPP+ 诱导的SH-SY5Y 细胞凋亡，其作用机制可能与JHDM1D-AS1 靶向调控SIRT1，进而影响帕金森细胞模型线粒体功能有关。

Mechanism Study of LncRNA JHDM1D-AS1 on Mitochondrial Function in MPP+-induced Parkinson’s Cell Models

Objective To investigate the effect and molecular mechanism of long non-coding RNA (LncRNA) JHDM1D antisense RNA 1(JHDM1D-AS1) on mitochondrial function in Parkinson’s cell model. Methods The SH-SY5Y cells were treated with 1-methyl-4-phenylpyridine ion (MPP+) to construct a Parkinson’s cell model, which was recorded as the MPP+ group, and the normal cells were used as the control group. JHDM1D-AS1 mimic and JHDM1D-AS1 siRNA were transfected into MPP+-induced SH-SY5Y cells, respectively, and the expression of JHDM1D-AS1 was detected by RT-PCR. Flow cytometry was used to analyze the effect of JHDM1D-AS1 expression on SH-SY5Y cell apoptosis, mitochondrial membrane potential and reactive oxygen species（ROS）content. The effect of JHDM1D-AS1 expression on SIRT1 protein expression was analyzed by Western blot experiments, and the targeting relationship between the two was verified by dual-luciferase reporter. The cells transfected with JHDM1D-AS1 mimic were further treated with SIRT1 activator Resveratrol to confirm the effect of JHDM1DAS1 on the mitochondria of Parkinson’s cells. Results The relative expression of JHDM1D-AS1 in the control group was 0.85 ± 0.21, and that in the MPP+ model group was 1.25 ± 0.33, compared to the control group，with a statistically significant difference (t=62.017, P<0.05). After transfection with JHDM1D-AS1 mimic and JHDM1D-AS1 siRNA sequences, the relative expression of JHDM1D-AS1 was 1.63 ± 0.38 and 0.72 ± 0.17 respectively, which was statistically significant compared with MPP+ group (F=112.035, P<0.05). The apoptosis rate of MPP+ group, JHDM1D-AS1 mimic and JHDM1D-AS1 siRNA group were 17.64%, 25.92% and 10.74%, respectively, and the difference was statistically significant (F=49.052, P<0.05). The green fluorescence intensity of MPP+ group was 22.20%, while that of JHDM1D-AS1mimic and JHDM1D-AS1 siRNA group was 43.97% and 10.65%, respectively, with significant difference (F=57.390, P<0.05). Flow cytometry results showed that the relative fluorescence intensity of MPP+ group was 27.58%±4.25%, JHDM1D-AS1 mimic and JHDM1D-AS1 siRNA group were 45.10%±6.05% and 14.82%±3.70 %, respectively, and the difference was statistically significant (F=25.794, P<0.05). The expression of SIRTI protein in the control group was 1.00±0.23, and the expression of SIRT1 in the MPP+ model group was 0.70±0.27, and the difference was statistically significant (t=35.740, P<0.05). After transfection with JHDM1D-AS1 mimic and JHDM1D-AS1 siRNA, the expression of SIRT1 was 0.44±0.16 and 1.34±0.22 respectively, which was statistically significant compared with MPP+ group (F=29.508, P<0.05). Target scan software predicted that there was a binding site between JHDM1DAS1 and SIRT1 sequence. Double luciferase assay showed that JHDM1D-AS1 overexpression could reduce the luciferase activity of WT-SIRT1. The mitochondrial membrane potential decreases when JHDM1D-AS1 was overexpressed. The mitochondrial membrane potential increases when SIRT1 activation was overexpressed. Mitochondrial membrane potential levels rebound after transfection of JHDM1D-AS1 mimic and activation of SIRT1. Conclusion Silencing the expression of JHDM1DAS1 could inhibit MPP+-induced apoptosis of SH-SY5Y cells, and its mechanism may be related to the targeted regulation of SIRT1 by JHDM1D-AS1, thereby affecting the mitochondrial function of the Parkinson’s cell model.