MicroRNA-7 Inhibits Neuronal Apoptosis In A Cellular Parkinson’s Disease Model By Targeting Bax And Sirt2

Parkinson’s disease (PD) is the second most common age-related neurodegenerative disease. MicroRNA-7 (miR-7) displays neuroprotective properties against PD. However, the biological roles of miR-7 and its underlying molecular mechanisms in PD remain unclear. We demonstrated herein that 1-methyl-4-phenylpyridinium ion (MPP+) confers toxic effects on dopaminergic neuron in a dose-dependent manner in a cellular PD model, although this phenomenon is attenuated by miR-7 treatment. Introduction of miR-7 inhibits MPP+- induced neuronal apoptosis as reflected by the reduced terminal transferase- mediated dUTP nick end labeling-positive rate, mitochondrial permeability potential, caspase 3 activity, and nucleosomal enrichment factor. Bax and sirtuin 2 (Sirt2) are the direct targets of miR-7. Moreover, the effects of miR-7 were counteracted by Bax and Sirt2 overexpression, respectively. The altered molecular expressions downstream of Bax and Sirt2 are also involved in miR-7 regulation of the MPP+-triggered neuronal apoptosis. These findings have implications on the potential application of miR-7 in PD treatment.Part one Establishment of a stable cellular Parkinson’s disease modelMethods1. SH-SY5Y cells were treated with various concentration of MPP+(0、1、 2、4 mM) for 24 hours, and ultimately the optimal concentration was selected for establishing a cellular PD model.2. The cellular viability of SH-SY5Y cells treated with MPP+ was determined using CCK-8 assay.3. Intracellular ROS production was assess by ROS detection assay.4. LDH activity, an indicator of cellular toxicity, was tested by LDH activity kits.Results1. The cellular viability of SH-SY5Y cells was decreased in a MPP+ dose-dependent manner.2.4 mM MPP+was chosen for the construction of a cellular PD model.3. When SH-SY5Y cells were stimulated with 4 mM MPP+, intracellular ROS significantly enhanced.4. When SH-SY5Y cells were challenged by 4 mM MPP+, LDH activity also remarkably elevated.Conclusions4 mM MPP+has great effect on the viability of SH-SY5Y cells, and increased intracellular ROS levels, upregulated LDH activity, which was selected for establishing an acute cellular PD model.Part Two The biological effects of introduction of miR-7 on MPP+-treated SH-SY5Y cellsMethods1. After SH-SY5Y cells objected to different concentration of MPP+ were transfected with miR-7 or miR-NC mimics, their proliferation and growth abilities were assessed by CCK-8 assay.2. The four treatment groups were as follows:(1) SH-SY5Y cells were transfected with miR-NC mimics; (2) SH-SY5Y cells were transfected with miR-7 mimics; (3) SH-SY5Y cells objected to 4 mM MPP+ were transfected with miR-NC mimics; (4) SH-SY5Y cells objected to 4 mM MPP+ were transfected with miR-7 mimics.3. ROS detection assay and LDH activity assay kits were used to determined cellular toxicity.4. FACS, TUNEL assay, JC-1 staining, caspase-3 activity assay and nucleon enrichment factor analysis were performed to evaluate cell apoptosis.Results1. miR-7 enhanced the viability of SH-SY5Y cells treated with various dose of MPP+.2. miR-7 reduced the toxicity of MPP+ to SH-SY5Y cells.3. miR-7 inhibited MPP+-enhanced SH-SY5Y cell apoptosis.ConclusionsmiR-7 treatment significantly inhibited MPP+-induced SH-SY5Y cell apoptosis, and simultaneously reduced the cellular toxicity of MPP+. Therefore, miR-7 may be potential agent for neuron protection.Part Three The prediction and validation of miR-7’s target genesMethodsBax and Sirt2 were predicted as miR-7’s target genes by employing TargetScan, miRanda, and PicTar.After constructing DLR reporter vector with wide type and mutate Bax and Sirt2 3’-UTR, they were transfected with SH-SY5Y cells with or without miR-7 or miR-NC, and luciferase activities were assayed.After SH-SY5Y cells objected to MPP+were transfected with miR-NC or miR-7 mimics, qRT-PCR and Western blot assays were performed to analyze the mRNA and protein expression of Bax and Sirt2.Results3’-UTRs of Bax and Sirt2 genes have the complementary sequences with miR-7.DLR assay showed that miR-7 significantly reduced luciferase activity of wide type Bax and Sirt2 3’-UTR, however, mutate Bax and Sirt2 3’-UTR didn’t.miR-7 significantly decreased the mRNA and protein expression of Bax and Sirt2.ConclusionsBax and Sirt2 were validated as miR-7’s target genes.miR-7 reduced the mRNA and protein expression of Bax and Sirt2 of SH-SY5Y cells objected to MPP+.Part Four The regulation of Bax and Sirt2 downstream molecules by miR-7MethodsAfter SH-SY5Y cells objected to various concentration of MPP+ were transfected with miR-7 mimics or Sirt2-expressing plasmid alone or in combination,Western blot assays were performed to analyze the protein expression of Sirt2 and Bim.Caspase-3 activity detection was used to assess cell apoptosis.After SH-SY5Y cells objected to various concentration of MPP+ were transfected with miR-7 mimics or Bax-expressing plasmid alone or in combination, Western blot assays were performed to analyze the protein expression of Bcl-2, Bax, Cl-caspase 3 and Cyt c.MMP, caspase-3 activity, and nucleon enrichment factor assays were performed to evaluate cell apoptosis.ResultsmiR-7 significantly inhibited the expression of Bax and Sirt2 in SH-SY5Y cells treated with MPP+. Ectopic expression of Bax or Sirt2 partly counteracted the protective effects of miR-7 on MPP+-stimulated SH-SY5Y cells.miR-7 reduced MPP+-induced SH-SY5Y cell apoptosis, however, ectopic expression of Bax or Sirt2 efficiently reversed the changes.The results from JC-1 staining, caspase-3 activity and nucleon enrichment factor assays showed that ectopic expression of Bax or Sirt2 counteracted the inhibitory effects of miR-7 on MPP+-induced SH-SY5Y cell apoptosis.miR-7 reduced the expression of Bim, Cl-caspase 3 and Cyt c in SH-SY5Y cells treated with MPP+, and upregulating Bcl-2. However, overexpression of Bax or Sirt2 showed the opposite tendencies.ConclusionsmiR-7 regulated the apoptotic signaling by modulating the expressions of Bax and Sirt2 as well as their downstream molecules, and ultimately inhibited MPP+-induced SH-SY5Y cell apoptosis, suggesting miR-7 as a potential target for PD therapy.