Deficiency of NEAT1 prevented MPP⁺-induced inflammatory response, oxidative stress and apoptosis in dopaminergic SK-N-SH neuroblastoma cells via miR-1277-5p/ARHGAP26 axis

Highlights

• NEAT1 was highly induced in response to MPP⁺ in SK-N-SH cells.

• Deficiency of NEAT1 suppressed MPP⁺-induced apoptosis, inflammatory response and oxidative stress in SK-N-SH cells.

• NEAT1 knockdown downregulated ARHGAP26 via targeting miR-1277-5p.

• Overexpression of miR-1277-5p triggered similar effect to NEAT1 deficiency in MPP⁺-induced SK-N-SH cells.

Abstract

Noncoding RNAs including long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) have been documented to play prominent role in neurodegenerative diseases including Parkinson’s disease (PD). This study intended to investigate the role of lncRNA nuclear enriched assembly transcript 1 (NEAT1) in MPP⁺-induced PD model in dopaminergic neuronblastoma SK-N-SH cells, as well as its mechanism through sponging miRNA (miR)-1277-5p. Real-time PCR and western blotting revealed that NEAT1 and ARHGAP26 were upregulated, and miR-1277-5p was downregulated in MPP⁺-treated SK-N-SH cells in a certain of concentration- and time- dependent manner. MPP⁺ induced apoptosis in SK-N-SH cells, as evidenced by decreased cell viability and Bcl-2 expression, and elevated apoptosis rate and levels of Bax and cleaved caspase-3, which were examined by MTT assay, flow cytometry and western blotting. Moreover, commercial assay kits indicated that inflammatory response and oxidative stress were provoked in response to MPP⁺, due to promoted contents of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, malondialdehyde, and lactate dehydrogenase, accompanied with suppressed superoxide dismutase and glutathione peroxidase levels. Notably, MPP⁺-induced apoptosis, inflammatory response and oxidative stress in SK-N-SH cells were mitigated by NEAT1 knockdown and/or miR-1277-5p overexpression. Moreover, silencing of miR-1277-5p could abrogate the suppression of NEAT1 deficiency on MPP⁺-induced cell injury. Similarly, upregulating miR-1277-5p-elicited neuroprotection in MPP⁺-induced SK-N-SH cells was reversed by ARHGAP26 restoration. Dual-luciferase reporter assay demonstrated a direct interaction between miR-1277-5p and NEAT1 or ARHGAP26. Collectively, NEAT1 upregulation might contribute to MPP⁺-induced neuron injury via NEAT1-miR-1277-5p-ARHGAP26 competing endogenous RNAs (ceRNAs) pathway.

Introduction

Parkinson’s disease (PD), the second most common neurodegenerative disorder after alzheimer's disease (Poewe et al., 2017). The incidence of PD is rapidly increased with age in China (Li et al., 2019), making PD as one main threat in aged people. Although the etiology of PD is still unclear, genetic factors and environmental factors (such as toxicants) have been declared to evoke the development of PD (Dunn et al., 2019). The common etiopathogenesis of PD is ascribed to the loss of dopaminergic neurons in substantia nigra pars compacta, and the accumulation of Lewy bodies in the brain (Poewe et al., 2017). Extensive evidences have indicated that inflammatory damage and oxidative injury are responsible to neurodegeneration and neuroinflammation in PD (Anderson et al., 2018, Puspita et al., 2017). N-methyl-4-phenylpyridinium (MPP⁺) is the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a kind of neurotoxin that selectively destroys dopaminergic neurons, eventually leading to the occurrence of PD (Jiang et al., 2019b). Therefore, clarifying the mechanism of MPP⁺-induced PD is important to clarify the pathogenesis of PD.

Noncoding RNAs including long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) are abnormally expressed in neurodevelopment and neurodegenerative diseases including PD (Majidinia et al., 2016, Oe et al., 2019). Growing evidences support a key role of lncRNAs and miRNAs in dopaminergic dysfunction in neurological pathologies, such as PD (Carrick et al., 2016). LncRNAs are longer than 200 nucleotides (nt), and are structurally similar to message RNAs (mRNAs), even though lncRNAs have little protein coding capacity (Lyu et al., 2019). Newest researches on the pathogenesis of diseases have elucidated that lncRNAs are emerging targets for diagnosis and drug treatment of PD (Lyu et al., 2019), and that the role of several lncRNAs have already been illuminated in the pathogenesis of PD (Fan et al., 2019, Majidinia et al., 2016). However, the research on contribution of lncRNAs in PD remains infant. LncRNA nuclear enriched assembly transcript 1 (NEAT1) is aberrantly-mostly upregulated in non-cancerous pathological conditions, including neurodegenerative diseases (Prinz et al., 2019). Furthermore, the de novo investigations have disclosed the close relationship among NEAT1, inflammatory response (Xie et al., 2019, Zhang et al., 2019b) and oxidative stress (Simchovitz et al., 2019, Sunwoo et al., 2017). Here, we intended to explore the role of NEAT1 in inflammatory injury and oxidative damage in PD.

MiRNAs are a class of transcripts (19–24 nt) that control the expression of multiple mRNAs at post-transcriptional level (Majidinia et al., 2016). Numerous documents have expounded the prominent role of miRNAs in PD (Goh et al., 2019), and cell-free miRNAs are considered as potential biomarkers for central nervous system disorders including PD (Doxakis, 2020, van den Berg et al., 2020). MiRNA (miR)-1277-5p is associated with accurate molecular classification of different cancers, such as kidney cancer, colorectal cancer, hepatocellular carcinoma (Cao et al., 2019, Motieghader et al., 2017, Youssef et al., 2011). Nevertheless, the function of miR-1277-5p is not well understood presently. Thus, we wondered the role of miR-1277-5p in MPP⁺-induced dopaminergic neurons.

The presented study focused on the expression of NEAT1 and miR-1277-5p in MPP^+-induced model of PD in dopaminergic neuroblastoma SK-N-SH cells. Furthermore, the role of both was also investigated in inflammatory injury and oxidative damage in response to MPP⁺. Notably, the molecular mechanism of NEAT1 was further clarified through functioning as a competing endogenous RNA (ceRNA) of miR-1277-5p to regulate Rho GTPase-activating protein 26 (ARHGAP26, also known as GRAF), a negative regulator of the Rho family (Wang et al., 2013).