MiR-142-5p Protects Against 6-OHDA-Induced SH-SY5Y Cell Injury by Downregulating BECN1 and Autophagy.

BACKGROUND: MiR-142-5p has been demonstrated to hold significant implications in neurological diseases. However, the impact and underlying regulatory mechanism of miR-142-5p in Parkinson's disease (PD) are still ominous.
METHODS: To simulate the PD, 6-hydroxydopamine (6-OHDA)-treated SH-SY5Y cell model was used in this study. Levels of messenger RNA and protein were tested by quantitative real-time polymerase chain reaction and Western blot analyses, respectively. The direct interaction between miR-142-5p and Beclin 1 (BECN1) was assessed by luciferase reporter assay. Furthermore, Cell Counting Kit-8 assay was performed to assess cytotoxicity of SH-SY5Y cell.
RESULTS: In consequence, a significant decrease of miR-142-5p was observed in 6-OHDA-induced SH-SY5Y cells. Over-/Low-expressed miR-142-5p resulted in a significant enhancement/inhibition on cell vitalities of 6-OHDA-treated SH-SY5Y cells, which might be modulated by repressing cellular autophagy through inhibiting level of BECN1 and LC3 II/LC3 I and elevating P62 level. Luciferase reporter assay showed that the BECN1 was the target gene of miR-142-5p. Additionally, the loss/gain of BECN1 rescued/blocked the effects of miR-142-5p on the viability of 6-OHDA-induced SH-SY5Y cells.
CONCLUSIONS: These results highlight that miR-142-5p functions as a neuroprotective regulator in 6-OHDA-induced neuronal SH-SY5Y cells simulating PD model in vitro via regulating autophagy-related protein BECN1 and autophagy to influence cell viability.

Introduction

Parkinson disease (PD) is ranked as the second most frequent, age-relevant, chronic neurodegenerative disease followed after Alzheimer disease (AD), which may lead to multiple negative impact on people’ lives, such as bradykinesia, resting-state tremor, and gait disorder.[1] As documented, autophagy is a lysosomal degradation pathway that removes aggregated proteins and damaged organelles, playing an essential role in survival, differentiation, development, and homeostasis.[2] Recently, increasingly studies have reported that autophagy dysregulation may be involved in the development of PD.[3,4] For instance, earlier reports demonstrated that autophagic vacuoles were accumulated in the brains of patients with PD.[5] However, how autophagy regulates the development of PD remains to be resolved.

In essence, microRNAs (miRNAs), identified as a class of small noncoding RNAs with about 20 nucleotides, can negatively regulate gene expression by degradation or posttranslational suppression of target messenger RNAs (mRNAs) in the 3′-untranslated region (3′-UTR). Over the past several years, it has been confirmed that that miRNAs play an important role in a variety of biological processes, such as cell differentiation, proliferation, and apoptosis.[6,7] As is known from the published studies, miRNAs are regarded as a crucial component in the synthesis of neuronal-committed progenitors as well as the differentiation and survival of immature neurons.[8,9] The critical inter-reliance between dopaminergic neurons and a functioning miRNA network is highlighted by suggesting that miR-133b regulating the maturation and function of midbrain dopaminergic neurons, as reviewed by Kim et al.[10] Multiple miRNAs have been reported to be expressed abnormally in major neurodegenerative diseases such as AD, PD, and Huntington disease.[11] Accordingly, potential miRNAs if they would be confirmed as neuroprotective activities contribute to revealing pathogenesis and raising several promising therapeutic strategy for neurodegenerative diseases. It’s worth noting that downregulation of miR-142 is identified in PD samples through detecting a subset of miRNAs with aberrant expression levels in the plasma of patients diagnosed with PD.[12] Of interest is that a thorough analysis of the regulatory network revealed miR-142 is a critical miRNA in the PD network and thus it may be proposed to be involved in PD progression.[13] Additionally, an upregulated expression level of miR-142-5p in neurons and macrophage/microglia nodules is confirmed for simian immunodeficiency virus encephalitis.[14] Notably, the abnormal expression of miR-142-5p contributes to the pathogenesis of AD by inducing synaptic dysfunction related to Aβ peptide 1-42 (Aβ42)-regulated pathophysiology.[15] However, whether miR-142-5p is involved in the pathogenesis of PD is still unclear.

In this study, we explored the functional implication of miR-142-5p in 6-hydroxydopamine (6-OHDA)-induced neurotoxicity imitating PD in vitro and underlying mechanism of action therein. This study provides a novel insight of potential biomarkers and therapeutic targets for PD diagnosis and treatment.

Materials and Methods

Cell Culture

Human neuroblastoma cell lines SH-SY5Y were obtained from the American Type Cell Collection (Manassas, Virginia). SH-SY5Y cell lines were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, California) Supplemented with 10% heat-inactivated fetal bovine serum, 100 μg/mL streptomycin, and 100 U/mL penicillin at 37°C in a humidified chamber with 5% CO2. After 48 hours of cultivation, cells were exposed to 6-OHDA (Sigma, St. Louis, Missouri) dissolved in saline (containing 0.2% Ascorbic acid) with different concentrations including 1, 50, and 100 mM for 24 hours.

Cell Transfection

MiR-142-5p antagomir, antagomir control, agomir, and agomir control were obtained from Shanghai GenePharma Co, Ltd (Shanghai, China). SH-SY5Y cell lines were seeded into a 6-well plate at a density of 1 × 106 cells/mL and then were transfected with 50 nM miR-142-5p agomir/antagomir or their controls by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s guidance. After transfection of 72 hours, the efficiency of transfection was used quantitative real-time polymerase chain reaction (qRT-PCR) to measure.

Cell Counting Kit-8 Assay

Viability of SH-SY5Y cells induced by 6-OHDA was evaluated using Cell Counting Kit-8 (CCK-8; Beijing Solarbio Science & Technology Co, Ltd, China) according to the manufacturer’s instruction. Samples after transfection for 72 hours were plated into 96-well plates at a density of 103 cells/well and then were incubated with 10 μL CCK-8 solution for 2 hours. Subsequently, the absorbance (optical density) of the wells in the plate was measured at 450 nm with a microplate reader.

Analysis of MiRNA Targets

To evaluate the target genes of miR-142 associated with PD, the predicted target lists obtained from TargetScan database (http://www.targetscan.org/) were used to confirm the target genes of miR-142-5p.[16] The transcripts of miR-142-5p with the conserved targets sites corresponding to 947 genes were selected. Indeed, recent studies proved that miRNAs can modulate autophagy by regulating multiple genes relevant to autophagy. It is well known that Beclin 1 (BECN1) is identified as a tightly well-documented related with autophagy. Notably, the binding sites between miR-142-5p and BECN1 gene were obtained through TargetScan database, and nucleotide sequences of target gene BECN1 (gene number: NM_019584.3) in 3′-UTR region were obtained from GenBank database (https://www.ncbi.nlm.nih.gov/genbank/).

Luciferase Reporter Assay

Dual-luciferase reporter assay system (Promega, Madison, Wisconsin, USA) was used to measure Luciferase activity based on the method described previously.[17] The complementary DNA (cDNA) fragment of BECN1 in 3′-UTR region involved in the miR-142-5p-binding site was amplified, which subsequently was subcloned into pGL3 luciferase promoter vector (Promega). Human neuroblastoma cell lines SH-SY5Y were seeded into 96-well plates at a density of 5 × 104 cells/well and cotransfected with agomir control and agomir, then Lipofectamine 2000 (Invitrogen) was used to construct luciferase reporters containing wild-type (WT) or mutant BECN1 3′-UTR. According to the instructions of manufacturer’s, dual-luciferase reporter assay kit (Promega) was used to measure the relative luciferase activities.

Quantitative Real-Time Polymerase Chain Reaction

Trizol was used to extract the total RNA and miRNeasy mini kit (Qiagen, Dusseldorf, Germany) was used to purify miRNA. All of the RNA was quantified by use of SmartSpec plus Spectrophotometer (Bio-Rad, Hercules, California, USA). Additionally, we used PrimeScript Reverse Transcriptase (Takara, Japan) and Bulge-Loop miRNA-specific reverse transcription primers (RiboBio, Guangzhou, China) to conduct the reverse transcription of RNA. Furthermore, quantitative PCRs were performed using SYBR R Premix Ex Taq II (Takara) to evaluate the expression of target gene BECN1 with GAPDH as a normalization control. Moreover, a small nuclear RNA U6 was used as normalization control, and the expression level of miR-142-5p was measured with Bulge-Loop primers (RiboBio) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The conditions of qRT-PCR reactions as follows: predegeneration and denaturation at 95°C for 3 minutes and 30 seconds, respectively; annealing treatment and extension at maintain for 30 seconds at 60°C and 72°C, respectively. These operations need to be cycled 40 times.

The sequences of primers were exhibited as follows: miR-142-5p F: 5′-AAAGT AGAAA GCACT AC-3′, R: 5′ -GAACA TGTCT GCGTA TCTC-3′; U6 F: 5′-CCCCT GGATC TTATC AGGCT C-3′, R: 5′-GCCAT CTCCC CGGAC AAAG-3′; BECN1 F: 5′-CCATG CAGGT GAGCT TCGT-3′, R: 5′-GAATC TGCGA GAGAC ACCAT C-3′; GAPDH F: 5′-TGTGG GCATC AATGGA TTTGG-3′, R: 5′-ACACC ATGTA TTCCG GGTCA AT-3′.

Western Blotting

RIPA lysis buffer (Beyotime, Nantong, China) was used to extract the total proteins. Next, bicinchoninic acid (BCA) assay was performed to measure the protein concentration, and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to resolve the proteins. Then Bio-Rad wet transfer system was applied to electrotransfer the resolved proteins to 0.22 µm polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat dry milk in phosphate-buffered saline at 4°C for 1 hour, and immunoblot analysis with rabbit polyclonal antibodies to BECN1 (1:1000, Cell Signaling Technology, Inc, CST, Danvers, Massachusetts), LC3-I/II (1:1000, CST), P62 (1:1000, CST), and β-actin (1:1000, CST). Subsequently, incubation of membranes was conducted using horseradish peroxidase-conjugated secondary anti-rabbit immunoglobulin G antibodies (1:5000, Proteintech, Rosemont, IL, USA) at room temperature for 1 hour in dark place.

Statistical Analysis

All of the data in present work were expressed as mean ± standard deviation. Statistical analyses were conducted with SPSS version 17.0 software (International Business Machines Corp, New York) and GraphPad Prism 5 (San Diego, California). Student t test was performed to analyze the difference between 2 groups and post hoc test with analysis of variance and Dunnett or Bonferroni were used to compare multiple groups. A value of P < .01 was considered statistically significant.

Results

MiR-142-5p Is Downregulated in 6-OHDA-Treated SH-SY5Y Cells

SH-SY5Y cells were treated with different concentration of 6-OHDA (1, 50, and 100 mM), CCK-8 assay was used to measure the cell viability. It can be seen from Figure 1A that cell viability of SH-SY5Y cells was significantly decreased after 50 or 100 mM 6-OHDA treatment (P < .01). Then in the following experiments, 100 mM 6-OHDA was used to treat SH-SY5Y cells. MiR-142-5p expression in 6-OHDA-treated SH-SY5Y cell was detected with qRT-PCR assay (Figure 1B). The results showed that expression of miR-142-5p was markedly reduced in 6-OHDA-treated SH-SY5Y cells at the concentration of 100 mM (P < .01), suggesting that miR-152-5p may be involved in the progression of PD.

Figure 1.

MiR-142-5p expression was downregulated in 6-OHDA-induced SH-SY5Y cells. A, The viability of SH-SY5Y cells was measured in different concentrations of 6-OHDA (1, 50, and 100 μM) with CCK-8 assay. B, qRT-PCR assay was used to evaluate the expression of miR-142-5p in 6-OHDA-induced SH-SY5Y cells. Experiments were repeated 3 times. Data are presented as mean ± standard deviation, N = 3. * P < .05 or ** P < .01 versus control. CCK-8 indicates Cell Counting Kit-8; qRT-PCR, quantitative real-time polymerase chain reaction; 6-OHDA, 6-hydroxydopamine.

Effect of MiR-142-5p on 6-OHDA-Induced SH-SY5Y Cells Damage

To explore the effect of miR-142-5p on 6-OHDA-induced neuronal injury, the expression of endogenous miR-142-5p in 6-OHDA-induced SH-SY5Y cells was negatively or positively regulated by miR-142-5p antagomir or agomir. Compared with the corresponding control, miR-142-5p antagomir or agomir could effectively decrease or increase the expression of miR-142-5p (Figure 2A). Cell Counting Kit-8 assay (Figure 2B) showed miR-142-5p agomir treatment significantly enhanced the viability of 6-OHDA-induced SH-SY5Y cell and miR-142-5p antagomir had the opposite roles (P < .01). These results indicated that the expression of miR-142-5p played a neuroprotective role in 6-OHDA-treated neuronal damage.

Figure 2.

MiR-142-5p overexpression improved the effects of 6-OHDA on SH-SY5Y cell viability. A, The expression of miR-142-5p after cell transfection was detected by qRT-PCR assay. B, the viability of 6-OHDA-induced SH-SY5Y cells with different transfection methods was determined using CCK-8 assay. Data are presented as mean ± standard deviation, N = 3. ** P < .01 versus control. Mock; ## P < .01 and && P < .01 versus 6-OHDA control group. Sham group was negative control with nothing addition. Mock in 6-OHDA group (including mock/antagomir control/antagomir/agomir control/agomir) was the addition of 6-OHDA only as the control of antagomir control/antagomir/agomir control/agomir subgroups. CCK-8 indicates Cell Counting Kit-8; qRT-PCR, quantitative real-time polymerase chain reaction; 6-OHDA, 6-hydroxydopamine.

MiR-142-5p Protects 6-OHDA-Induced SH-SY5Y Cells From Damage Through Inhibiting Autophagy

It has been confirmed that autophagy dysfunction is one precipitating factor for the pathogenesis of PD.[18] Additionally, the involvement of miR-142 in autophagy has been reported, as reviewed from Zhai et al study.[19] To evaluate whether the functional role of miR-142-5p in 6-OHDA-treated neuronal injury was related with autophagy, some key autophagy-related proteins, including BECN1, LC3-I, LC3-II, and P62, were detected by Western blot assays after different treatment (Figure 3A and B). Consequently, we found that the protein levels of autophagy-related BECN1, LC3-I, LC3-II, and P62 were significantly altered after SH-SY5Y cells were stimulated with 6-OHDA. That is, the expressions of BECN1 and LC3-II/I were upregulated, and P62 expression was downregulated (P < .01, Figure 3B-D). Whereas after 6-OHDA-induced SH-SY5Y cells were treated with agomir, the protein level of autophagy marker P62 that reflects the intensity of autophagy was enhanced, the other autophagy marker LC3-II level was remarkably reduced, LC3-I conversely was notably increased, and thus the LC3-II/I ratio that reflects the information of autophagy was reduced. Additionally, BECN1 involved in autophagy and other biological processes[20] was found to have significantly reduced the protein level in agomir-transfected PD cell model (P < .01). The results demonstrate that miR-142-5p protects 6-OHDA-induced SH-SY5Y cells from damage through inhibiting autophagy.

Figure 3.

Effects of miR-142-5p on the protein of BECN1 in 6-OHDA-induced SH-SY5Y cells after agomir/agomir control transfection were detected by qRT-PCR and Western blot assay. A, The proteins bands of BECN1, LC3-II/I, and P62 were presented by Western blot detection. B-D, The quantitative analysis values of these proteins levels were expressed by histogram. Data are presented as mean ± standard deviation, N = 3. ** P < .01 versus control. Sham; ## P < .01 versus 6-OHDA control group. qRT-PCR indicates quantitative real-time polymerase chain reaction; 6-OHDA, 6-hydroxydopamine.

BECN1 Is the Target Gene of MiR-142-5p

To further explore the underlying mechanism of 6-OHDA-treated neuronal injury, the downstream genes of miR-142-5p were predicted by TargetScan analysis. We found that BECN1, an autophagy-related gene, was potentially predicted be the target genes of miR-142-5p (Figure 4A). Then a dual-luciferase reporter assay was conducted to verify the prediction. Figure 4B showed that the overexpression of miR-142-5p strikingly reduced the luciferase activity of WT BECN1 gene by comparing with the agomir control group (P < .01). There was no effect on the mutated BECN1 gene in agomir-transfected cells, which indicated that miR-142-5p can directly regulate the expression of BECN1 in 6-OHDA-treated SH-SY5Y cells.

Figure 4.

Direct interaction between miR-142 and BECN1 was identified by luciferase report assay. A, Schematic diagram of miR-142-5p target site in the 3′-UTR of BECN1 mRNA (wild-type) and mutation of BECN1 in the binding sites. B, Dual-luciferase reporter assay was used to evaluate the interaction between miR-142-5p and the 3′-UTR of BECN1. Data are presented as mean ± standard deviation, N = 3. ** P < .01 versus control. UTR indicates untranslated region.

MiR-142-5p Improves 6-OHDA-Treated SH-SY5Y Cells Injury by Targeting BECN1

The cytoprotective effects of miR-142-5p on the 6-OHDA-induced SH-SY5Y cell damage have been demonstrated by the above analysis. Moreover, the direct regulation of miR-142-5p on target gene BECN1 was also confirmed. Therefore, in order to explore whether BECN1 is the key target that mediates the biological processes of miR-142-5p in 6-OHDA-treated SH-SY5Y cells, the knockdown and overexpression of BECN1 were constructed. Consequently, it can be clearly found from Figure 5A and B that the mRNA and protein level of BECN1 after siRNA1/2 or pcDNA-BECN1 treatment was altered correspondingly (P < .01). Further, the effect of BECN1 on 6-OHDA-treated SH-SY5Y cells was measured by CCK-8 assays (Figure 5C). It can be seen from Figure 5C that the beneficial effect of miR-142-5p agomir on the viability of 6-OHDA-induced SH-SY5Y cells could be blocked by upregulating the expression of BECN1 (P < .01). Conversely, the superimposed destructive effect of miR-142-5p antagomir on 6-OHDA-induced SH-SY5Y cells viability could be rescued by downregulating the expression of BECN1 (P < .01). Based on the above results, we concluded that miR-142-5p may play a neuroprotective role in the progression of PD by targeting BECN1 gene.

Figure 5.

Effects of BECN1 on 6-OHDA-induced SH-SY5Y cells. A, qRT-PCR assay was used to confirm the expression of BECN1 mRNA after knockdown and pcDNA-BECN1 methods. B, Protein level of BECN1 in siBECN1 and pcDNA-BECN1 groups was determined by Western blot assay and was quantified. C, The viability of SH-SY5Y cells treated by transfection, silence of BECN1, enhancement of BECN1 was examined with CCK-8 assay. Data are presented as mean ± standard deviation, N = 3. *P < .05 or **P < .01 versus control. Mock. ## P < .01 versus antagomir/agomir control group. Sham group was negative control with nothing addition. Mock in 6-OHDA group (including mock/antagomir control/ antagomir/agomir control/agomir) was the addition of 6-OHDA only as the control of antagomir control/antagomir/agomir control/agomir subgroups. CCK-8 indicates Cell Counting Kit-8; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; 6-OHDA, 6-hydroxydopamine.

Discussion

MicroRNAs, a class of small noncoding RNAs, posttranscriptionally regulate gene expression of many metazoan by binding to partially complementary sites in mRNAs targets and play an important roles in various biological processes by interacting with target genes, such as differentiation, apoptosis, proliferation, and autophagy.[21-23] Increasingly studies report that miRNAs can be considered as key regulators of gene expression involved in neurodegenerative disorders, such as AD and PD.[24,25] Abnormal function of several miRNAs in PD has been presented. For example, it has been suggested that the downregulation of miR-7 expression in a PD mouse model decreased the levels of SNCA gene and thus led to cell protection from oxidative stress.[26] Further studies have demonstrated that the protective effect of miR-7 on cells was contributed by the remission of nuclear factor-κB suppression caused by the reduction of target mRNA RelA expression.[27] In addition, the expression of miR-34b and miR-34c in patients with PD was reported to be downregulated and the early downregulation of miR-34b/c with mitochondrial regulation function was demonstrated to play potential functional role in the development of PD.[28] It has been reported that miR-142-5p plays an important role in inflammation, cell apoptosis, and oxidative stress.[29-31] Particularly in AD, the inhibition of miR-142-5p was found to prevent the decrease of postsynaptic density protein 95 level in SH-SY5Y neuronal cells treated with Aβ42, which indicated that miR-142-5p may be a potential target that improves the synaptic signaling in AD.[15] Therefore, miR-142-5p might also play potential functional role in the pathogenesis and development of PD.

In this study, we investigated that the functional effect of miR-142-5p on the 6-OHDA-treated neural SH-SY5Y cell impairment by regulating the downstream target BECN1, an autophagy-related key regulator, which was identified by bioinformatic analysis. Since autophagy dysfunction is involved in the pathogenesis of PD, the BECN1 may be a promising strategy of neurotherapeutics by mediating the oxidative stress and inflammation in PD.[20] Additionally, experimental studies have suggested that 6-OHDA can induce the neuron loss in vivo.[32] In supporting of previous reports, the effect of 6-OHDA on SHSY5Y cells was performed with CCK-8 assay, the expression of miR-142-5p in 6-OHDA-treated SH-SY5Y cells was measured by qRT-PCR assay. It can be found from Figure 1 that the viability of 6-OHDA-treated SH-SY5Y cells was inhibited and expression levels of miR-142-5p in 6-OHDA-treated SH-SY5Y cells was significantly downregulated. Whereas the overexpression of miR-142-5p markedly enhanced the viability of 6-OHDA-treated SH-SY5Y cells (Figure 2), illustrating miR-142-5p functions as a neuroprotective player in PD. Furthermore, interaction between miR-142-5p and its target gene BECN1 was evaluated by bioinformatic prediction, luciferase reporter assay, and Western blot. The results show that miR-142-5p may be a promising therapeutic candidate of PD by targeting BECN1.

BECN1 is an essential component of the class III phosphatidylinositol 3-kinase complexes and can serve as a key regulator in cellular homeostasis pathway, like autophagy, through interacting with multiple different protein partners.[33] It has been reported that the deficiency and malfunction of BECN1 protein could induce neurodegenerative disorders, such as Huntington, AD, and Lewy body disease, illuminating that BECN1 acts as an essential gene in pathogenesis of diseases.[34,35] Additionally, the overexpression of BECN1 has been determined to activate autophagy and reduce the accumulation of SNCA.[36] Therefore, BECN1 can be considered as a potential therapeutic target of neurodegenerative disorders. The downregulation of BECN1 in AD has been demonstrated. In present work, we demonstrated that BECN1 was a direct target for miR-142-5p using dual-luciferase reporter assay (Figure 4). The overexpression of miR-142-5p significantly abrogated protein levels of BECN1 by qRT-PCR and Western blot assays (Figure 3). Meanwhile, the autophagy marker, LC3-II/I ratio, was decreased, and conversely p62 protein was enhanced, which indicated that the autophagy in 6-OHDA-treated SH-SY5Y cell model of PD was inhibited. Furthermore, the knockdown and overexpression of BECN1 in miR-142-5p-transfected cells showed that the overexpression of BECN1 suppressed the viability of 6-OHDA-induced SH-SY5Y cell model of PD and thus abated the neuroprotective effect of miR-142-5p overexpression (Figure 5). Therefore, BECN1 can function as an autophagy regulator to mediate the neurons viabilities in PD.

In summary, our studies revealed that the potential protective effect of miR-142-5p on 6-OHDA-treated SH-SY5Y cell injury as an in vitro model of PD. The results show that the overexpression of miR-142-5p inhibits the expression levels of BECN1 and thus enhances the viability of 6-OHDA-induced SH-SY5Y cell model of PD. Therefore, it can be inferred that miR-142-5p may be a potential candidate for PD diagnosis and treatment by targeting BECN1.

Conclusion

The results of our data demonstrate that upregulated level of miR-142-5p alleviates 6-OHDA-induced SH-SY5Y cell injury by abrogating cell viability, which is modulated through targeting BECN1. Hence, our findings provide insight into the understanding of miR-142-5p/BECN1 as a significant regulator in the pathophysiology of PD through 6-OHDA-induced cell injury in vitro.