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Ropinirole alters gene expression profiles in SH-SY5Y cells: a whole genome microarray study.

Introduction

Parkinson's disease (PD) is a progressive neurological disorder with primary symptoms of bradykinesia, tremor, and rigidity, and patients with advanced disease also show postural instability. It is the second most common neurodegenerative disorder after Alzheimer's disease, and is responsible for significant morbidity as well as shortened life expectancy. It also places a substantial economic burden on the patient, their family, and the society (1). Therefore, any therapy proven to modify the course of PD would be extremely valuable.

To date, treatments for PD have been confined to symptomatic therapies, which have focused on motor deficits and the loss of dopaminergic neurons in the substantia nigra. However, the past few years have seen important advances in the development of new drugs for PD, and importantly how existing drugs are used as part of a long-term strategy for disease management (2).

Ropinirole (ROP) is a novel dopamine receptor agonist with a high affinity for all dopamine D2 subfamily receptors, but the highest affinity for the D3 receptor subtype (3). It has been demonstrated to have neuroprotective effects and has been used for clinical PD therapy (4). Researchers have made many attempts to clarify the potential mechanism of ROP action over recent years. For instance, it has been thought to increase the concentration of glutathione, catalase, and superoxide dismutase (5). Moreover, both bromocriptine and ROP were shown to reduce hydroxyl radical generation in the rodent striatum after infusion of the neurotoxin 1-methyl-4-phenylpyridinium (6). However, the exact mechanisms appear complicated and controversial and require further investigation. The advent of microarray chips provides an entirely new approach to the molecular characterization of neurodegenerative diseases and their models (7).

In the present study, therefore, we used genome-wide Affymetrix microarrays to identify genes that were regulated following ROP treatment of SH-SY5Y cells to illustrate the potential mechanisms of its effects.

Material and Methods

Cell culture

The human neuroblastoma cell line SH-SY5Y, sub-cloned from the SK-N-SH cell line, is often used as a model of human dopaminergic neurons, and thus was used in the current study. Cells were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, USA) and maintained at 37[degrees]C under a humidified 5% C[O.sub.2] atmosphere. ROP stock was freshly made in water prior to each experiment, and cells were divided into two groups (control and treated groups). Cells of treated groups were exposed for 2 h to 10 [micro]M ROP, which is thought to be a clinically relevant dose (8). Control cells received no ROP treatment.

Total RNA extraction and microarray experiments

Total RNA fractions were isolated from cultured cells after specific treatment using the SV total RNA isolation system (Promega, USA). Total RNA samples were spectrophotometrically scanned from 220 to 320 nm; A260/A280 was typically >1.9. Formaldehyde agarose gel electrophoresis was used as a quality control for total RNA. RNA samples were then used to generate biotinylated cRNA targets for the Affymetrix GeneChip Human genome U133 Set. Six microarray chips were prepared, including three biological replicates for control and treated groups. All experiments were performed according to manufacturer protocols (Affymetrix Inc., USA).

After hybridization, arrays were stained in the GeneChip Fluidics Station 450 and scanned on the Affymetrix Scanner 3000. Fluorescent signal intensities were analyzed using the Gene Chip Operating System (Affymetrix). Ratios comparing treated and control groups were calculated to represent fold-changes in gene expression. Regulated genes were shown to be consistent across all biological replicate sets. Annotation and categorization of the regulated genes were based on gene ontology performed by the Database for Annotation, Visualization and Integrated Discovery (DAVID; https://david.ncifcrf. gov/summary.jsp). KEGG and BIOCARTA functional pathways were evaluated for regulated genes, and DAVID 2007 was used to detect chromosomal loci containing ROP-modulated genes.

TaqMan[R] real-time PCR assay

Total RNA was isolated as described above and reverse transcription was performed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, USA). PCR amplification was performed using TaqMan Gene Expression Assays and the TaqMan Universal PCR Master Mix according to the manufacturer's instructions (Applied Biosystems). Assay IDs of genes CALM3, EPS15, RIPK5, and PIK3C2B were Hs00270914_m1, Hs00179978_m1, Hs00418647_m1, and Hs00153248_m1, respectively. Amplification was conducted on duplicate samples using the ABI 7900 Detection System according to the manufacturer's instructions. GAPDH was used as an endogenous control to normalize all assays. Relative quantification of gene expression levels was determined using the Comparative Ct method.

Western blot analysis

Proteins were extracted using RIPA Lysis Buffer (Beyotime Institute of Biotechnology, China) with Phosphatase Inhibitor Cocktail Tablets (Roche, Switzerland) according to the manufacturer's instructions. Briefly, 100 mg of protein was run on a 12% denaturing polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane. After incubation with an anti-PIK3C2B primary antibody (Abcam, USA), the membrane was washed and incubated with a corresponding horseradish peroxidase-labeled secondary antibody (BD Pharmingen, USA). Detection was performed using an ECL kit (Amersham Pharmacia Biotech, Japan) according to the manufacturer's instructions. Absorbance was analyzed using Image-Pro Plus software (Media Cybernetics, USA). PIK3C2B protein levels were was normalized to those of [beta]-actin and compared among groups.

Statistical analysis

Data are reported as means [+ or -] SE. Statistical analysis was performed using analysis of variance and the Student's t-test. Genes were deemed significantly different between groups if P<0.05, and if fold-changes were greater than 1.5 or less than 0.67. P values were corrected by the Benjamini-Hochberg false discovery rate method using R software.

Results

ROP modulated gene expression in SH-SY5Y cells

We identified a total of 113 genes as differentially regulated by ROP treatment, of which 48 were upregulated and 65 were downregulated. Among the 113 genes, 101 were known genes and 12 were expressed sequence tags. As shown in Figure 1, GOTERM_Molecular Function_ALL revealed that most of these genes had functions in protein and RNA binding, and enzyme inhibitor activity. Table 1 lists 20 genes representative of the complete list. Further pathway analysis (KEGG and BIOCARTA functional pathways) revealed that only the phosphatidylinositol 3-kinase (PI3K) signaling pathway was over-represented, including genes CALM3, INPP4A, and PIK3C2B. Notably, PIK3C2B expression was strongly promoted by ROP treatment in this pathway. We also identified a number of modulated genes that are located near PIK3C2B on chromosome 1, including KLHL17, USP24, C1ORF149, ID3, MTHFR, KIAA0090, ADAMTSL4, SERBP1, RIPK5, EPS15, PIGR, ZFYVE9, and ZMYM6. Of these, EPS15 expression was clearly induced by ROP (Table 2).

TaqMan real-time PCR and Western blot

To confirm this observed regulation of gene expression by ROP treatment, we performed TaqMan real-time PCR of a number of selected genes. CALM3 was chosen because it is involved in the PI3K signaling pathway; PIK3C2B was selected for its prominent elevation and potential role in PD; and EPS15 and RIPK5 were selected as representative loci on chromosome 1 and their putative relationship with PD. As shown in Figure 2, a strong increase in the mRNA levels of PIK3C2B and EPS15 was detected following ROP treatment, which supported microarray data. A change in expression of CALM3 and RIPK5 was also confirmed. Western blotting showed that PIK 3C2B protein levels were 2.5 times higher in the ROP-treated group than the control group (Figure 3).

We validated our microarray data in HeLa cells by performing cell culture, TaqMan real-time PCR, and Western blotting as described previously. As shown in Figure 4, similar expression patterns were observed in HeLa cells to those seen in SH-SY5Y cells.

Discussion

PD is a neuropathological disorder involving the degeneration of dopaminergic neurons in the substantia nigra, and subsequent loss of their terminals in the striatum. The ensuing loss of dopamine causes most of the debilitating motor disturbances associated with PD. Current PD medications treat the symptoms of the disease, focusing on halting or retarding the degeneration of dopaminergic neurons. Recently, there has been considerable interest in neuroprotection as a therapeutic strategy for PD, and several drugs such as ROP have been proposed as candidate agents (9). However, the molecular mechanism of neuroprotection is elusive.

In the present study, we treated SH-SY5Y cells with ROP and applied whole-genome microarray to screen changes in gene expression with the aim of uncovering the underlying molecular mechanism. Using bioinformatics, we identified genes that were differentially regulated after ROP treatment, which are known to function in protein and RNA binding, and enzyme inhibitor activity. We also observed that the PI3K signaling pathway was overrepresented and that PIK3C2B expression was distinctly increased in this pathway.

The PI3K family is evolutionarily conserved and is implicated in many biological processes including cell survival, proliferation, inflammation, adhesion, glucose metabolism, chemotaxis, and cancer. It can be classified into three distinct sub-groups (I, II, and III) based on substrate specificity and sequence homology. PIK3C2B is a family member of class II proteins, which contain a C2 domain and PX domain (10,11). Although diverse biological roles have been assigned to class I and class III PI3Ks, the functions of class II PI3Ks are still unknown. However, PIK3C2B has recently been implicated in cell growth, cell migration, and differentiation (12-14). Moreover, activation of a major neuroprotective signaling pathway, the PI3K/Akt pathway, can prevent cell death in a PD model of SH-SY5Y cells (9).

Intriguingly, we observed the distinct promotion of PIK3C2B transcript and protein expression levels in SH-SY5Y cells following ROP treatment. This indicated that ROP might exert neuroprotective effects through the PI3K pathway, and that PIK3C2B might play a role in this process. Additionally, we previously observed that the PI3K/Akt pathway modulates the expression of Nurr1, which is a transcription factor essential for the differentiation and maturation of central dopaminergic cells (15). This suggested that ROP might induce PIK3C2B and modulate Nurr1 to exert neuroprotection. However, the present study found no direct evidence of Nurr1 modulation by either ROP or PIK3C2B. Further investigations may shed new light on the mechanism of ROP neuroprotection and the role of PIK3C2B in PD.

Nine loci in the human genome have previously been linked to PD. Mutations in alfa-synuclein, parkin, DJ-1, and, arguably UCH-L1 genes have been associated with familial PD (16). Recently a locus on chromosome 1 was linked to common late-onset PD in the Icelandic population (16). Meanwhile, linkage studies have also defined susceptibility regions for late-onset PD on chromosomes 1 and 2 (17). We observed that ROP regulated several genes located on chromosome 1, suggesting that this might be its main way of exerting neuroprotective effects. Only three of these genes, USP24, MTHFR, and EPS15, have been associated with PD in earlier studies (17-19). For instance, in vitro experiments showed that EPS15 enhanced the ubiquitin ligase activity of PARKIN, and PARKIN-mediated EPS15 ubiquitination is crucial in promoting the PI3K/Akt signaling pathway (19,20).

In the present study, we observed that ROP distinctly increased the expression of EPS15. Considering the role of EPS15 and PI3K/Akt in neuronal survival, our observation is likely to further our understanding of the role of ROP in PD therapy. Despite other chromosomal 1 genes having no known link with PD, their underlying biological functions may nevertheless provide new implications for disease. RIPK5, a member of the RIP serine/threonine kinase family, was previously reported to induce both caspase-dependent apoptosis and caspase-independent cell death (21). Considering the important role of cell death pathways in PD (22), future work may identify a novel role for RIPK5 in the pathogenesis of PD.

In conclusion, we used genome-wide microarray analysis to identify genes that were regulated after ROP treatment. Pathway analysis suggested that ROP mainly modulated the PI3K signaling pathway in SH-SY5Y cells. Further extensive investigation of PIK3C2B and other loci on chromosome 1 may open up a new avenue to understand the pathology of PD and provide novel pharmaceutical targets to improve patient care.

http://dx.doi.org/ 10.1590/1414-431X20154857

References

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M.Z. Zhu [1], W.D. Le [2,3] and G. Jin [2,4,5]

[1] School of Public Health, Shanghai University of Traditional Chinese Medicine, Shanghai, China institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Science/Shanghai Jiao Tong

[2] University School of Medicine, Shanghai, China

[3] Department of Neurology, Baylor College of Medicine, Houston, TX., USA

[4] ShanghaiBio Corporation, North Brunswick, NJ, USA

[5] Shanghai Biochip Co., Ltd and National Engineering Center for Biochip at Shanghai, Shanghai, China

Correspondence: G. Jin: <jasonggjin@gmail.com> | W. Le: <weidongl@sibs.ac.cn>

Received March 27, 2015 | Accepted September 1, 2015

Caption: Figure 2. TaqMan[R] real-time PCR confirmation of microarray data in SH-SY5Y cells. CALM3, EPS15, RIPK5, and PIK3C2B underwent TaqMan real-time PCR in SH-SY5Y cells. The y-axis represents the fold-change in expression after ROP treatment, and microarray and TaqMan data are plotted on the x-axis. There were no significant differences between the microarray and TaqMan data (P>0.05; Student's t-test).

Caption: Figure 3. Elevation of PIK3C2B protein expression by ropinirole (ROP) treatment in SH-SY5Y cells. [beta]-actin was used as the loading control. PIC3C2B protein levels were significantly increased by treatment with 10 [micro]M ROP. * P<0.05, 10 [micro]M ROP compared to the control group (Student's t-test).

Caption: Figure 4. Validation of microarray data in HeLa cells. A, CALM3, EPS15, RIPK5, and PIK3C2B underwent TaqMan[R] real-time PCR in HeLa cells. The y-axis represents the fold-change in expression after ROP treatment, and microarray and TaqMan data are plotted on the x-axis. * P<0.05, TaqMan data compared to microarray data (Student's t-test). B, Western blot analysis in HeLa cells. [beta]-actin was used as the loading control. PIC3C2B protein levels were elevated by treatment with 10 [micro]M. * P<0.05, 10 [micro]M ROP compared to control (Student's t-test).
Table 1. Representative 20 genes of all 113 regulated by ropinirole.

Probeset ID     Gene name              Gene symbol

234278_at       Epidermal growth       EPS15
                factor receptor
                pathway substrate 15

242560_at       Fanconi anemia,        FANCD2
                complementation
                group D2

204484_at       Phosphoinositide-3-    PIK3C2B
                kinase, class 2,
                beta polypeptide

231830_x_at     RAB11 family           RAB11FIP1
                Interacting protein
                1 (class I)

235395_at       SEC63-like (S.         SEC63
                cerevisiae)

222297_x_at     Ribosomal protein      RPL18
                L18

234082_at       Chromosome 21 open     C21ORF116
                reading frame 116

229659_s_at     Polymeric              PIGR
                immunoglobulin
                receptor

232808_at       Hypothetical protein   ANTXR1
                FLJ10601

233059_at       Potassium              KCNJ3
                inwardly-rectifying
                channel, subfamily
                j, member 3

210076_x_at     Serpine1 MRNA          SERBP1
                binding protein 1

227721_at       C3 and PZP-like,       CPAMD8
                alpha-2-
                macroglobulin domain
                containing 8

227404_s_at     Early growth           EGR1
                response 1

239289_x_at     KIAA1018               MTMR15

244360_at       F-box and leucine-     FBXL17
                rich repeat protein
                17

239419_at       Protein tyrosine       PTPRA
                phosphatase,
                receptor type A

229792_at       Kelch-Like 17          KLHL17
                (Drosolphila)

219480_at       Snail homolog 1        SNAI1
                (Drosophila)

226864_at       Protein kinase         PKIA
                (camp-dependent,
                catalytic) inhibitor
                alpha

201694_s_at     Early growth           EGR1
                response 1

Probeset ID     Molecular            Fold-      P       FDR
                function             change

234278_at       protein binding       2.59    0.0177   0.0431

242560_at       protein binding       2.57    0.0191   0.0431

204484_at       protein binding       2.01    0.0452   0.0475

231830_x_at     protein binding       1.94    0.0229   0.0431

235395_at       heat shock            1.94    0.0471   0.0431
                protein binding

222297_x_at     RNA binding           1.91    0.0118   0.0431

234082_at       unclassified          1.91    0.0074   0.0431

229659_s_at     protein binding       1.89    0.0216   0.0431

232808_at       protein binding       1.78    0.0029   0.0431

233059_at       ion channel           1.77    0.0187   0.0431
                activity

210076_x_at     RNA binding           0.51    0.0047   0.0431

227721_at       enzyme inhibitor      0.54    0.0151   0.0431
                activity

227404_s_at     transcription         0.57    0.0269   0.0431
                activator activity

239289_x_at     unclassified          0.58    0.0299   0.0471

244360_at       unclassified          0.58    0.0437   0.0435

239419_at       catalytic activity    0.58    0.0039   0.0431

229792_at       protein binding       0.58    0.0103   0.0443

219480_at       protein binding       0.58    0.0080   0.0431

226864_at       enzyme inhibitor      0.58    0.0004   0.0431
                activity

201694_s_at     transcription         0.59    0.0285   0.0150
                regulator activity

FDR: False discovery rate. The Student's t-test was used for
statistical analysis.

Table 2. Ropinirole-regulated genes located on chromosome 1.

Probeset ID     Gene name                   Gene symbol

210076_x_at     Serpine1 MRNA               SERBP1
                binding protein 1

229792_at       Kelch-like 17               KLHL17
                (Drosophila)

226071_at       ADAMTS-like 4               ADAMTSL4

228517_at       Chromosome 1 open           C1ORF149
                reading frame 149

212395_s_at     KIAA0090                    KIAA0090

207826_s_at     Inhibitor of DNA            ID3
                binding 3, dominant
                negative
                helix-loop-helix
                protein

213698_at       Zinc finger,                ZMYM6
                MYM-type 6

214663_at       Receptor interacting        RIPK5
                protein kinase 5

208446_s_at     Zinc finger, fyve           ZFYVE9
                domain containing 9

239035_at       5,10-                       MTHFR
                methylenetetrahydrofolate
                reductase (NADPH)

212381_at       Ubiquitin specific          USP24
                peptidase 24

229659_s_at     polymeric                   PIGR
                immunoglobulin
                receptor

204484_at       Phosphoinositide-3-         PIK3C2B
                kinase, class 2,
                beta polypeptide

234278_at       Epidermal growth            EPS15
                factor receptor
                pathway substrate 15

Probeset ID     Molecular function        Fold-      P       FDR
                                          change

210076_x_at     RNA binding                0.51    0.0047   0.0431

229792_at       protein binding            0.58    0.0103   0.0443

226071_at       protease binding           0.61    0.0495   0.0498

228517_at       unclassified               0.61    0.0440   0.0443

212395_s_at                                0.64    0.0414   0.0471

207826_s_at     transcription cofactor     0.67    0.0381   0.0459
                activity

213698_at       nucleic acid binding       0.67    0.0091   0.0431

214663_at       protein binding            0.67    0.0137   0.0443

208446_s_at     signal transducer          1.54    0.0276   0.0431
                activity

239035_at       oxidoreductase activity    1.55    0.0479   0.0492

212381_at       ubiquitin-specific         1.64    0.0086   0.0431
                protease activity

229659_s_at     protein binding            1.89    0.0216   0.0431

204484_at       protein binding            2.01    0.0452   0.0475

234278_at       protein binding            2.59    0.0177   0.0431

FDR: False discovery rate. The Student's t-test was used
for statistical analysis.

Figure 1. Molecular function categories of genes
regulated by ropinirole (ROP). GOTERM_Molecular
Function_ALL revealed that most of these genes
functioned in protein and RNA binding, and in
enzyme inhibitor activity.

  Category                    Term                 Genes      %

GOTERH_MF_ALL   protein binding                              50
GOTERH_MF_ALL   enzyme inhibitor activity                     6
GOTERH_MF_ALL   binding                                      71
GOTERH_MF_ALL   RNA binding                                   9
GOTERH_MF_ALL   protein domain specific binding               3

  Category         D      P-Value   Benjamini

GOTERH_MF_ALL    43.9     0.0003     0.2017
GOTERH_MF_ALL     5.3     0.0122     0.9924
GOTERH_MF_ALL    62.3     0.0243     0.9735
GOTERH_MF_ALL     7.9     0.0272     0.9326
GOTERH_MF_ALL     2.6     0.0971     0.9326
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Author:Zhu, M.Z.; Le, W.D.; Jin, G.
Publication:Brazilian Journal of Medical and Biological Research
Article Type:Report
Date:Feb 1, 2016
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