CircSNCA downregulation by pramipexole treatment mediates cell apoptosis and autophagy in Parkinson’s disease by targeting miR-7

SNCA is related to the mechanism of PPX treatment of PD

There are 30 genes concerned with PD and 581 with Alzheimer’s Disease (AD) [10], among which 16 genes are identical (Figure 1A-B). All of these genes are listed in Table 1. STITCH network analysis [11] was conducted between these 16 genes and PPX (Figure 1C). According to the findings of Wang et.al [12] that pramipexole treatment ameliorated SNCA/α-synuclein accumulation, SNCA directly responded to PPX treatment in this study. Except for PPX, the PPI network revealed that SNCA was closely associated with apoptosis-related genes such as BCL2, CASP3, BAX, TP53 and PTEN (Figure 1D).

1-Methyl-4-phenylpyridinium (MPP+)-induced neurotoxicity in SH-SY5Y cells is widely applied as the cell model of PD [13]. After induction of SH-SY5Y cells with 2.5 mM of MPP+ for 12 h, different concentrations (0, 10, 50, or 100 μM) of PPX were added into the mixture for 12, 24 or 36 h. Cell viability was measured by MTT assay, and MPP+ decreased cell viability significantly; however, PPX rescued this situation (Figure 2A). The conditions of 100 μM PPX and 12 h incubation were continually applied in the subsequent experiments (Figure 2B). Predictive genes related to PD were detected by qRT-PCR. SNCA shared the same change tendency with CASP3, BAX, PTEN and P53 (pro-apoptotic genes) but displayed the opposite tendency with BCL2 (anti-apoptotic gene) (Figure 2B). When treated with MPP+, the SNCA mRNA relative expression level was increased sharply compared to the

NC group, while that of cells treated with both MPP+ and PPX was lower but still higher than NC (Figure 2B). The western blot results showed that MPP+ induced increases in SNCA, CASP3, BAX, PTEN and P53 levels and induced a reduction in anti-apoptosis protein BCL2 (Figure 2C). According to the results, PPX treatment could decrease SNCA expression in MPP+-induced PD together with pro-apoptotic genes and increase the expression of anti-apoptotic genes.

CircSNCA expression was inhibited by PPX

SNCA mRNA and circSNCA are homology-dependent genes. Hsa_circ_0070441 (143 bp) matures from the CDS region of SNCA mRNA, and hsa_circ_0127305 (114 bp) matures from the 3’UTR of SNCA (Figure 3A). According to qRT-PCR analysis, both had higher expression levels after being treated with MPP+, while the former responded more drastically. When being treated with both MPP+ and PPX, the hsa_circ_0127305 level decreased but was still higher than that of NC. However, there was no significant difference between the MPP+ and MPP+ & PPX groups for hsa_circ_0070441 (Figure 3B). Targeted miRNAs of hsa_circ_0127305, miR-580 and miR-7 were predicted using Circular RNA Interactome algorithm [14]. The relative expression of miR-580 was not detected, and miR-7 was significantly reduced in the MPP+ group compared with NC group. The level of miR-7 was the highest in the MPP+ & PPX group compared with the MPP+ group (Figure 3C). We also tested the circSNCA RNA level under different concentrations of PPX and treatment times (Figure 3D and 3E). The circSNCA level decreased with the increase in the PPX concentration (Figure 3D). For the treatment time, after the first 4 h after PPX treatment, no significant change was evident; however, 8 h after PPX treatment, the circSNCA level decreased, and 12 h after the treatment, the level of circSNCA decreased to the lowest level and remained stable thereafter (Figure 3E).

Endogenous competition mechanism exists in the circSNCA/miR-7/SNCA network

CircSNCA and SNCA 3’UTR had the same target sites of the miR-7 seed region (Figure 4A-a). Double luciferase reporter assays were performed to detect the relationship between circSNCA and miR-7 or miR-7 and SNCA mRNA (Figure 4A-b and c). The dual luciferase reporter gene assay results demonstrated that only when circSNCA-WT and SNCA 3’UTR–WT were co-transfected with miR-7 was there a sharp reduction in luciferase activity (Figure 4A-b and c). Specific probes for detecting circSNCA (green dots) and miR-7 (red-dots) were transfected into SH-SY5Y cells to evaluate the space sites of circSNCA and miR-7 (Figure 4B). Both green dots and red dots were located in the cytoplasm of SH-SY5Y, with strong space overlap. These two experiments implied that there was endogenous competition between circSNCA and SNCA mRNA for miR-7 binding (Figure 5A). To test this hypothesis, we either overexpressed or knocked down circSNCA to investigate how circSNCA regulated miR-7 expression (Figure 5B and 5C). With circSNCA overexpression, the miR-7 level decreased, while with circSNCA knockdown, the miR-7 level increased (Figure 5D and 5E). Compared to the siRNAs (Si-Circ-1, Si-Circ-2, Si-Circ-3) for circSNCA, Si-Circ-1 showed the best effects on circSNCA knockdown. Western blot showed that in the MPP+ group, SNCA expression was enhanced with circSNCA overexpression, declined with circSNCA knockdown, and slightly increased with both circSNCA overexpression and PPX treatment, compared to the NC group. Similar results were observed in terms of CASP3, BAX, PTEN and P53 expression, while opposite results were observed for BCL2 (Figure 5F). It could be speculated and concluded that PPX had a negative regulation effect on the expression of SNCA and pro-apoptotic proteins and a positive regulatory effect on anti-apoptotic proteins. CircSNCA could attenuate the therapeutic effects of PPX in an in vitro PD model.

Furthermore, autophagy-associated protein, LC3B, was also detected by western blot. The LC3B-I level showed no significant change to circSNCA overexpression/ knockdown or PPX treatment. However, the LC3B-II level was low with circSNCA overexpression, high with circSNCA knockdown and slightly low with both circSNCA overexpression and PPX treatment (Figure 6A). To conclude, MPP+ induced an increase in circSNCA in a PD cell model, while PPX reversed it (Figure 6B). The upregulation of circSNCA could sponge and degrade miR-7 through the target sequences and Ago2, which may lead to attenuated inhibition of miR-7 on SNCA mRNA and the increased expression of SNCA. CircSNCA upregulation also positively correlated with the increasing expression levels of pro-apoptotic proteins (CASP3, BAX, PTEN and P53) and the decreasing levels of anti-apoptotic protein BCL2 and autophagy-associated protein LC3B-II.

Bioinformatics retrieval

DiGSeE (http://210.107.182.61/geneSearch/) is a search platform for genetic bases of human diseases. “Parkinson” and “Alzheimer’s Disease” were used as keywords during co-existing gene selection. The interactions between these genes and PPX were determined and plotted via STITCH (http://stitch.embl.de/) and protein-protein interactions were analyzed on STRING (https://string-db.org/), with the calculation performed by Dijkstra algorithm.

Reagents and antibodies

PPX was purchased from Tocris Bioscience. MPP+ was purchased from Sigma (St. Louis, MO, USA). The primary antibodies for immunoblot analysis are listed as follows: SNCA (CST, Danvers, MA, USA, 2642), LC3BI/II (Abcam, ab51520), CASP3 (Abcam, ab2302), BAX (Abcam, ab32503), PTEN (Abcam, ab32199), P53 (Abcam, ab1431), BCL2 (Abcam, ab32124) and β-actin (Abcam, ab8227). SiRNAs for knockdown of circSNCA were synthesized by GenePharma company (Shanghai, China). pLCDH-ciR (GeneSeed, China) was used to overexpress circSNCA. FITC-labeled circSNCA probe and Cy3-labeled miR-7 probe were synthesized by Sangon Biotech. The transfection reagent utilized was Lipofectamine 2000 (Invitrogen, Shanghai, China).

Cell lines and cell culture

SH-SY5Y cells (BNCC338056, BeNa Culture Collection, Beijing, China) were grown in high-glucose Dulbecco’s-modified eagle medium (DMEM-H) with 10% fetal bovine serum (FBS) containing glutamine and sodium pyruvate, in a 5% CO₂ humidified incubator at 37°C.

MTT assay

After 12 h treatment of 2.5 mM MPP+ with 10, 50, or 100 μM PPX, the viability of SH-SY5Y cells was identified via MTT assay. 1 mg MTT was added to each milliliter of medium and incubated at 37°C for 4 h. After 4 h, the medium in the plate was discarded, and in each well, 200 μL dimethyl sulfoxide (DMSO) was added before 1-min shaking for dissolution in a microplate reader (Bio-Rad Model 680; Bio-Rad, Hercules, CA, USA). The absorbance of cells in each well was measured at 570 nm, and the cell growth curve was drawn based on an average of five wells. The experiment was repeated in triplicate.

Western blot

SH-SY5Y cells were lysed in radio-immunoprecipitation assay (RIPA) buffer (BioVision, Milpitas, CA, USA). Total protein in supernatants was quantified using the BCA-200 protein assay kit (Pierce Biotechnology, Rockford, IL, USA). The protein was separated with 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (500 mA). The membrane was sealed in Tris Buffered Saline Tween (TBST) with 5% skim milk at room temperature for 1 h, and subsequently incubated with primary antibodies at 4°C overnight. After TBST-washing three times, the membrane was incubated for 1 h at room temperature with secondary antibody. Protein bands were visualized using enhanced chemiluminescence (Santa Cruz Biotechnology; Santa Cruz, CA, USA). Quantity One software (Bio-Rad) was used for image analysis. The results were analyzed by Image-Pro Plus 5.0 (Media, Cybernetics, USA). β-actin was included as the internal control.

Reverse transcription and quantitative PCR (qRT-PCR)

The total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA). The obtained RNA was reverse transcribed to cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo, Shanghai, China), and Power SYBR Green PCR Master Mix (Thermo, Shanghai, China) was used for determination. PCR program: predenaturation at 95^oC for 10 min, followed by 40 cycles of denaturation at 95^oC for 15 s, annealing at 60^oC for 30 s, and extension at 72^oC for 30 s. Subsequently, the dissolution curve of PCR products was generated. With GAPDH expression as the standard for mRNA and U6 expression as the standard for miRNA, relative mRNA and miRNA expression was calculated by the 2-^ΔΔCt method. The primer sequences are supplied in Table 2.

Dual luciferase reporter gene assay

Luciferase reporter gene recombinant plasmids were inserted with the sequences of wild-type (WT) and mutated type (MUT) circSCNA, WT and MUTSCNA 3’-untranslated region (3’-UTR). MiR-7 mimics or control were co-transfected with WT and MUT circSCNA into the 293 cell line (BeNa Culture Collection, Beijing, China) using Lipofectamine 2000 (Invitrogen). Luciferase Dual Assay Kit (Thermo Fisher Scientific) was used for dual-luciferase reporter gene assay 48 h after cells were transfected.

Immunofluorescent localization

3×10⁴ SH-SY5Y cells were plated onto slides for 24 h of growth in advance of the probe transfection experiments. 20 nM probes of FITC-labeled circSNCA or Cy3-labeled miR-7 (Sangon Biotech) were co-transfected into SH-SY5Y cells for 36 h. After culture incubation, the cells were digested by trypsin and fixed onto slides. The nuclei were stained with DAPI, and the images were collected using fluorescence microscopy (Carl Zeiss, Jena, Germany).