MicroRNA miR-103a-3p targets NPAS3 to regulate progression of Alzheimer’s disease

Purpose: This study aimed at investigating miR-103a-3p expression, functional roles and underlying mechanism in regulating Alzheimer’s progression. Methods: RT-qPCR was used to assessed miR-103a-3p and NPAS3 expression in human neuroblastoma cells. Cell transfection of overexpressed or knocked down genes and CCK-8 assay measured cell viability while RT-qPCR was used to detect proliferation and apoptosis in biomarkers, Ki87 and PCNA, caspase-8 and caspase-3, respectively. Furthermore, luciferase assay was used to evaluate the luciferase activity while western blotting analysis was applied to determine protein biomarkers regarding proliferation and apoptosis. Results: Expression of miR-103a-3p decreased but NPAS3 increased in AD cell lines. Overexpressed miR-103a-3p attenuated cell viability and NPAS3 bound miR-103a-3p to regulate AD progression. The inhibitory effect of miRNA on cell viability in AD was reversed by NPAS3. Conclusion: miR-103a-3p/NPAS3 might help to enrich knowledge on treatment of AD.


INTRODUCTION
Alzheimer's disease (AD) is an irremediable brain ailment that mostly affects 60-year-old or older people. Aberrant plasma amyloid beta peptides 1-42 (Aβ42) have been reported as AD biomarkers which induce vascular dysfunction, impaired synaptic transmission and plasticity [1][2][3][4]. The dysregulation of microRNAs (miRNAs) presents an opportunity to explore therapeutic miRNAs in AD [5][6][7]. Studies have focused on seeking potential AD therapy from a biomedicine perspective to identify molecular therapeutic targets in AD [8].
The microRNA, MiR-103a-3p, acts as a tumor suppressor or an oncogene in cancer contexts [9]. As such, the roles of miR-103a-3p have been investigated in several disorder including cancers and other diseases such as glioma stem cells [10], bladder carcinoma [11], and glioma angiogenesis [12]. However, the role of miR-103a-3p in AD are yet to unveiled. The gene, Neuronal PAS Domain Protein 3 (NPAS3) is related to multiple human psychiatric and neurodevelopmental disorders [13]. It is known to regulate neural cell viability, affecting proliferation of neural cells through VGF [14] and was recognized in major mental issues including AD [15]. However, its molecular mechanism in AD is unknown.
In this study, it was hypothesized that miR-103a-3p could regulate NPAS3 expression in AD which might provide a basis for AD therapy. Although recent reports have highlighted the vital role of miR-103a-3p in development and advancement of a variety of cancers, diseases and even disorders [10][11][12]17,18], miR-103a-3p functions have never been addressed before in AD pathogenesis and progression. Therefore, this study is aimed at investigating functional roles of miR-103a-3p in AD in vitro and the interactions and co-effects of miR-103a-3p and NPAS3 in regulating AD progression.

EXPERIMENTAL Cell culture and transfection
The human neuroblastoma cell line, SH-SY5Y (ATCC, Beijing, China) was maintained in RPMI-1640 with 10 % fetal bovine serum (FBS) (Gibco, Life Technologies, China) and 100 µg/mL penicillin streptomycin with density of 2 × 105 cells per mL. After 48hrs, the substrate was superseded with Dulbecco's Modified Eagle's Medium (DMEM) with 10 % FBS coupled with 5 µM rat astrocytes (RA) for neuronal segregation. The cells were then maintained in a humidified atmosphere at 37˚C with 5 % CO2 in an incubator. Cell transfection of SH-SY5Y cells was done using Lipofectamine 2000 (Beyotime, Shanghai, China) to transfect miR-103a-3p mimics or negative control mimics (Guangzhou Fulengen Co. Ltd., China) following the manufacturer's guidelines. Then, the cells were treated with 10 µM amyloid beta Aβ42 oligomer at different time periods (0h-72h) and prepared as previously described [2].

RT-qPCR
Total RNA was isolated from cell free fractions of cerebrospinal fluid and plasma samples using Beyozol mixture (#R0011, Beyotime, Shanghai, China). Then reverse transcription of 1 μg RNA was done for each specimen to cDNA using BeyoRT™ cDNA First Chain Synthesis Kit (#D7166, Beyotime, Shanghai, China) following guidelines provided by the manufacturer. Beyofast™ SYBR Green QPCR Mix and associated mRNA qRT-PCR detection kit (Beyotime, China) were used to measure miR-103a-3p and NPAS3 expression, respectively using an Applied Biosystems Vii7 RT-qPCR instrument (ABI, Vernon, CA, USA) with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control with 2 -ΔΔCT method. The primers synthesized by Genelily BioTech Co., Ltd (Shanghai, China) used in this study are summarized in Table 1. All experiments were conducted in triplicates. Sequences

CCK-8 assay
The transfected cells were planted into 96-well multiplying plates and cultured between 0 to 48 hours. Cell viability was assessed by Cell Counting Kit (#C0037, Beyotime, Shanghai, China), following standard protocol specified by manufacturer. Absorbance was measured using the microplate reader (Molecular Devices, CA, USA) at 450 nm.

Western blotting assay
Western blot was to detect the protein expression of cell cycle and apoptosis-related biomarkers, including Ki67, PCNA, caspase-3 and caspase-8. Human neuroblastoma cell line, SH-SY5Y, was exposed to Aβ-42 IL-1β at different times and then the proteins were extracted and washed twice with cold PBS and lysed in sample loading buffer with mix of 1.5% sodium dodecylsulfate (SDS), 10% glycerol, 5 mM β-mercaptoethanol, bromophenol blue and 75 mM Tris (pH 7.0). Cell lysates were split by SDS-PAGE using 12% gel and the proteins were moved onto a polyvinylidene fluoride membrane. Additionally, the membranes were nurtured and probed with the following antibodies: Ki67, PCNA, caspase-3 and caspase-8 enlisted in the Table 2 under the temperature of 4°C overnight. The immunoblots were established and seen by ECL Western blot medium (Thermo Fisher Scientific, Shanghai, China) using U6 and GAPDH as internal control. The analysis of each group was repeated three times and the image J detection system was employed to determine the concentration of the bands.

Luciferase assay
The target sequence NPAS3 with the wild type (WT) or mutant type (MT) miR-103a-3p binding locations were synthesized and cloned into a pGL3Vector (Promega, USA), to construct WT and MT NPAS3 plasmids. These WT or MT NPAS3 plasmids were transfected into SH-SY5Y along with NC mimics or miR-103a-3p mimic (Sigma-Aldrich) using Lipofectamine 8000. After 48h, luciferase assay was conducted with Dual-Luciferase Reporter kit (Promega) following protocol by the manufacturer.

Statistical analysis
The experiments were conducted separately three times and the data presented as mean and standard error (SE). Student's t-test, and ANOVA analyses were conducted as appropriate. At 95% confidence interval, p<0.05 was considered to be significant.

MicroRNA, miR-103a-3p, was poorly expressed but promoted cell viability in ADmimic cells
The results of gene expression determined in SH-SY5Y treated with Aβ42 at different time periods (0h-72h) using RT-qPCR. indicated downregulated miR-103a-3p in the human neuroblastoma cell line treated with Aβ42 compared with the control group (Figure1A, p<0.05). However, the decrease, improved with time and the lower expression was observed after 72 hours. Furthermore, following CCK-8 assay, miR-103a-3p in AD-like cells was significantly increased with time compared with the untreated cells ( Figure 1B, p<0.05). However, the highest level of expression was observed after 72 hours. To confirm proliferation, RT-qPCR was to assess proliferation of the biomarkers (Ki67 and PCNA) which showed an increase with time (Figure1C, P< 0.05). However, the decrease of caspase-3 and caspas-8 was observed in Aβ42 treated SH-SY5Y cells for AD over time (0h-72h) compared the untreated SH-SY5Y cells ( Figure 1D, p< 0.05). The Aβ42 treated SH-SY5Y cell line was adopted for further experiments. However, no significant difference was observed for 48h and 72h treated cells hence further experiments were conducted for treated cells from 0h-48h.

MicroRNA, miR-103a-3p, overexpression suppresses cell viability of AD cells
When AD cells were transfected with either control mimics or mimics of miR-103a-3p at varying times (12h-48) to explore changes in cell viability, RT-qPCR confirmed the transfection efficacy and results showed significant increased trend for mimics group at different times (12h-48h) (Figure 2A, p<0.05). Using CCK-8 analysis to evaluate the influence of miR-103a-3p on cellular proliferation in AD, the results showed decreased cell viability for miR-103a-3p mimics transfected cells compared to mimics-NC group after 12h (Figure2B, P<0.05). After 24h, results demonstrated a significantly lower cell viability when miRNA was enhanced in AD cells ( Figure  2C, p<0.05). Lastly, after 48h the cell viability was significantly the lowest for miR-103a-3p mimics transfected cells compared to mimics-NC group ( Figure 2D, p<0.05). These data implied that overexpressed miRNA inhibited cell viability of AD cells.

MicroRNA, miR-103a-3p, directly targeted NPAS3 to regulate AD progression
When TargetScan (http://www.targetscan.org/ vert_72/) was referred to in search for the putative target gene of miR-103a-3p as well as predicted binding, the predicted binding positions is shown in Figure 3A. Thereafter, luciferase reporter vector having Wild Type or Mutant Type miR-103a-3p bonded sites in NPAS3 were established in order to confirm the connection. Subsequently, control mimics or miR-103a-3p mimic were transfected to Wild Type or Mutant Type in order to confirm the luciferase activity which indicated remarkable reduced luciferase activity for WT-NPAS3 in miR-103a-3p mimic transfected Aβ42 treated SH-SY5Y cells compared with control mimics (Figure 3B, P<0.05). However, no influence was noticed in rest groups (Figure3B, P<0.05).). As determined by Real Time-qPCR, the expression level of NPAS3 also dramatically increased with time (0h-48h) in the AD-like cells ( Figure 3C, P<0.05).

DISCUSSION
The RT-qPCR assay in this study has shown that downregulated expression of miR-103a-3p in the human neuroblastoma cell line treated with Aβ42 significantly increased with increasing time compared with the untreated SH-SY5Y cells for AD. It was further demonstrated that this decrease promoted cell viability increased with time after performing CCK-8 assays and validating with proliferation biomarkers and apoptosis biomarkers. As such downregulated miR-103a-3p expression in AD-mimic cells enhanced cell viability and a notable pathological apoptosis. However, miR-103a-3p overexpression suppressed cell viability of AD cells. This was confirmed when the CCK-8 assay was performed after upregulating miR-103a-3p with observed significant reduced cell viability at varying time increments. The proliferation biomarkers confirmed the reduced cellular viability by both Ki67 and PCNA. Apoptosis was increased when miR-103a-3p was upregulated in both caspase-8 and caspase-3. These results implied that miR-103a-3p was down regulated and its overexpression restrained cell viability of AD cells.
It is widely believed that miRNAs can exert their functions by regulating the expression of target genes [19]. Targescan predicted putative binding positions between miR-103a-3p and NPAS3. It was confirmed that miR-103a-3p directly targeted the 3'-UTR of NPAS3 to regulate NPAS3 expression and AD progression. Additionally, the expression level of NPAS3 was found to increase with time in AD cell lines suggesting that miR-103a-3p/NPAS3 interplay could be a potential novel treatment target for AD. Furthermore, the restoration experiment demonstrated the interplay between miR-103a-3p and NPAS3. Thus, when miR-103a-3p was overexpressed, it abolished the proliferation ability of NPAS3 which was also verified by CCK-8 assay, proliferation biomarkers, apoptosis biomarkers and western blotting.

CONCLUSION
The results of this study indicate that miR-103a-3p is crucial to the regulation of AD proliferation by moderating NPAS3 expression, which is responsible for the proliferation of neural cells and dementia and may potentially contribute to AD progression. These outcomes add to the knowledge related to the slow development of AD and opens the door to a new therapeutic approach for AD.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors.

Open Access
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