This article is mentioned in:

Introduction

Alzheimer's disease (AD) is a fatal incurable neurodegenerative disease that is prevalent worldwide (1). The progressive disease is characterized by the accumulation of plaques formed of short β-amyloid (Aβ) peptides, neurofibrillary tangles inside neurons, loss of neurons in the hippocampus and cerebral cortex, and widespread brain atrophy. AD is the most common form of dementia in the elderly, accounting for 50–70% of the late-onset dementia, with 17–20 million affected worldwide (2). Although several key proteins, such as amyloid precursor protein (APP), β-secretase 1, Tau and presenillin-1 (PSEN1), have been identified to be implicated in AD pathogenesis, the etiology and pathogenesis of AD are not well understood.

MicroRNAs are a group of small (~22 nucleotides) endogenous noncoding RNAs, that function as suppressors of gene expression. By binding to the 3′-UTR of their target genes, miRNAs can induce mRNA degradation or translational repression (3,4). miRNAs are abundant in the brain and crucially important in neurodevelopment and synaptic plasticity (5,6). Increasing evidence has revealed that alterations in miRNA expression may contribute to the initiation and progression of AD (7–11). However, the mechanism underlying the role of microRNAs in AD has not been fully elucidated.

p27Kip1 is encoded by cyclin-dependent kinase inhibitor 1B and functions to negatively control cell cycle progression (12,13). Expression of p27Kip1 can normally inhibit the phosphorylation of pRb and therefore arrest cell proliferation at G1. A number of studies have shown that disordered expression of cell-cycle markers, including p27Kip1, contributes to the pathogenesis of AD (14,15). However, p27Kip1 gene expression regulation is not completely understood. The present study hypothesized that the expression of p27Kip1 may be regulated by certain miRNAs, and aimed to elucidate the interactions between miR-222 and p27Kip1 in AD

Materials and methods

Animal model

The APPswe/PSΔE9 mice model was purchased from the Jackson Laboratory (Ben Harbor, ME, USA). Briefly, the transgenic mice were established by co-injection of the APPswe and PS1ΔE9 vectors (16). The expression of the transgenes is under the control of the mouse prion protein promoter, which can drive high protein expression in the neurons and astrocytes of the central nervous system (17,18). The APPswe transgene encodes a mouse-human hybrid transgene containing the mouse sequence in the extracellular and intracellular regions, and a human sequence within the Aβ domain with Swedish mutations K594N/M595L. The PS1ΔE9 transgene encodes the exon-9-deleted human PSEN1. Polymerase chain reaction (PCR) amplification of tail DNA was performed to genotype the mice for the presence of the transgene. The PCR reaction mixture of 20 µl contained 100 ng genomic DNA, 2 µl 10X buffer, 0.4 µl dNTPs (10 mM), 0.5 µl forward and reverse primers (20 µM), and 0.2 U Taq DNA polymerase (Sangon Biotech, Shanghai, China). The PCR was performed with pre-denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 54°C for 30 s (for APP) or 52°C for 40 s (for PS1), elongation at 72°C for 30 s (for APP) or 60 s (for PS1), and a final extension at 72°C for 10 min. Primer sequences were as follows: Forward: 5′-GACTGACCACTCGACCAGGTTCTG-3′ and reverse: 5′-CTTGTAAGTTGGATTCTCATATCCG-3′ for APP (product length, 344 bp); and forward: 5′-AATAGAGAACGG CAGGAGCA-3′ and reverse: 5′-GCCATGAGGGCACTAATCAT-3′ for presenilin 1 (product length, 608 bp). The transgenic mice and age-matched controls were housed under controlled illumination (12 h light/dark cycle) in a specific pathogen-free environment (room temperature, 20–22°C; humidity, 55–60%). Food and water were available ad libitum. Eight six month-old transgenic mice and eight age-matched controls were used in this study. The mice were sacrificed by inhalation of 5% isoflurane (RWD Life Science, Shenzhen, China) prior to cervical dislocation. Using small scissors, the head was severed and the skin separated in order to fracture the skull. Using small tweezers, small sections of skull were carefully removed in order to expose the brain. The brain tissue samples were immediately frozen in liquid nitrogen, and stored at −80°C until protein and RNA extraction was performed. All animal protocols were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (19). Formal approval of this study was obtained from the Committee on the Ethics of Animal Experiments at The Second Hospital of Shandong University (Jinan, China).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA from brains and cultured cells was isolated using the mirVanaTM miRNA Isolation kit (AM1560, Ambion, Austin, TX, USA) in accordance with the manufacturer's instructions. The RNA was treated with DNase I (AM1906, Ambion) to eliminate genomic DNA contamination. RNA (1 µg) was reverse transcribed into miRNA cDNA and total cDNA using the miScript Reverse Transcription kit (218061, Qiagen, Hilden, Germany), followed by qPCR on a Lightcycler (Roche Diagnostics, Mannheim, Germany) machine using the miScript SYBR Green PCR kit (218073, Qiagen), according to the manufacturer's instructions. Primers for mature miR-222 and U6snRNA were purchased from Qiagen (MS00007609 and MS00007497, Hilden, Germany). The primer sequence were as follows: Forward: 5′-CAGGTCTCCAAGACGACATAGA-3′ and reverse: 5′-CGCCTTTTCGATTCATGTACTGC-3′ for p27Kip1 (20); and forward: 5′-CATGGGTGTGAACCATGAGA-3′ and reverse: 5′-TGTGGTCATGAGTCCTTCCA-3′ for GAPDH. All reactions were run in triplicate. Relative expression levels for p27Kip1 mRNA and miR-222 were determined using the 2−ΔΔCq method.

Cell culture

The SH-SY5Y and HEK-293T cell lines were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and routinely cultured in Dulbecco's modified Eagle's medium (11965-092, Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen Life Technologies), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in a humidified air atmosphere of 5% CO2 at 37°C.

miR-222 overexpression in cultured cells

Overexpression of miR-222 was performed using an miR-222 expression vector, which was constructed using BLOCK-iTTM Pol II miR RNAi Expression Vector kit with EmGFP (K4936-00, Invitrogen Life Technologies) according to the manufacturer's instructions. The top oligo sequence for miR-222 is 5′-TGCTGAGCTACATCTGGCTACTGGGTGTTTTGGCCACTGACTGACACCCAGTACAGATGTAGCT-3′, and the bottom oligo sequence for miR-222 is 5′-CCTGAGCTACATCTGTACTGGGTGTCAGTCAGTGGCCAAAACACCCAGTAGCCAGATGTAGCTC-3′. The negative control vector was provided by Invitrogen Life Technologies, which contains an insert that can form a hairpin structure just as a regular pre-miRNA, but is predicted not to target any known vertebrate gene. The expression vector or control vector was transfected into the SH-SY5Y cell line with Lipofectamine 2000 reagent (11668-019, Invitrogen Life Technologies). To generate the stable cell line that can constitutively express miR-222, blasticidin (15205, Sigma-Aldrich) was added 24 h after transfection at concentration of 3 µg/ml for 10 days. Resistant cells were analyzed by fluorescent microscopy (TE2000-U; Nikon Corporation, Tokyo, Japan).

miR-222 knockdown in cultured cells

The inhibition of miR-222 in SH-SY5Y cells was produced with the locked nucleic acid (LNA) oligonucleotides for specific in vitro knockdown. The LNA oligonucleotides (exhibiting high affinity to complementary nucleic acids and able to efficiently silence their target miRNAs in vitro and in vivo) were purchased from Exiqon (Vedbaek, Denmark) and transfected with Lipofectamine 2000 reagent (11668-019, Invitrogen Life Technologies, Carlsbad, CA, USA) at a final concentration of 80 nM. The cells were collected 48 h after transfection, and the levels of miR-222 and p27Kip1 were analyzed.

Dual-luciferase reporter assay

The analysis by the miRNA target prediction algorithm TargetScan (http://www.targetscan.org/) revealed that p27Kip1 is a candidate target gene of miR-222. The 3′-UTR of human p27Kip1 was amplified by PCR using the following primers 5′-TAAGAATATGTTTCCTTGTTTATCAGAT-3′ and 5′-AATAGCTATGGAAGTTTTCTTTATTGAT-3′. The PCR products were directionally cloned downstream of the Renilla luciferase stop codon in the psiCHECKTM-2 vector (C-8021, Promega Corporation, Madison, WI, USA). The 3′-UTR of the mutant vector of p27Kip1 was also constructed by the overlap extension PCR method using the following primers: 5′-CTCTAAAAGCGTTGGAGCATTATGCAATTAGG-3′ and 5′-CCTAATTGCATAATGCTCCAACGCTTTTAGAG-3′. The wild-type or mutant p27Kip1 3′-UTR psiCHECK-2 plasmid was transiently co-transfected with miR-222 expression vector or negative control vector into HEK-293T cells. Cell lysates were harvested 48 h after transfection and then firefly and Renilla luciferase was measured with the Dual Luciferase Reporter Assay system (E1910, Promega Corporation) following the manufacturer's instructions. Renilla luciferase activities were normalized to firefly luciferase activities to also control transfection efficiency.

Western blot analysis

The cells and tissues were homogenized in chilled radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) and stood for 30 min on ice. After centrifugation at 17,530 × g for 30 min at 4°C, the supernatants were collected as protein samples. The protein concentration was detected by the BCATM Protein Assay kit (Pierce, Biotechnology Inc., Rockford, IL, USA). Proteins (40 µg) were separated on 12% SDS-PAGE gels (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 2 h, and incubated overnight at 4°C with mouse monoclonal anti-human/mouse primary antibody against p27Kip1 (1:250; cat. no. ab82264, Abcam, Cambridge, MA, USA) and rabbit polyclonal anti-human/mouse primary antibody against GAPDH (1:500; cat. no. D261392; Sangon Biotech). The membranes were then washed in Tris-buffered saline (TBS) for three times, and incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-mouse/rabbit secondary antibody (1:1,000; cat. no. A0216/A0208; Beyotime Institute of Biotechnology). Subsequent to another three washes in TBS, signals from the bound secondary antibody were generated using the enhanced chemiluminescence solution (Amersham, Piscataway, NJ, USA) and were detected by exposure of the membranes to X-ray films (Kodak, Rochester, NY, USA). The relative signal intensity was quantified by densitometry with UVIPhoto and UVISoft UVIBand Application V97.04 (Uvitech, Cambridge, UK).

Statistical analysis

Statistical analyses were performed with the statistical software package, SPSS, version 17.0 (SPSS, Inc., Chicago, IL, USA). The results are expressed as the mean ± standard deviation of three independent experiments. The differences between groups were analyzed by Student's t-test. P-values are two-sided, and P≤0.05 was considered to indicate a statistically significant difference.

Results

miR-222 is downregulated in APPswe/PSΔE9 mice versus control mice

Eight 6-month-old transgenic mice and eight age-matched controls were used in this study. RT-qPCR was performed to evaluate the miR-222 expression level in 8 APPswe/PSΔE9 mice and 8 controls. All reactions were run in triplicate. As shown in Fig. 1A, there was a significantly decreased level of miR-222 in the APPswe/PSΔE9 mice compared with the control group.

Figure 1 Expression of miR-222 and p27Kip1 in the cerebral cortex of APPswe/PSΔE9 mice and controls. (A) The level of miR-222 in the cerebral cortex of APPswe/PSΔE9 mice and controls was determined by RT-qPCR. Data were normalized based on the detection of U6snRNA in the various samples. Statistical analysis was performed using Student's t-test. *P<0.05. (B) GAPDH and p27Kip1 proteins were measured from cerebral cortex of APPswe/PSΔE9 (pap) mice and controls using western blot analysis. (C) Semi-quantitative analyses of western blots for p27Kip1 protein. The data are presented as the mean ± standard deviation. *P<0.05. (D) p27Kip1 mRNA in cerebral cortex of APPswe/PSΔE9 mice and controls was measured by RT-qPCR. The data were normalized based on the detection of GAPDH in the various samples. miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Reduced miR-222 is correlated with high level of p27Kip1 protein in APPswe/PSΔE9 mice and control mice

To investigate the potential interaction of miR-222 and p27Kip1 in vivo, western blot analysis was performed on the total protein extracts from the cerebral cortex of 6-month-old APPswe/PSΔE9 mice and controls. It was demonstrated that the level of p27Kip1 protein was increased in the APPswe/PSΔE9 mice compared with age-matched controls (Fig. 1B and C), which is inversely associated with the expression of miR-222. In addition, the mRNA level of p27Kip1 was also detected by RT-qPCR in the same mice and no significant difference was observed (Fig. 1D). It is well-known that miRNAs execute post-transcriptional regulation by inducing mRNA degradation or translational repression. The present data indicated that low miR-222 expression may positively regulate p27Kip1 expression by promoting the translation of p27Kip1 mRNA.

3′UTR of p27Kip1 mRNA is targeted by miR-222 in HEK-293T cells

To investigate whether p27Kip1 is a target gene of miR-222 the alignment of miR-222 with the 3′-UTR of p27Kip1 was analyzed by the miRNA target prediction algorithm TargetScan (http://www.targetscan.org/). It was shown that there was one putative binding site in the 3′-UTR of p27Kip1, which has conserved binding sites for miR-222. To experimentally validate the potential interaction, a luciferase reporter con taining the 3′-UTR of human p27Kip1 was cotransfected into HEK-293T cells with the miR-222-expression vector or negative control vector. The luciferase activity was measured 48 h post transfection. As shown in Fig. 2, the overexpression of miR-222 produced a highly significant reduction (54%, P<0.05) in luciferease activity of the reporter plasmid compared with the controls. In addition, when the predicted binding site of miR-222 in the 3′-UTR was mutated by overlapping PCR, the repression of luciferease activity caused by miR-222 overexpression was abrogated. Collectively, the data suggest that p27Kip1 is a predicted target of miR-222, and the inhibitory effect of miR-222 is due to direct interaction with the putative binding site of the 3′-UTR of p27Kip1.

Figure 2 Interaction of miR-222 with the 3′UTR of p27Kip1 mRNA. p3′UTR-p27 or p3′UTRmut-p27 luciferase constructs containing a wild-type or a mutated p27Kip1 3′UTR were cotransfected into HEK-293T cells with the miR-222 expression vector (P-222) or negative control vector. Luciferase activity was determined 48 h after transfection. The ratio of normalized sensor to control luciferase activity is shown. Error bars represent the standard deviation and were obtained from three independent experiments. *P<0.05. miR, microRNA; UTR, untranslated region.

miR-222 regulates p27Kip1 expression in SH-SY5Y cells

To further determine whether miR-222 can directly regulate p27Kip1, the changes in the p27Kip1 level in SH-SY5Y cells following miR-222 overexpression were analyzed. To do this, the stable transfectant cell line of miR-222, which showed a 3.5-fold increase in miR-222 levels compared with controls (Fig. 3A), was generated. In addition, western blot analysis was performed on the same cells to evaluate the protein level of p27Kip1. The results showed that there was a significant reduction in the p27Kip1 protein level (86%, P<0.05) in the stable transfection cell line of miR-222, as compared with cells transfected with the control vector (Fig. 3B and C). Furthermore, miR-222 expression was knocked down by trans-fecting SH-SY5Y cells with LNA antisense oligonucleotides targeting miR-222 and the effects on p27Kip1 production were analyzed. There was a 2.7-fold decrease of miR-222 level in SH-SY5Y cells transfected with anti-222 LNA compared with cells transfected with LNA against a microRNA not expressed in these cells (Fig. 3A). As expected, the reduction of miR-222 was accompanied by an increase in p27Kip1 protein of 2.1-fold (Fig. 3B and 3C). It is well-understood that repression of gene expression by miRNAs may be due to translational repression or mRNA degradation. To test the relative contribution of the two mechanisms, the p27Kip1 mRNA level was detected and no significant difference was observed following overexpression or knockdown of miR-222. The data demonstrated that miR-222 may regulate p27Kip1 expression by interfering with translation of p27Kip1 mRNA, rather than inducing mRNA degradation.

Figure 3 Effects of ectopic miR-222 expression and miR-222 knockdown in SH-SY5Y cells. (A) The level of miR-222 was measured by RT-qPCR. Total RNA was extracted from SH-SY5Y cells transfected with miR-222 expression vector (P-222), negative control vector (control), LNA antisense oligonucleotides targeting miR-222 (LNA-222), or LNA oligos against a microRNA not expressed in these cells (LNA-control) *P<0.05. (B) Representative western blots for p27Kip1 in the same SH-SY5Y cells utilized for A. (C) Semi-quantitative analyses of western blots for p27Kip1 in the same SH-SY5Y cells utilized for A. Data are presented as the mean ± standard deviation *P<0.05. (D) The level of p27Kip1 mRNA was measured by RT-qPCR. Total RNA was extracted from the same SH-SY5Y cells utilized in A. Ct values of p27Kip1 were normalized with GAPDH which was served as the reference gene. Error bars represent standard deviation and were obtained from three independent experiments. miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Discussion

miRNAs are small non-coding RNA molecules, ~ 22 nucleotides in length, that regulate gene expression by repressing translation or degrading target mRNAs. miRNAs are abundantly expressed in the brain, where they regulate diverse neuronal and glial functions (4). Numerous types of miRNAs have been recognized to be correlated with neurodegenerative diseases (21–23). miR-222 is a member of the to the miR-221/222 family and has been identified to be overexpressed in several types of cancer (24–27). Growing evidence has indicated the important roles of miR-222 in tumor proliferation, drug resistance, apoptosis and metastasis (28–30). However, for miR-222, the possible roles and associated target genes in AD is unclear. In the current study, it was observed that miR-222 is downregulated in APPswe/PSΔE9 mice compared with age-matched controls. In addition, the overexpression of miR-222 or its knockdown produced the predictable opposite effects on p27Kip1 expression. Through the luciferase assay, it was demonstrated that miR-222 can directly target p27Kip1 by interaction with the putative binding site of the 3′-UTR of p27Kip1.

Previous studies have reported that the dysregulation of cell-cycle markers is important in AD pathogenesis (31–39). p27Kip1, also termed cyclin-dependent kinase inhibitor 1B, can prevent cell-cycle progression from the G1 to S phase by binding to CDK2 and cyclin E complexes. Extensive evidence has suggested that the overexpression of p27Kip1 is correlated with the pathogenesis of AD. However, p27Kip1 gene expression regulation is not completely understood. The present study analyzed the expression of p27Kip1 in the cerebral cortex of APPswe/PSΔE9 mice and demonstrated that the protein level of p27Kip1 is upregulated in these mice compared with age-matched controls. However, there was no significant difference in mRNA level of p27Kip1 between the model mice and controls. This indicates that p27Kip1 mRNA may be regulated through a post-transcriptional mechanism. Sequence analysis by Targetscan suggested that the 3′-UTR of p27Kip1 mRNA contained a predicted binding site for miR-222. An inverse correlation between the expression of miR-222 and p27Kip1 was identified in the model mice and controls. Additionally, there was an inverse correlation between the p27Kip1 and miR-222 expression levels in SH-SY5Y cells. Collectively, the results indicated that p27Kip1 is a direct target of miR-222 and the downregulation of miR-222 may contribute to the dysregulation of the cell cycle in AD due to dysregulation of the expression of p27Kip1.

In conclusion, it was confirmed that miR-222 was down-regulated in AD, in association with p27Kip1 upregulation. miR-222 can directly target p27Kip1 by binding to the 3′-UTR of p27Kip1 inducing the translational repression of p27Kip1. Therefore, downregulation of miR-222 may be correlated with the dysregulation of the cell cycle in AD by affecting the expression of p27Kip1.

Acknowledgments

The authors would like to thank Dr Wei Tong and Dr Xueli Liu for technical assistance, and Dr Jiamei Li, Dr Lingling Zhang, Miss Haixia Shi and Miss Xiaoying Li for their help with research. The present study was supported by grants from the Shandong Provincial Natural Science Foundation of China (grant no. ZR2015PH038).

References