MiR-29c-3p May Promote the Progression of Alzheimer's Disease through BACE1

The aim of this study was to explore the specific role of miR-29c-3p in Alzheimer's disease (AD). Animal models of AD were established by injecting streptozotocin (STZ) into mice through the lateral ventricle, while cell models of AD were induced by 10 μM β-amyloid (Aβ). We detected miR-29c-3p and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) contents and measured AD cell proliferation and apoptosis. A low miR-29c-3p level and a high BACE1 level were detected in the brain tissue of AD animal models and AD cell models. Aβ-processed cells had markedly lower proliferation activity, higher apoptosis, increased phosphorylation of tau protein was over phosphorylated, but the overexpression of miR-29c-3p or the silencing of BACE1 significantly enhanced the cell proliferation activity and reduced cell apoptosis by regulating the contents of related proteins. Inhibition of miR-29c-3p or overexpression of BACE1 aggravated Aβ-induced side effects. We used Targetscan7.2 to predict the downstream target genes of miR-29c-3p. Then, we detected that there were target binding sites between miR-29c-3p and BACE1. The rescue experiment identified BACE1 as a functional target for miR-29c-3p. AD leads to decreased miR-29c-3p level and increased BACE1 level. MiR-29c-3p has specific binding sites with the 3′-untranslated region (3′-UTR) of BACE1 and thus negatively regulates the BACE1 level, thereby affecting the progression of AD.


Introduction
Alzheimer's disease (AD), a neurodegenerative disease common in old people, mainly manifests as impaired cognitive functions, social disorders, abnormal behaviors, and memory loss [1,2]. People with AD account for more than 80% of cases of dementia in people over 65 worldwide [3]. Currently, the number of AD patients in the United States exceeds 5 million, with an expected increase to 16 million by 2050 [4]. Decades of ongoing investigations of AD have not figured out its etiology. So far, there is no optimal treatment strategy for AD. e occurrence of AD is related to β-amyloid (Aβ) plaques and neurofibrillary tangles (containing tau protein) in the cerebral cortex and subcortical regions, so there is the speculation that the reducing or eliminating of this substance from the brain may prevent or reverse the progression of AD disease [4,5].
MicroRNAs (miRNA), a class of endogenous noncoding RNAs with 18-24 nucleotides, play vital roles in the mammalian brain [6]. MiRNAs are highly expressed or specifically expressed in the nervous system to affect neural development and synaptic functions and participate in processes such as memory formation and regulation of protein synthesis, as well as to induce specific neurodegenerative diseases [7][8][9]. MiR-29c-3p is abnormally expressed in a variety of diseases [10]. With markedly lower expression in hepatocellular carcinoma, miR-29c-3p can inhibit tumors by targeting DNA methyltransferase 3B (DNMT3B) and the Hippo signaling pathway involving large tumor suppressor kinase 1 (LATS1) [11]. So far, little has been known about the effect of miR-29c-3p on AD progression. One of the main pathological changes caused by AD is senile plaque deposition in the brain, which mainly consists of Aβ that comes from amyloid precursor protein (APP) after being sequentially cleaved by c-secretase and β-site amyloid precursor protein cleaving enzyme 1 (BACE1), also called β-secretase 1, a key gene for Aβ accumulation in the brain of AD patients [12,13]. MiR-29C is involved in AD progression and reduces the accumulation of Aβ by decreasing the expression of BACE1 [14]. We need to study further to check if miR-29c-3p has the same function as miR-29c in AD patients.
Here, we assessed the function of miR-29c-3p in the proliferation and apoptosis of Aβ-induced AD cells, aiming to figure out the specific mechanisms of miR-29c-3p in AD.

Animal Models.
We purchased male SPF C57BL/6J mice (20-25 g) from the Department of Laboratory Animal Science of China Medical University. After adaptive feeding for 1 week, we randomly assigned mice to the AD group (10 mice) and the control group (10 mice). Mice in the control group were reared normally, while mice in the AD group were made into AD models by injecting streptozotocin (STZ) through the lateral ventricle [15].
is study was carried out after obtaining approval from the ethics committee of our hospital, in strict accordance with guidelines issued by the Care and Use of Laboratory Animal and the National Institutes of Health (NIH).

Western Blot Detection.
e protein in cells was lysed by RIPA buffer (Cell Signal Technology, Inc., Danvers, MA, USA). We quantified the protein concentration with the BCA kit (Beyotime Biotechnology, Shanghai, China). e electrophoretic separation of proteins was performed using the 12% SDS-PAGE gel. en, we transferred the protein to a PVDF membrane manufactured by Millipore and blocked the membrane in skimmed milk-PBS solution at room temperature for 1 hour. After the incubation of the membrane and the primary antibody (Abcam, Branford, CT, USA) at 4°C for one night, the primary antibody was washed off and then the horseradish peroxidase-labeled goat antirabbit secondary antibody (Abcam, Branford, CT, USA) was added to the membrane and incubated at 37°C for 1 hour. Finally, the membrane was subjected to a PBS washing, followed by color development using ECL luminescent reagent ( ermo Fisher Scientific, Waltham, MA, USA). e relative protein expression � gray value of the strip to be tested/gray value of the internal reference protein.

Detection of Cell Proliferation Activity.
We employed an MTT kit ( ermo Fisher Scientific Co., Ltd., Hangzhou, China) to test the PC12 cell proliferation activity. Cells were incubated at 37°C with 5% CO 2 for 24 h, 48 h, 72 h, and 96 h. en, the medium was discarded and 20 μL of MTT was added to per well for incubation for 4 hours. Next, we added 150 μl of dimethyl sulfoxide to the well and shook the plate for 5-10 minutes to make sure the purple crystals were completely dissolved. Multiskan ™ GO full-wavelength microplate reader ( ermo Fisher Scientific Co., Ltd., Hangzhou, China) was employed to test the absorbance at 450 nm to detect cell proliferation. e experiment was repeated 3 times.

Detection of Cell Apoptosis.
We purchased an Annexin-V-FITC/PI Apoptosis Detection Kit ( ermo Fisher Scientific, Waltham, MA, USA) to test cell apoptosis. en, we washed cells transfected with different plasmids with the cold phosphate saline buffer and trypsinized cells and collected them into a centrifuge tube. Twenty μl of Annexin-V-FITC labeling solution, 1 ml of buffer, and 20 μl of PI reagent were mixed together for incubation at room temperature for 5 minutes away from light, and then, cell apoptosis was detected on the FACScan flow cytometer (Becton Dickinson, USA).

Dual-Luciferase Reporter (DLR) Assay.
e prediction of the binding sites between miR-29c-3p and BACE1 was performed on TargetScan. FragmentS of either the wild type (BACE1-wt) or mutant (BACE1-mut) BACE1 3′-untranslated region (3′-UTR) containing the predicted binding sites were cloned on the vector. After the DNA sequencing for verification, we transfected miR-29c-3p mimics or miR-NC into PC12 cells using a Lipofectamine ™ 2000 kit (Invitrogen, USA). We harvested cells 48 hours later and conducted the DLR assay on DLR ® (Promega Corporation).

Statistical Analysis.
e statistical analysis was performed on the SPSS20.0 and the data visualization on the GraphPad 7. e intergroup comparison was analyzed by the independent t-test and the comparison between multiple groups by the one-way analysis of variance, with the LSD ttest as the post hoc test. P < 0.05 indicates a statistical difference.

Effect of AD on MiR-29c-3p and BACE1 Levels.
e qRT-PCR detection revealed a low miR-29c-3p level and a high BACE1 level in brain tissues of mouse models (P < 0.05), a markedly reduced miR-29c-3p level, and markedly increased levels of BACE1 mRNA and protein in Aβ-processed PC12 cells (P < 0.05). More details are shown in Figure 1.

Function of MiR-29c-3p in the Apoptosis and Viability of
Aβ-Induced PC12 Cells. We transfected PC12 cells with miR-29c-3p inhibitor, miR-NC, and miR-29c-3p mimics separately. More details are shown in Figure 2. Aβ-processed cells had markedly lower cell viability and increased tau protein phosphorylation than cells from the control group (P < 0.05). MiR-29c-3p mimics relieved the toxic side effects caused by Aβ, enhanced cell viability, and reduced the level of tau protein phosphorylation, while miR-29c-3p inhibitor further inhibited cell viability (P < 0.05). Aβ-processed cells had markedly higher cell apoptosis, higher expression levels of apoptosis-related proteins (caspase-9, caspase-3, and Bax), and lower expression and content of Bcl-2 than cells from the control group (P < 0.05). e overexpression of miR-29c-3p attenuated the effects of Aβ, while the inhibition of miR-29c-3p accelerated the Aβ-induced cell apoptosis.

Effect of BACE1 on the Apoptosis and Viability of Aβ-
Induced PC12 Cells. We transfected sh-BACE1, vector, and si-BACE1 into PC12 cells separately, as shown in Figure 3. Si-BACE1 relieved the toxic side effects caused by Aβ, enhanced cell viability, while sh-BACE1 further inhibited cell viability and reduced tau protein phosphorylation (P < 0.05). Cells in the si-BACE1 group had markedly lower cell apoptosis rate, lower content of caspase-9, caspase-3, and Bax, and higher content of Bcl-2 than cells from the control group (P < 0.05). Cells in the sh-BACE1 group had stronger apoptosis than cells in the Aβ group.

3.4.
e Targeting Relationship between MiR-29c-3p and BACE1. We made a prediction of the target genes of miR-29c-3p on Targetscan7.2 and discovered targeted binding sites between the two genes. DLE assay revealed that the fluorescence activity of BACE1-Wt was significantly reduced. Transfection of miR-29c-3p inhibitor resulted in higher BACE1 protein level, while transfection of miR-29c-3p mimics resulted in lower BACE1 protein level (P < 0.05) (Figure 4).

Discussion
e pathogenesis of AD is caused by a variety of complex factors, such as age, heredity, and neurotransmission barriers [17,18], bringing severe economic and living burdens to patients and society. e main pathological characteristics of AD disease include vascular amyloidosis, intracellular abnormal aggregation of phosphorylated tau protein, the formation of neurofibrillary tangles (NFTs), senile plaques formed by β-amyloid (Aβ) deposition, and neuron cells loss in the hippocampal area and cerebral cortex [19][20][21]. e accumulation and sedimentation of tau protein is an important factor responsible for neuronal degeneration and death, as well as the onset of AD [22]. e abnormal hyperphosphorylation of tau protein is caused by increased protein kinase activity or decreased phosphatase activity [23]. erefore, reducing the abnormal hyperphosphorylation of tau protein is the key to treating AD.
In this study, the viability of PC12 cells processed by Aβ was markedly reduced, while the cell apoptosis was markedly increased, which is consistent with the results of previous researches [24,25]. MiRNAs are closely related to the onset of AD and can affect cell differentiation, development, proliferation, and apoptosis by regulating the corresponding target genes [26,27]. e abnormal expression of miRNAs in tissue or serum plays can affect the development and progression of diseases [28]. MiRNAs may affect the development and functions of the central nervous system [29]. MiR-29c-3p, a tumor suppressor [30], is remarkably downregulated in many solid tumors, including breast cancer [31], gallbladder cancer [32], and gastric cancer [33]. Sørensen et al. [34] proposed that the miR-29c-3p level is reduced in the cerebrospinal fluid of AD patients. Here, we detected a low expression level of miR-29c-3p in the brain tissue of AD mice and PC12 cell models of AD, which indicates that miR-29c-3p may affect the pathogenesis of AD. We transfected Aβprocessed PC12 cells with miR-29c-3p inhibitor, miR-NC, and miR-29c-3p mimics, separately. en, we discovered     PC12 cell apoptosis by targeting SIRT1 [36]. Based on the results of this study and previous findings, we speculate that miR-29c-3p can reduce the cytotoxicity caused by Aβ and induce AD lesions. BACE1, a key enzyme to perform APP degradation to produce Aβ, has a markedly lower protein expression level in AD mice than in wild mice of the same age [16]. BACE1 protein levels and enzyme activity were remarkably higher in the brain samples of AD patients than in the brain samples of non-AD patients [37]. Many existing clinical drugs for AD aim to target decrease the protein level and enzyme activity of BACE1 to reduce Aβ production and secondary neuropathological changes [38]. Here, we detected a high BACE1 level in AD and found that BACE1 silencing relieved cytotoxicity caused by Aβ, decreased cell apoptosis, increased cell viability, and reduced the phosphorylation of tau protein. Many studies have shown that miRNAs play a part in the occurrence and development of AD through regulating BACE1. Feng et al. [39] proposed that miR-124 can reduce Aβ-induced inhibition of cell viability and decrease the apoptosis of SH-SY5Y cells by regulating BACE1. Bioinformatics analysis revealed that BACE1, a gene closely related to Aβ production in the course of AD, is a potential functional target gene for miR-29c-3p. In this study, the DLR assay verified the target gene of miR-29c-3p. BACE1-Wt fluorescence activity was significantly reduced, but BACE1-Mut had no significant change.
e results of plasmid transfection revealed that miR-29c-3p overexpression inhibited the activity of BACE1, indicating miR-29c-3p has a negative regulatory effect on BACE1. e results of rescue experiments demonstrated that sh-BACE1 significantly reversed the therapeutic effect of miR-29c-3p mimics, identifying BACE1 as a functional target for miR-29c-3p.

Conclusion
is study mainly explored the underlying mechanism of miR-29c-3p in AD, aiming to provide new treatment targets for AD. However, this study is subject to some limitations. Given the complicated regulatory mechanism of AD, we did not figure out the specific regulatory pathways of AD. Besides, we will explore and the role of miR-29c-3p in the life and behavior of AD mice in the future to perfect this study.
In summary, AD can decrease the expression of miR-29c-3p and increase the expression of BACE1. MiR-29c-3p has specific binding sites with the 3′-untranslated region (3′-UTR) of BACE1 and thus negatively regulates the BACE1 level, thereby affecting the progression of AD.

Data Availability
e datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.