miR-153-3p suppresses the differentiation and proliferation of neural stem cells via targeting GPR55

Alzheimer's disease is the most frequent neurodegenerative disease and is characterized by progressive cognitive impairment and decline. NSCs (neural stem cells) serve as beneficial and promising adjuncts to treat Alzheimer's disease. This study aimed to determine the role of miR-153-3p expression in NSC differentiation and proliferation. We illustrated that miR-153-3p was decreased and GPR55 was upregulated during NSC differentiation. IL-1β can induce miR-153-3p expression. Luciferase reporter analysis noted that elevated expression of miR-153-3p significantly inhibited the luciferase value of the WT reporter plasmid but did not change the luciferase value of the mut reporter plasmid. Ectopic miR-153-3p expression suppressed GPR55 expression in NSCs and identified GPR55 as a direct target gene of miR-153-3p. Ectopic expression of miR-153-3p inhibited NSC growth and differentiation into astrocytes and neurons. Elevated expression of miR-153-3p induced the release of proinflammatory cytokines, such as TNF-α, IL-1β and IL-6, in NSCs. Furthermore, miR-153-3p inhibited NSC differentiation and proliferation by targeting GPR55 expression. These data suggested that miR-153-3p may act as a clinical target for the therapeutics of neurodegenerative diseases.


INTRODUCTION
AD (Alzheimer's disease) is the most frequent neurodegenerative disease and is characterized by progressive cognitive impairment and decline [1][2][3][4]. However, available therapeutic methods for AD are still lacking because of the unclear pathogenesis and etiology of this disease [5][6][7]. NSCs (neural stem cells) are multipotent and self-renewing cells found in the central nervous system of adults and developing mammals [8][9][10]. These stem cells differentiate into oligodendrocytes, astrocytes and neurons and serve as beneficial and promising adjuncts to treat neurological diseases such as spinal cord injuries, Parkinson's disease, brain trauma and AD [11][12][13][14]. However, many challenges must be solved before the clinical use of NSCs.
In our study, we illustrated that miR-153-3p inhibited NSC differentiation and proliferation and proinflammatory cytokine release by targeting GPR55 expression in NSCs.

Cell culture and transfection
NSCs were separated and cultured as described previously [33]. Cells were separated from five rat embryos and placed in medium supplemented with N2, bFGF and EGF. Our study was approved by The Affiliated Yan'An Hospital of Kunming Medical University. The miR-153-3p control, inhibitor and their control plasmids were obtained from GenePharma (China) and transfected into cells with Lipofectamine 3000 at a concentration of 10 nmol/l.

Dual luciferase assay
The wild-type 3'-UTR and mutant 3'-UTR of GPR55 containing the predicted binding site of miR-153-3p were amplified by PCR and inserted into the pMIR-REPORT luciferase plasmid. Lipofectamine-2000 was utilized for transfection with miR-153-3p control or mimic and the wild-type and mutant 3'-UTRs of GPR55 as described previously. Luciferase activity was detected using a luciferase reporter kit (Promega, USA).

ELISA
After treatment, the cell culture supernatant was obtained to measure the levels of proinflammatory cytokines such as TNF-α, IL-1β and IL-6 by using ELISA kits (Cambridge, UK).

Proliferation assay
Cells were plated in 96-well dishes and were allowed to continue growing for 0, 1, 2 and 3 days after treatment. Cell growth was detected using Cell Counting Kit-8 (Dojindo, China), and the cells were incubated with CCK-8 reagent (10%) for 3 hours at 37° C. The absorbance was measured using a microplate reader at 450 nm.

Immunofluorescence
Cells were fixed in paraformaldehyde (4%) for half an hour at room temperature and then washed in PBS (phosphate-buffered saline) 3 times. After 1 hour of blocking in Triton X-100 (0.2%) and goat serum (3%) in PBS, the cells were incubated with anti-nestin, anti-Tuj1 and anti-GFAP (1:400; Millipore, USA) at 4° C overnight. After washing 3 times in PBS, the cells were incubated with secondary antibodies. The cells were visualized with fluorescence microscope.

Statistical analysis
Experimental statistics were presented as means±standard deviation. Statistical significance (P<0.05) was analyzed by ANOVA or Student's t-test using the SPSS software system (Chicago, USA).

GPR55 is a direct gene target of miR-153-3p
qPCR illustrated that miR-153-3p was overexpressed in NSCs after treatment with the miR-153-3p mimic compared with that in the miR-NC group (Figure 2A, p<0.001). This result suggested that the efficiency of miR-153-3p was high. By searching bioinformatic TargetScan 7.2 (http://www.targetscan.org/vert_72/), we identified one potential target site between miR-153-3p and the GPR55 3'-UTR ( Figure 2B). We also showed that these sequences were conserved among different species ( Figure 2B). Ectopic miR-153-3p expression suppressed GPR55 expression in NSCs ( Figure 2C, p<0.01). Luciferase reporter analysis noted that elevated expression of miR-153-3p significantly inhibited the luciferase value of the WT reporter plasmid but did not change the luciferase value of the mut reporter plasmid ( Figure 2D, p<0.01).

miR-153-3p inhibits NSC differentiation and proliferation by targeting GPR55 expression
Because GPR55 is an NSC regulator, we speculated that miR-153-3p might act on these functions by regulating GPR55 expression. To prove this hypothesis, several gain and loss function experiments were performed. The  Figure 5I, p<0.05) expression compared with the vehicle group in miR-153-3p-treated NSCs. These data were also confirmed using immunocytochemical staining ( Figure 5E). These results showed that miR-153-3p inhibited NSC differentiation and proliferation by targeting GPR55 expression.

DISCUSSION
Previous studies have illustrated that miRNAs regulate NSC differentiation, neuronal maturation and proliferation [10,34,35]. For example, Wu et al. [30]. illustrated that miR-374b modulates NSC differentiation and growth by regulating Hes1. Chen et al. [31]. noted that miR-132 acts as a moderator of neurite outgrowth, cell differentiation and self-renewal of NSCs. Xue et al. [36]. indicated that miR-145 protects NSC function by regulating the MAPK signaling pathway to remediate rat cerebral ischemic stroke. Channakkar et al. [37]. showed that miR-137 modulates NSC fate via the regulation of mitochondrial dynamics. However, the potential functional role of miR-153-3p in the fate of NSCs remains unclear. miR-153-3p modulates cisplatin resistance and cell growth through Nrf-2 in esophageal carcinoma [38]. Li et al. [39]. illustrated that miR-153-3p modulates ovarian carcinoma progression by regulating MCL1 expression. Sun et al. [40]. indicated that miR-153-3p promotes glioma cell radiosensitivity by modulating BCL2. A previous study showed that IL-1β induces miR-153 expression in beta cells and that IL-1β plays critical roles in the fate of NSCs [41][42][43]. In the present study, we noted that miR-153-3p is decreased during NSC differentiation and that IL-1β induces miR-153-3p expression in NSCs. Ectopic expression of miR-153-3p inhibited NSC growth and differentiation into astrocytes and neurons. Elevated expression of miR-153-3p induced the release of proinflammatory cytokines, such as TNF-α, IL-1β and IL-6, in NSCs. These results indicated that miR-153-3p plays critical roles in the cell differentiation and self-renewal of NSCs.
GPR55 is a lipid-sensing receptor that plays important roles in cell mobilization, invasion and cell cycle progression in tumor development. Wang et al. [44]. indicated that CID16020046 (GPR55 antagonist) protects against inflammation induced by ox-LDL in HAECs (aortic endothelial cells). Saliba et al. [45]. illustrated that several compounds with antagonistic activities of GPR55 suppress PGE2 release in microglia. Recently, Hill et al. [46]. showed that GPR55 activation promotes NSC proliferation and differentiation into neuronal cells. Moreover, they found that a GPR55 agonist defends against neurogenesis rate reductions in NSCs induced by IL-1β. GPR55 activation suppresses inflammatory cytokine expression in NSCs [47]. In our  study, we searched for bioinformatic targets and identified one potential target site between miR-153-3p and the GPR55 3'-UTR. Luciferase reporter analysis noted that the elevated expression of miR-153-3p significantly inhibited the luciferase value of the WT reporter plasmid but did not change the luciferase value of the mut reporter plasmid. Moreover, we showed that ectopic expression of miR-153-3p suppresses GPR55 expression in NSCs. Furthermore, miR-153-3p inhibited NSC differentiation and proliferation by targeting GPR55 expression. However, more experiments must be performed on human NSCs in the future. These results provide novel insights into the modulation of GPR55 and its cell function in the development of NSCs.
In summary, our results noted the involvement of miR-153-3p in modulating the differentiation and growth of NSCs. It also illustrated that miR-153-3p inhibits NSC differentiation and proliferation and proinflammatory cytokine release by targeting GPR55 expression in NSCs. These data suggest that miR-153-3p acts as a clinical target for neurodegenerative disease therapeutics.

AUTHOR CONTRIBUTIONS
Xiaolin Dong and Yanping Li performed experiments, Xiaolin Dong, Hui Wang, Liping Zhan, Qingyun Li, Yang Li, Gang Wu, Huan Wei, Yanping Li, design of the study, conceived and drafted the manuscript, analysed the data, Xiaolin Dong and Yanping Li wrote and revised the paper.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest.

FUNDING
Our study was supported by the Special fund for the applied basic research of Yunnan Science and Technology Department associated with Kunming Medical University. NO. 2019FE001(-102).

ETHICAL STATEMENT
The study was approved by The Affiliated Yan'An Hospital of Kunming Medical University.