MiR-384 Regulates Reelin by Inhibiting ADAMTS4 in Neuronal Cell Lines

MicroRNAs (miRNAs) are important regulators of gene expression at the post-transcriptional level. The present study aims to investigate the role of miR-384 in Reelin by regulating ADAMTS4 in neuronal cell lines. Brain tissues from Aβ1-42 induced mouse model of Alzheimer’s disease and the control group were collected. RT-PCR, Western blotting and immunohistochemistry were performed to detect the levels of ADAMTS4 and miR-384 in tissues. Luciferase reporter assay, Western blotting and in vitro assay were used to validate that ADAMTS4 was the target gene of miR-384. Neuronal cell line, Neuro-2a, was selected for transfection assay. ADAMTS4 was significantly down-regulated in hippocampi of Alzheimer’s disease mouse model, and negatively correlated with miR-384. Then, ADAMTS4 was identified as a direct target of miR-384. Over-expressing of miR-384 in Neuro-2a showed that ADAMTS4 and the cleaved Reelin fragments were down regulated, and proliferation of neuronal cell lines (Neuro-2a and SH-SY5Y) were inhibited through DAB-1 pathway. In conclusion, these results revealed that miR-384 may play a regulatory role in Reelin via inhibiting ADAMTS4 in neuronal cell lines.


Immunohistochemistry (IHC)
The hippocampus tissues were fixed by 4% paraformaldehyde for 7 d, embedded in paraffin, and sectioned to 2-5 μm sections on glass slides. The sections were incubated with anti-ADAMTS4 (SRP00350, Saierbio, Shanghai, China) for 48 h firstly and then with HRP-conjugated goat-anti-rabbit antibody (Proteintech, Shanghai, China). Next, the sections were stained by DAB staining kit (DA1010, Solarbio, Beijing, China) following the instruction, and photographed under microscopy.

Results
miR-384 and ADAMTS4 are negatively correlated in Normal/AD mouse brain The expression of ADAMTS4 in different tissues was detected by RT-PCR analysis, and the result showed that ADAMTS4 was highly expressed in liver and brain (Fig. 1A). Comparing between the Aβ induced AD mice group and the normal group, it was indicated that expression of ADAMTS4 was inhibited on both mRNA and protein levels in AD mice brains ( Fig. 1C and D).
Likewise, the result of IHC showed that ADAMTS4 was down-regulated in hippocampi of AD mice (Fig. 1E).
Reelin proteolytic cleavage generates Reelin fragments of different molecular weights, with the 180 kDa N-terminal fragment as the predominant. Thus, the cleaved 180 kD Reelin fragment was detected. As compared with the control group, the cleaved 180 kD Reelin fragment was inhibited in AD group (Fig. 1D). The expression of miR-384 was detected, and it was up-regulated AD mice brains (Fig. 1B), which was reversely correlated to ADAMTS4. These results suggested a probability of the interaction between miR-384 and ADAMTS4.

miR-384 regulates ADAMTS4 by single targeting site
The conserved miR-384 targeting sites of ADAMTS4 ( Fig. 2A) were predicted using TargetScan [21]. Plasmids pSi2-hUTR and pSi2-mUTR were constructed, containing human and mouse target sites, respectively. To verify the predicted sites, mutant plasmids pSi2-hm1 and pSi2-hm2 were also constructed. Comparison of Gaussia luciferase and firefly luciferase activities of transfected HEK293 cells showed that miR-384 inhibited Gaussia luciferase through human and mouse 3'-UTR of ADAMTS4, and didn't inhibit through mutated targeting sites (Fig. 2B). MiR-384 mimics with different concentration were transfected into HEK293, and levels of miR-384 increased correspondingly (Fig. 2C). Meanwhile, the mRNA level of ADAMTS4 was significantly decreased (Fig. 2D). The result of Western blot showed that the protein level of ADAMTS4 was down-regulated by the transfection of miR-384 at a dosage dependent manner. Besides, the cleaved 180 kD Reelin fragment was decreased after miR-384 transfection (Fig. 2E). These results indicated that miR-384 regulates ADAMTS4 through the predicted targeting site.

miR-384 inhibits Reelin signaling and neuronal cell proliferation
In canonical Reelin signaling pathway, Reelin exerts its functions by bingding to two receptors, apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VLDLR), and inducing phosphorylation of the intracellular protein Dab1 which activates multiple signal transduction pathways, such as Akt, PI3K and CrK/CrkL. Thus, the concentration of Dab1 and Akt can be used as an indicator of Reelin signaling.
To evaluate the possible functions of miR-384 on Reelin signaling, Neuro-2a cells were transfected with miR-384 mimics, and the cells with over-expressed miR-384 were obtained ( Fig.   3A). RT-PCR and Western blot analyses showed that the mRNA and protein levels of ADAMTS4 in Neuro-2a were significantly down-regulated by over-expressing miR-384 ( Fig. 3B and E).
Moreover, the 180kD Reelin fragments were significantly decreased, and phosphorylation Dab-1 and Akt were up-regulated, without alteration in total Dab-1 and Akt (Fig. 3E). Also, proliferation assays of Neuro-2a and SH-SY5Y were performed to determine the role of miR-384 in neuronal cell proliferation. Over-expressing miR-384 in the cells revealed a significant inhibition in cell proliferation ( Fig. 3C and D). These results suggested that miR-384 may inhibit Reelin signaling and neuronal cell proliferation.

Discussion
ADAMTS4, a member of ADAMTSs family, has been shown to cleave Reelin, a large secreted glycoprotein which is considered to be involved in the pathogenesis of AD [9][10][11][12]14]. Using TargetScan, miR-384 was predicted to be a potential regulator of ADAMTS4. In this study, the relationship between miR-384 and ADAMTS4 was investigated, and the effect they may have on Reelin signaling and neuronal cell proliferation were tested. First, the expression levels of ADAMTS4 and miR-384 in AD mouse group and control group were measured, and the results showed that miR-384 and ADAMTS4 were negatively correlated in the groups. Then, the targeting site of ADAMTS4 by miR-384 was validated using dual-luciferase reporter assay system. Furthermore, the role of miR-384 on Reelin signaling and neuronal cell proliferation was explored, and it was found that miR-384 inhibited Reelin signaling and neuronal cell proliferation.  [13,20,24]. Therefore, more work is needed to achieve a better understanding of Reelin and its signaling mechanisms, which may throw a light on clinical therapies against cognitive deficits in AD.

Conclusion
In conclusion, the findings of the present study showed that miR-384 may play a regulatory role in ADAMTS4-Reelin pathway by targeting a single conserved site of ADAMTS4, and over-expression of miR-384 inhibits Reelin signaling.