Department of Biomedical Sciences, Graduate School for Biomedical Science and Engineering, Hanyang University, Seoul, Republic of Korea.
Biomedical Research Institute, Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Republic of Korea.
Cell Mol Life Sci. 2019 Nov;76(22):4569-4580. doi: 10.1007/s00018-019-03290-3. Epub 2019 Sep 5.
Hippocalcin (HPCA) is a neuron-specific calcium-binding protein predominantly expressed in the nervous system. In the present study, we demonstrate that HPCA regulates neuronal differentiation in SH-SY5Y cells. We observed that the expression level of HPCA was increased during neuronal differentiation. Depletion of HPCA inhibited both neurite outgrowth and synaptophysin (SYP) expression, whereas overexpression of HPCA enhanced neuronal differentiation. Interestingly, we also found that the expression of HPCA mRNA was modulated by miR-24-3p. Using a dual-luciferase assay, we showed that co-transfection of a plasmid containing the miR-24-3p binding site from the 3'-untranslated region (3'UTR) of the HPCA gene and an miR-24-3p mimic effectively reduced luminescence activity. This effect was abolished when miR-24-3p seed sequences in the 3'UTR of the HPCA gene were mutated. miR-24-3p expression was decreased during differentiation, suggesting that the decreased expression level of miR-24-3p might have upregulated mRNA expression of HPCA. As expected, upregulation of miR-24-3p by an miRNA mimic led to reduced HPCA expression, accompanied by diminished neuronal differentiation. In contrast, downregulation of miR-24-3p by an antisense inhibitor promoted neurite outgrowth as well as levels of SYP expression. Taken together, these results suggest that miR-24-3p is an important miRNA that regulates neuronal differentiation by controlling HPCA expression.
海帕钙蛋白（Hippocalcin，HPCA）是一种主要在神经系统中表达的神经元特异性钙结合蛋白。在本研究中，我们证明 HPCA 调节 SH-SY5Y 细胞中的神经元分化。我们观察到 HPCA 的表达水平在神经元分化过程中增加。HPCA 的耗竭抑制了轴突生长和突触小泡蛋白（Synaptophysin，SYP）的表达，而过表达 HPCA 则增强了神经元分化。有趣的是，我们还发现 HPCA mRNA 的表达受 miR-24-3p 调节。通过双荧光素酶报告基因实验，我们表明转染包含 HPCA 基因 3'非翻译区（3'UTR）中 miR-24-3p 结合位点的质粒和 miR-24-3p 模拟物可有效降低荧光素酶活性。当 HPCA 基因 3'UTR 中的 miR-24-3p 种子序列发生突变时，这种效应被消除。miR-24-3p 的表达在分化过程中降低，表明 miR-24-3p 表达水平的降低可能上调了 HPCA 的 mRNA 表达。正如预期的那样，miR-24-3p 模拟物的上调导致 HPCA 表达降低，伴随着神经元分化减弱。相比之下，miR-24-3p 反义抑制剂的下调促进了轴突生长和 SYP 表达水平。总之，这些结果表明 miR-24-3p 是一种重要的 miRNA，通过控制 HPCA 的表达来调节神经元分化。
