Abstract
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.
Highlights
MicroRNAs are small, highly conserved noncoding RNA molecules of approximately 22 nucleotides
Using an miRNA Target Prediction algorithm, we found that only miR-24-3p had two potential binding sites to 3′-untranslated region (3′UTR) of HPCA among several miRNAs. miR-24-3p, which belongs to the miR-24 family, is a well-known cancer-associated miRNA that is differentially expressed in the initiation and progression in multiple kinds of tumors
To determine the involvement of HPCA in regulating human neuronal differentiation, SH-SY5Y cells were induced to differentiate in the presence of 50 μM retinoic acid (RA) for 5 or 7 days
Summary
MicroRNAs (miRNAs) are small, highly conserved noncoding RNA molecules of approximately 22 nucleotides. They can modulate gene expression through complementary base pairing of the seed sequence (the 2–8 nucleotides from the 5′ end of the miRNA) located in the 3′-untranslated. Our previous study showed that HPCA expression was gradually increased during neuronal differentiation [16]. We assumed that an miRNA would be associated with HPCA expression during neuronal differentiation. Using an miRNA Target Prediction algorithm, we found that only miR-24-3p had two potential binding sites to 3′UTR of HPCA among several miRNAs. miR-24-3p, which belongs to the miR-24 family, is a well-known cancer-associated miRNA that is differentially expressed in the initiation and progression in multiple kinds of tumors. There is no evidence of any association with neurons in miR-24-3p
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