MicroRNA-5572 Is a Novel MicroRNA-Regulating SLC30A3 in Sporadic Amyotrophic Lateral Sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease caused by the loss of motor neurons. Although the pathogenesis of sporadic ALS (sALS) remains unclear, it has recently been suggested that disorders of microRNA (miRNA) may be involved in neurodegenerative conditions. The purpose of this study was to investigate miRNA levels in sALS and the target genes of miRNA. Microarray and real-time RT-PCR analyses revealed significantly-decreased levels of miR-139-5p and significantly increased levels of miR-5572 in the spinal cords of sALS patients compared with those in controls. We then focused on miR-5572, which has not been reported in ALS, and determined its target gene. By using TargetScan, we predicted SLC30A3 as the candidate target gene of miR-5572. In a previous study, we found decreased SLC30A3 levels in the spinal cords of sALS patients. We revealed that SLC30A3 was regulated by miR-5572. Taken together, these results demonstrate that the level of novel miRNA miR-5572 is increased in sALS and that SLC30A3 is one of the target genes regulated by miR-5572.

Keywords: amyotrophic lateral sclerosis; microRNA; microarray; spinal cord.

Figure 1

Global analysis of microRNA (miRNA) expression in sporadic amyotrophic lateral sclerosis (sALS) was performed using spinal cord samples from patients with sALS. (A) The number or ratio of altered miRNAs in sALS is presented. More than twofold changes in miRNA levels were calculated from the result of microarray analysis. (B) Decreases in miRNA levels in the microarray were confirmed by real-time RT-PCR analysis. All data are presented as mean ± standard error. Statistical significance between control (n = 5) and sALS (n = 4) group in each miRNA was determined by Student’s t-test (* p < 0.05). (C) Increases in miRNA levels in the microarray were confirmed by real-time RT-PCR analysis. All data are presented as mean ± standard error. Statistical significance between control (n = 5) and sALS (n = 4) group in each miRNA was determined by Student’s t-test (* p < 0.05). (D) Re-plotted individual data (control; n = 5 and sALS; n = 4) of miR-139-5p/U6 ratio in Figure 1B are presented as box and scatter plot. Statistical significance was determined by Student’s t-test (* p < 0.05). (E) Re-plotted individual data (control; n = 5 and sALS; n = 4) of miR-5572/U6 ratio in Figure 1B are presented as box and scatter plot. Statistical significance was determined by Student’s t-test (* p < 0.05).

Figure 2

Determination of SLC30A3 was performed using spinal cord samples from patients with sALS. (A) The levels of SLC30A3 in the spinal cord were determined by Western blotting analysis. Samples from five patients in each experimental group (control; n = 5 and ALS; n = 5) were examined. (B) SLC30A3 in the spinal cords of sALS patients was quantified (control; n = 5 and ALS; n = 5). Data are presented as box and scatter plot calculated from the band intensity of the Western blot. Statistical significance was determined by Student’s t-test (* p < 0.05).

Figure 3

Determination of regulation of miR-5572 on SLC30A3 expression. (A) The level of SLC30A3 mRNA was determined in HEK293 cells transfected with negative control (NC) (n = 5) and miR-5572 mimic (n = 5) by real-time RT-PCR analysis. All data are presented as box and scatter plot. Statistical significance was determined by Student’s t-test (* p < 0.05). (B) The level of SLC30A3 protein was determined in HEK293 cells transfected with NC (n = 4) or miR-5572 mimic (n = 4) by Western blotting analysis. All data are presented as box and scatter plot. Statistical significance was determined by Student’s t-test (* p < 0.05).

Figure 4

Reporter gene assay for the functional analysis of 3′-UTR regulated by miRNA. (A) The reporter construct contained the inserted DNA fragment of human SLC30A3 3′-UTR wild type (WT) or mutant (Mut) downstream of luc2 in the pmirGLO Dual-Luciferase miRNA Target Expression Vector. (B) Reporter gene assay was performed using CHO cells co-transfected with NC or miR-5572 mimic, and the reporter vector cloned with WT 3′-UTR of SLC30A3 or mutant 3′-UTR of SLC30A3 (n = 3 in each experimental group). All data are presented as box and scatter plot. Statistical significance was determined by two-way ANOVA followed by post hoc Bonferroni’s test (* p < 0.05).