A novel function of microRNA let-7d in regulation of galectin-3 expression in attention deficit hyperactivity disorder rat brain

Abstract

In this study we investigated the locomotor activity and non-selective attention in spontaneously hypertensive rats (SHR) with control Wistar-Kyoto (WKY) rats, which were employed as an attention deficit hyperactivity disorder (ADHD) model. In open-field test and làt maze, SHR rats were found to be much more spontaneously active than WKY rats. As compared with WKY rats, a lower level of galectin-3 was observed in SHR brain prefrontal cortex (PFC), which was the major affected brain area of ADHD. Through miRNA microarray screening, rno-let-7d was noted to be solely upregulated in SHR PFC. Interestingly, rno-let-7d had a binding site at galectin-3 mRNA and was shown to regulate galectin-3 3' untranslated region (UTR) directly. Mutation of galectin-3 3'UTR by one nucleotide of the seed sequence prevented rno-let-7d regulation of the 3' UTR completely. Although rno-let-7d did not directly regulate tyrosine hydroxylase (TH) 3'UTR, the level of galectin-3 was important for cAMP response element binding protein, the major transcript factor for TH gene. Either overexpression or downexpression of galectin-3 could result in modulation of TH expression in both PC12H and PC12L cells. In conclusion, our data suggested a novel function of rno-let-7d in regulation of galectin-3 and in ADHD development. Rno-let-7d, which is increased in the PFC of SHR brain, negatively regulated galectin-3, which is coupled with TH expression regulation.

Figure 1

Behavioral task comparison between spontaneously hypertensive rats (SHR) and Wistar–Kyoto (WKY) rats. A. Comparison of the total number of square crossings in 1 h open‐field test; B. Comparison of the number of rearings among 15 minutes groups in 1‐h open‐field test; C. Number comparison of corner crossings among 10‐minute groups in 30‐minute làt‐maze test; D. The number comparison of corner rearings among 10‐minute groups in 30‐minute làt‐maze test. Data were given as an average of the number of crossings or rearings with standard errors, where n = 5. **P < 0.01.

Figure 2

Comparison of galectin‐3 expression between ADHD and WKY rats. A. Galectin‐3 protein was analyzed by Western blot in prefrontal cortex (PFC), striatum (STR), midbrain (MB), cerebellum (CB), aorta (AO), liver (LV); B. Gray density analysis of the bands in A. The bands on X‐ray films was scanned and analyzed by the software Image J (Wayne Rasband, National Institute of Health, USA). Data were given as an average of relative density with standard errors, where n = 5. **P < 0.01; C. Immunostaining of the PFC slice was with galectin‐3 antibody, and cells were viewed in 400× magnifications. PFC slices of the brain were incubated with rabbit anti‐galectin‐3 antibody, HRP‐conjugated goat anti‐rabbit immunoglobulin and DAB sequentially, and counterstained with methyl green. The image was the representative of three independent experiments. D. Analysis of galectin‐3 mRNA level in PFC, STR and MB by RT–PCR. Abbreviations: ADHD = attention deficit hyperactivity disorder; DAB = diaminobenzidine tetrahydrochloride; HRP = horseradish peroxidase; RT–PCR = reverse transcriptase–polymerase chain reaction; SHR(S) = spontaneously hypertensive rats; WKY(W) = Wistar–Kyoto rats.

Figure 3

Rno‐let‐7d was upregulated in SHR prefrontal cortex. A. Foldchanges of all differentially expressed micro RNAs in the microarray results of the prefrontal cortex in SHR and WKY rats. B. Schematic representation of the interaction between rno‐let‐7d seeding sequence and the binding site on the wild‐type Lgals3‐3′UTR. Abbreviations: CDS = Coding Sequence; SHR = spontaneously hypertensive rat; UTR = untranslated region; Wt = wild type; WKY = Wistar–Kyoto rats.

Figure 4

Rno‐let‐7d regulation of galectin‐3 expression. A. Validity of rno‐let‐7d expression plasmid was confirmed by PCR in both CBRH‐7919 and PC12L cells 48 h after transfection. The result was similar in two cells and the figure was the representative of three independent experiments. Reaction without reverse transcriptase (RT‐) was used as the negative control. The expression rno‐let‐7d was also confirmed by real‐time PCR (right panel). B. Luciferase activity analysis in CBRH‐7919 cells after co‐transfection of rno‐let‐7d precursor with luciferase report plasmid. The plasmid containing scramble sequence was as a control. Data were shown as an average of relative luciferase units which were normalized to the control, with standard errors (**P < 0.01), where n = 6. C. Schematic representation of the interaction between rno‐let‐7d seeding sequence and the binding site on the mutant‐type Lgals3‐3′UTR (C→G). Abbreviations: Mut = mutant type, the report plasmid included the mutant‐type Lgals3‐3′UTR; PCR = polymerase chain reaction; UTR = untranslated region; Wt = wild type, the report plasmid included the wild‐type Lgals3‐3′UTR.

Figure 5

Galectin‐3, TH and rno‐let‐7d expressions in PC12 cells. A. Galectin‐3 and TH protein levels were detected by Western blot. Galectin‐3 and TH expressions were analyzed in both PC12L and PC12H cells at 48 h after passage, which showed that the expressions were higher in PC12L cells than in PC12H cells. Galectin‐3 and TH expressions in PC12L cells were then investigated at 6 days after incubation with 1 µmol/L RA. B. Galectin‐3 and TH mRNA levels were simultaneously detected by RT–PCR. C. Immunofluorescence staining of galectin‐3 in PC12L and PC12H cells, in 200× magnifications. D. Mature rno‐let‐7d was detected in PC12L and PC12H cells. Reactions without reverse transcriptase (RT‐) were as a negative control. E. Mature rno‐let‐7d was detected by either stem‐loop RT‐PCR (left panel) or real‐time PCR (right panel) in PC12L cells before and after 48 h incubation with 1 µmol/L RA. Reaction without reverse transcriptase (RT‐) was used as a negative control. The results were the representative of three independent experiments. F. In PC12L cells and CBRH‐7919 cells, galectin‐3 protein expression was reduced after transient transfection with rno‐let‐7d precursor. G. Density analysis of Western blot results in F. Density of the bands on X‐ray films was scanned, quantified by Image J (Wayne Rasband, National Institute of Health, USA). Data were shown as an average of relative density with standard errors (**P < 0.01), where n = 5. H. Immunohistochemistry observation of TH in PC12L and PC12H cells. Magnification: 200×. Abbreviations: DAPI = 4′,6‐diamidino‐2‐phenylindole; PC12L = low differentiated pheochromocytoma cells; PC12H = high differentiated pheochromocytoma PC12 cells; RA =  retinoic acid; RT–PCR = reverse transcriptase–polymerase chain reaction; TH = tyrosine hydroxylase.

Figure 6

Observation of TH expression after rno‐let‐7d transfection. A. In PC12L cells, TH expression was detected by Western blot 72 h after transient transfection with rno‐let‐7d precursor. B. The band density in A was analyzed by the software Image J (Wayne Rasband, National Institute of Health, USA). Data were shown as an average of relative density with standard errors, where n = 3. (**P < 0.01). C. TH mRNA levels were detected by RT‐PCR in PC12L cells transfected with rno‐let‐7d precursor. D. Relative luciferase activity in CBRH‐7919 and PC12L cells was shown 48 h after co‐transfection with rno‐let‐7d precursor and report plasmids. The results were representative of three independent experiments. Abbreviations: PC12L = low differentiated pheochromocytoma cells; RA = retinoic acid; RT–PCR = reverse transcriptase–polymerase chain reaction; TH = tyrosine hydroxylase.

Figure 7

Association of galectin‐3 expression with TH regulation. A. Galectin‐3 and TH protein levels in PC12H cells were detected by Western blot 48 h after incubation with 100 ng/mL NGF. B. Galectin‐3 and TH mRNA levels were observed at the same time, indicating that their change was in a same tendency as the protein level. C. Galectin‐3 and TH protein levels were compared after 48 h transfection with galectin‐3 expression plasmid or interference plasmid. D. Galectin‐3 and TH mRNA levels were detected at the same time with (C). E. CREB transcription factors were detected by real‐time PCR after galectin‐3 and rno‐let‐7d transfection. Abbreviations: CREB = cAMP response element binding; NGF = nerve growth factor; PCR = polymerase chain reaction; TH = tyrosine hydroxylase.