Post-transcriptional regulation of IGF1R by key microRNAs in long-lived mutant mice - PubMed (original) (raw)

Post-transcriptional regulation of IGF1R by key microRNAs in long-lived mutant mice

Ruqiang Liang et al. Aging Cell. 2011 Dec.

Abstract

Long-lived mutant mice, both Ames dwarf and growth hormone receptor gene-disrupted or knockout strains, exhibit heightened cognitive robustness and altered IGF1 signaling in the brain. Here, we report, in both these long-lived mice, that three up-regulated lead microRNAs, miR-470, miR-669b, and miR-681, are involved in posttranscriptional regulation of genes pertinent to growth hormone/IGF1 signaling. All three are most prominently localized in the hippocampus and correspond to reduced expression of key IGF1 signaling genes: IGF1, IGF1R, and PI3 kinase. The decline in these genes' expression translates into decreased phosphorylation of downstream molecules AKT and FoxO3a. Cultures transfected with either miR-470, miR-669b, or miR-681 show repressed endogenous expression of all three genes of the IGF1 signaling axis, most significantly IGF1R, while other similarly up-regulated microRNAs, including let-7g and miR-509, do not induce the same levels of repression. Transduction study in IGF1-responsive cell cultures shows significantly reduced IGF1R expression, and AKT to some extent, most notably by miR-681. This is accompanied by decreased levels of downstream phosphorylated forms of AKT and FoxO3a upon IGF1 stimulation. Suppression of IGF1R by the three microRNAs is further validated by IGF1R 3'UTR reporter assays. Taken together, our results suggest that miR-470, miR-669b, and miR-681 are all functionally able to suppress IGF1R and AKT, two upstream genes controlling FoxO3a phosphorylation status. Their up-regulation in growth hormone signaling-deficient mutant mouse brain suggests reduced IGF1 signaling at the posttranscriptional level, for numerous gains of neuronal function in these long-lived mice.

© 2011 University of Louisville. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

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Figures

Fig. 1

Fig. 1. Expression of key miRNAs in Ames dwarf and littermate wild-type mouse brain

A graphical representation of expression levels using qualitative PCR of key miRNAs identified by MM Chips, and respective fold change values within the same age groups among Ames dwarf and littermate controls. D2, D24, D33, C2 and C24: D represents dwarf and C represents littermate WT control mice ages 2, 24 and 33 months respectively. Panel (A) shows key up-regulated miRNAs identified by MM Chips, and panel (B) shows key down-regulated miRNAs. (*p <0.01, **p < 0.0001; all histograms represent average ± std. dev.; n = 3, three samples from each age group of each genotype.)

Fig. 2

Fig. 2. In situ detection of lead miRNAs in Ames dwarf and GHRKO mouse brains

In situ hybridization (ISH) detection of miRNAs (miR-470, −669b and −681) in brain tissues from Ames dwarf mice (D) at 2, 24, and 33 months, depicted as D2, D24, and D33 respectively, and littermate controls (C) at 2 and 24 months, labeled as C2 and C24 age groups (A, B, C). (D) Increased hybridization signal of these miRNAs in Ames dwarf mice across all age groups, as compared to WT littermates, may be observed. (*p < 0.01, **p < 0.0001; all histograms represent average ± std. dev.; n = 3, three samples from each age group of each genotype.) (E) In situ hybridization (ISH) detection of these three miRNAs in brain tissues from 2 month old GHRKO mice and littermate controls. (F) A graphical representation of densitometric analysis of their expression in cortex and hippocampus is shown. (*p < 0.01, **p < 0.0001; all histograms represent average ± std. dev.; n = 3, three samples from each age group of each genotype.)

Fig. 3

Fig. 3. Altered GH signaling in Ames dwarf and GHRKO mouse brains

In Ames dwarf mouse brain tissues (A), age-dependent decreased levels of IGF1 (14 kDa), IGF1R (95 kDa beta subunit), PI3K(85kDa), pAKT(T308) and pFoxO3a (S253) are observed, compared to littermate WT control mice, but not in total pan-AKT (56 kDa) and FoxO3a (detected as a 100 kDa band). Similar decrease of these proteins in 2 month old GHRKO mouse brain tissues are observed compared to littermate WT controls (B), while total pan-AKT and total FoxO3a are not significantly changed. (C) A graphical representation of densitometric analysis of Western blots of Ames dwarf mouse brain. (*p < 0.01, **p < 0.0001; all histograms represent average ± std. dev.; n = 3, three samples from each age group of each genotype.) The age groups used are Ames dwarf at 2, 24, and 33 months, with D2, D24, and D33 labels, and the wild type control at 2 and 24 months, represented as C2 and C24 respectively. (D) A graphical representation of densitometric analysis of Western blots of GHRKO mouse brain. (*p < 0.01, **p < 0.0001; all histograms represent average ± std. dev.; n = 3, three samples from each age group of each genotype.)

Fig. 4

Fig. 4. MiRNA-dependent altered expression of key genes of GH signaling

(A) Western blot analysis of total cell proteins at different time points after serum-deprived WI-38 cells are stimulated by IGF1. As a result of IGF1 stimulation, maximum phosphorylated Akt is seen at 90 minutes. Graphical representation of densitometric data showing the effect of IGF1 signaling at different time points in the form of a histogram is shown with normalized values. β-actin was used as an internal control. (B) Western blot analysis of miRNA (miR-470, −669b and −681) suppression of endogenous expression of GH/IGF axis genes in serum-starved and IGF1-stimulated WI-38 cells. Significant repression of IGF1R (represented by the 95 kDa beta subunit), pAKT (T308) and pFoxO3a (S253) by three key miRNAs (miR-470, −669b, and −681) is observed. No significant change in total AKt expression was observed except in miR-681 transduced cells. No significant change in total FoxO3a expression was observed. Graphical representation of densitometric data in the form of a histogram is shown with normalized values. β-actin was used as an internal control. (*p < 0.01, **p < 0.0001; all histograms represent average ± std. dev.; n = 3, three samples from each of three experiments.)

Fig. 5

Fig. 5. MiRNA-induced repression of IGF1R 3′UTR in HEK-293 cells

(A) HEK-293 cells co-transfected with one of three key mouse miRNAs (miR-470, −669b, or −681) and IGF1R 3′UTR reporter, showing that the 3′UTR of IGF1R is repressed by the transfected miRNAs, but not by scrambled control and IGF1R 3′UTR co-transfected cells. This indicates that these miRNAs suppress the target (red fluorescence) protein through the 3′UTR of IGF1R. This effect is absent when a plasmid carrying a scrambled sequence is used (indicated with arrows). (B) A graphical representation of densitometric analysis of color intensity is shown. (*p < 0.01, **p < 0.0001; all histograms represent average ± std. dev.; n = 3, three samples from each of three experiments.)

Fig. 6

Fig. 6. MiRNA-mediated altered overlapping somatotropic signaling in long-lived mice

This schematic diagram shows the common miRNA-based regulation in both long-lived mouse models (Ames dwarf and GHRKO mice). It depicts the role of three key miRNAs (miR-470, −669b, and −681) targeting a key gene, IGF1R, of the GH/IGF1 axis, and altering GH signaling, thus contributing to longevity.

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