Harshini Sarojini

Murine microRNAs implicated in liver functions and aging process.

Small non-coding microRNAs (miRNAs) are involved in gene regulation in various cellular and developmental processes, including mechanisms of aging. Here, the mouse liver was used as a paradigm for the study of miRNAs implicated in the aging process in mammals. Expression profiling of 367 murine miRNAs (Sanger Version 8.2) was assessed in livers from 4 to 33 months old mice, and their predicted targets were compared with proteomic profiling data generated from the same animals. Gradual increases in the levels of miR-669c and miR-709 were observed from mid-age of 18-33 months, while miR-93 and miR-214 were significantly up-regulated only in extremely old liver. In contrast, we did not identify any miRNAs showing significant down-regulation with age. Interestingly, the up-regulated miRNAs targets are associated with detoxification activity and regeneration capacity functions known to decline in old liver. In particular, three up-regulated miRNAs may contribute to the aging-related decline in oxidative defense by targeting various classes of glutathione S-transferases. Other proteins in decline in old liver and targeted by the up-regulated miRNAs are involved in mitochondrial functions or maintenance. Taken together, we identified the up-regulation of key miRNAs that may participate in the decline of regeneration and oxidative defense mechanisms in aging liver.

Increased expression of miR-34a and miR-93 in rat liver during aging, and their impact on the expression of Mgst1 and Sirt1

Age-dependent loss of oxidative defense is well recognized in rodent models, although the control mechanism is still obscure; a few studies have shown how microRNAs, a non-coding RNA species, regulate the expression of their target genes at the post-transcriptional level. In the current study, miR-34a and miR-93 are observed to increase in middle- and old-age rat liver, compared to young rats; the up-regulation of these two miRNAs is determined by qPCR through a grind-and-find approach, and histochemical in situ hybridization. Three commonly used miRNA target prediction programs suggest four candidate targets of miR-34a and miR-93: Sp1, Nrf2 (Nfe2l2), Sirt1 and Mgst1; their expression is found to be reduced inversely to the up-regulation of the two miRNAs by Western blotting of protein extracts, as well as immunofluorescence staining of intact liver tissues. Furthermore, the suppression of the four proteins by miR-34a/miR-93 is examined in HEK 293 cells by transfection and co-transfection; miR-34a represses all four proteins expression, whereas miR-93 affects only Sp1, Sirt1 and Mgst1. Taken together, our study suggests a model of post-transcriptional repression, not only of genes involved in oxidative stress regulation and oxidative stress defense proteins, such as Sirt1 and Mgst1, but also of upstream transcription factors (TFs) regulating their activation, since Sp1 is the TF for both Sirt1 and Mgst1, and Nrf2 is the TF of Mgst1. Thus, up-regulation of both miR-34a and miR-93 constitutes an inescapable repression of two vital oxidative defense genes, by targeting not only the targets, but also transcription factors controlling their activation, a double dampening regulation at the post-transcriptional level.

Stepwise up-regulation of MicroRNA expression levels from replicating to reversible and irreversible growth arrest states in WI-38 human fibroblasts

MicroRNAs (miRNAs) are small non‐coding RNAs that regulate diverse genetic expression networks through their control of mRNA stability or translation. Their role in aging mechanisms has been proposed in various model systems. In this report, the expression profiling of 462 human miRNAs in the reversible growth arrest state of quiescence, and irreversible states of replicative senescence and hydrogen peroxide‐induced premature senescence, are compared to young replicating lung fibroblasts. Greater numbers of up‐regulated than down‐regulated miRNAs are observed when cells stop proliferating, particularly in premature senescence, somewhat less in replicative senescence, and less still in quiescence. Several altered miRNA expressions are shared by the three growth arrest states, including the up‐regulation of miR‐34a, ‐624, ‐638 and miR‐377, and the down‐regulation of miR‐365 and miR‐512‐5p. miRNAs up‐regulated in both permanent growth arrest states but not in quiescence include let‐7g, miR‐26a, ‐136, ‐144, ‐195 and miR‐200b. In each of the growth arrest states, miR‐34a and let‐7f have the most robust up‐regulation in H2O2‐induced premature senescence, followed by miR‐638 and miR‐663 in replicative senescence, and finally, miR‐331‐3p and miR‐595 in quiescence. Our comprehensive evaluation of miRNA target correlations with known biomarkers for replicative senescence suggests that miRNAs may repress pathways controlling not only cell cycle traverse and proliferation, but also insulin‐like signaling, DNA repair and apoptosis, all of which are cellular functions deficient in senescent human fibroblasts. J. Cell. Physiol. 221: 109–119, 2009.

Secretome from mesenchymal stem cells induces angiogenesis via Cyr61.

It is well known that bone marrow‐derived mesenchymal stem cells (MSCs) are involved in wound healing and regeneration responses. In this study, we globally profiled the proteome of MSCs to investigate critical factor(s) that may promote wound healing. Cysteine‐rich protein 61 (Cyr61) was found to be abundantly present in MSCs. The presence of Cyr61 was confirmed by immunofluorescence staining and immunoblot analysis. Moreover, we showed that Cyr61 is present in the culture medium (secretome) of MSCs. The secretome of MSCs stimulates angiogenic response in vitro, and neovascularization in vivo. Depletion of Cyr61 completely abrogates the angiogenic‐inducing capability of the MSC secretome. Importantly, addition of recombinant Cyr61 polypeptides restores the angiogenic activity of Cyr61‐depleted secretome. Collectively, these data demonstrate that Cyr61 polypeptide in MSC secretome contributes to the angiogenesis‐promoting activity, a key event needed for regeneration and repair of injured tissues. J. Cell. Physiol. 219: 563–571, 2009.

Changes in MicroRNA expression patterns in human fibroblasts after low-LET radiation

Exposure to radiation provokes cellular responses controlled in part by gene expression networks. MicroRNAs (miRNAs) are small non‐coding RNAs which mostly regulate gene expression by degrading the messages or inhibiting translation. Here, we investigated changes in miRNA expression patterns after low (0.1 Gy) and high (2.0 Gy) doses of X‐ray in human fibroblasts. At early (0.5 h) and late (6 and 24 h) time points, irradiation caused qualitative and quantitative differences in the down‐regulation of miRNA levels, including miR‐92b, 137, 660, and 656. A transient up‐regulation of miRNAs was observed after 2 h post‐irradiation following high doses of radiation, including miR‐558 and 662. MicroRNA levels were inversely correlated with targets from mRNA and proteomic profiling after 2.0 Gy of radiation. MicroRNAs miR‐579, 608, 548‐3p, and 585 are noted for targeting genes involved in radioresponsive mechanisms, such as cell cycle checkpoint and apoptosis. We suggest here a model in which miRNAs may act as “hub” regulators of specific cellular responses, immediately down‐regulated so as to stimulate DNA repair mechanisms, followed by up‐regulation involved in suppressing apoptosis for cell survival. Taken together, miRNAs may mediate signaling pathways in sequential fashion in response to radiation, and may serve as biodosimetric markers of radiation exposure. J. Cell. Biochem. 105: 824–834, 2008.

MicroRNA regulation in Ames dwarf mouse liver may contribute to delayed aging

The Ames dwarf mouse is well known for its remarkable propensity to delay the onset of aging. Although significant advances have been made demonstrating that this aging phenotype results primarily from an endocrine imbalance, the post‐transcriptional regulation of gene expression and its impact on longevity remains to be explored. Towards this end, we present the first comprehensive study by microRNA (miRNA) microarray screening to identify dwarf‐specific lead miRNAs, and investigate their roles as pivotal molecular regulators directing the long‐lived phenotype. Mapping the signature miRNAs to the inversely expressed putative target genes, followed by in situ immunohistochemical staining and in vitro correlation assays, reveals that dwarf mice post‐transcriptionally regulate key proteins of intermediate metabolism, most importantly the biosynthetic pathway involving ornithine decarboxylase and spermidine synthase. Functional assays using 3′‐untranslated region reporter constructs in co‐transfection experiments confirm that miRNA‐27a indeed suppresses the expression of both of these proteins, marking them as probable targets of this miRNA in vivo. Moreover, the putative repressed action of this miRNA on ornithine decarboxylase is identified in dwarf mouse liver as early as 2u2003months of age. Taken together, our results show that among the altered aspects of intermediate metabolism detected in the dwarf mouse liver – glutathione metabolism, the urea cycle and polyamine biosynthesis – miRNA‐27a is a key post‐transcriptional control. Furthermore, compared to its normal siblings, the dwarf mouse exhibits a head start in regulating these pathways to control their normality, which may ultimately contribute to its extended healthspan and longevity.

PEDF from mouse mesenchymal stem cell secretome attracts fibroblasts

Conditioned medium (secretome) derived from an enriched stem cell culture stimulates chemotaxis of human fibroblasts. These cells are classified as multipotent murine mesenchymal stromal cells (mMSC) by immunochemical analysis of marker proteins. Proteomic analysis of mMSC secretome identifies nineteen secreted proteins, including extracellular matrix structural proteins, collagen processing enzymes, pigment epithelium‐derived factor (PEDF) and cystatin C. Immunodepletion and reconstitution experiments show that PEDF is the predominant fibroblast chemoattractant in the conditioned medium, and immunofluorescence microscopy shows strong staining for PEDF in the cytoplasm, at the cell surface, and in intercellular space between mMSCs. This stimulatory effect of PEDF on fibroblast chemotaxis is in contrast to the PEDF‐mediated inhibition of endothelial cell migration, reported previously. These differential functional effects of PEDF toward fibroblasts and endothelial cells may serve to program an ordered temporal sequence of scaffold building followed by angiogenesis during wound healing. J. Cell. Biochem. 104: 1793–1802, 2008.

Up-regulating sphingosine 1-phosphate receptor-2 signaling impairs chemotactic, wound-healing, and morphogenetic responses in senescent endothelial cells.

Vascular endothelial cells (ECs) have a finite lifespan when cultured in vitro and eventually enter an irreversible growth arrest state called “cellular senescence.” It has been shown that sphingolipids may be involved in senescence; however, the molecular links involved are poorly understood. In this study, we investigated the signaling and functions of sphingosine 1-phosphate (S1P), a serum-borne bioactive sphingolipid, in ECs of different in vitro ages. We observed that S1P-regulated responses are significantly inhibited and the S1P1-3 receptor subtypes are markedly increased in senescent ECs. Increased expression of S1P1 and S1P2 was also observed in the lesion regions of atherosclerotic endothelium, where senescent ECs have been identified in vivo. S1P-induced Akt and ERK1/2 activation were comparable between ECs of different in vitro ages; however, PTEN (phosphatase and tensin homolog deleted on chromosome 10) activity was significantly elevated and Rac activation was inhibited in senescent ECs. Rac activation and senescent-associated impairments were restored in senescent ECs by the expression of dominant-negative PTEN and by knocking down S1P2 receptors. Furthermore, the senescent-associated impairments were induced in young ECs by the expression of S1P2 to a level similar to that of in vitro senescence. These results indicate that the impairment of function in senescent ECs in culture is mediated by an increase in S1P signaling through S1P2-mediated activation of the lipid phosphatase PTEN.

Prosaposin in the secretome of marrow stroma-derived neural progenitor cells protects neural cells from apoptotic death

J. Neurochem. (2010) 112, 1527–1538.

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

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.