Principal Investigator Harvey Lodish
Minh Le, a former graduate student working jointly with our lab and that of Bing Lim in the Genome Institute of Singapore, has elucidated the role of miR-125b in neurogenesis. miR-125 is a homolog of lin-4, which is important for developmental timing in C. elegans. The expression of miR-125b is upregulated during embryogenesis and enriched in the nervous system of vertebrate species. Minh, together with Huangming Xie, Beiyan Zhou, and Moon Um in my lab, first obtained the expression profile of microRNAs during neuronal differentiation of the human neuroblastoma cell line SH-SY5Y; six microRNAs were significantly upregulated during differentiation induced by all-trans-retinoic acid and brain-derived neurotrophic factor. She then demonstrated that ectopic expression of either miR-124a or miR-125b increased the percentage of differentiated SH-SY5Y cells with neurite outgrowth. miR-125b is also upregulated during differentiation of human neural progenitor ReNcell VM cells, and Minh showed that miR-125b ectopic expression significantly promoted neurite outgrowth of these cells. To identify the targets of miR-125b regulation, Minh profiled the global changes in gene expression following miR-125b ectopic expression in SH-SY5Y cells. More than 50% of the downregulated mRNAs contain the seed match sequence of miR-125b; transcripts with stronger seed matches were repressed to a greater extent. Importantly, TargetScan 5.1 predicted 188 of the downregulated transcripts to be direct targets of miR-125b. Pathway analysis suggests that a subset of miR-125b-repressed targets antagonize neuronal genes in several neurogenic pathways, thereby mediating the positive effect of miR-125b on neuronal differentiation. Minh further confirmed the binding of miR-125b to the microRNA response elements of nine selected mRNA targets and validated the binding specificity for three targets. Together, these data reveal for the first time the important role of miR-125b in human neuronal differentiation.
Furthermore, Minh, together with Shyh-Chang Ng and Cathleen The, demonstrated that miR-125b is indispensable for zebra fish embryogenesis, particularly for the survival of neural cells during development. She identified p53, a key tumor suppressor, as a bona fide target of miR-125b in both zebra fish and humans. miR-125b-mediated downregulation of p53 is strictly dependent on the binding of miR-125b to a microRNA-response element in the 3’ UTR of p53 mRNA. Overexpression of miR-125b represses the endogenous level of p53 protein and suppresses apoptosis in human neuroblastoma cells and human lung fibroblast cells. By contrast, knockdown of miR-125b elevates the level of p53 protein and induces apoptosis in human lung fibroblasts and in the zebra fish brain. In zebra fish this phenotype can be rescued significantly by either ablation of endogenous p53 function or by ectopic expression of miR-125b. Interestingly, miR-125b is downregulated when zebra fish embryos are treated with gamma-irradiation or camptothecin, corresponding to the rapid increase in p53 protein in response to DNA damage. Ectopic expression of miR-125b suppresses both the increase of p53 and stress-induced apoptosis.
Minh and Shyh-Chang then used both gain- and loss-of-function screens for miR-125b targets in humans, mice and zebrafish, and validated these targets with the luciferase assay and a novel miRNA pull-down assay. They demonstrated that miR-125b directly represses 20 novel targets in the p53 network. These targets include both apoptosis regulators like Bak1, Igfbp3, Itch, Puma, Prkra, Tp53inp1, Tp53, Zac1,and also cell-cycle regulators like cyclin C, Cdc25c, Cdkn2c, Edn1, Ppp1ca, Sel1l,in the p53 network. They found that although each miRNA-target pair was seldom conserved, miR-125b regulation of the p53 pathway is conserved at the network-level. Their results led us to propose that miR-125b buffers and fine-tunes p53 network activity by regulating the dose of both proliferative and apoptotic regulators, with implications for tissue stem cell homeostasis and oncogenesis.