Identification of MicroRNAs Regulating Reprogramming Factor LIN28 in Embryonic Stem Cells and Cancer Cells

Abstract

LIN28 (a homologue of the Caenorhabditis elegans lin-28 gene) is an evolutionarily conserved RNA-binding protein and a master regulator controlling the pluripotency of embryonic stem cells. Together with OCT4, SOX2, and NANOG, LIN28 can reprogram somatic cells, producing induced pluripotent stem cells. Expression of LIN28 is highly restricted to embryonic stem cells and developing tissues. In human tumors, LIN28 is up-regulated and functions as an oncogene promoting malignant transformation and tumor progression. However, the mechanisms of transcriptional and post-transcriptional regulation of LIN28 are still largely unknown. To examine microRNAs (miRNAs) that repress LIN28 expression, a combined in silico prediction and miRNA library screening approach was used in the present study. Four miRNAs directly regulating LIN28 (let-7, mir-125, mir-9, and mir-30) were initially identified by this approach and further validated by quantitative RT-PCR, Western blot analysis, and a LIN28 3-UTR reporter assay. We found that expression levels of these four miRNAs were clustered together and inversely correlated with LIN28 expression during embryonic stem cell differentiation. In addition, the expression of these miRNAs was remarkably lower in LIN28-positive tumor cells compared with LIN28-negative tumor cells. Importantly, we demonstrated that these miRNAs were able to regulate the expression and activity of let-7, mediated by LIN28. Taken together, our studies demonstrate that miRNAs let-7, mir-125, mir-9, and mir-30 directly repress LIN28 expression in embryonic stem and cancer cells. Global down-regulation of these miRNAs may be one of the mechanisms of LIN28 reactivation in human cancers.LIN28 is an evolutionarily conserved RNA-binding protein with two RNA-binding domains (a cold shock domain and retroviral type CCHC zinc finger motif), which was first characterized as a critical regulator of developmental timing in Caenorhabditis elegans (1, 2). The mammalian homologs of the C. elegans lin-28 gene (LIN28 and LIN28B) are important in processes such as embryogenesis (3), skeletal myogenesis (4), germ cell development (5, 6), and neurogliogenesis (7, 8). Genome-wide association studies have implicated the LIN28B locus in controlling both height and the timing of menarche in humans (9 -13). This finding has been successfully phenocopied in a transgenic mouse model that expresses inducible LIN28 (14). Increasing evidence suggests that LIN28 may also be a master regulator controlling the pluripotency of ES cells (15)(16)(17)(18). LIN28, together with OCT4, SOX2, and NANOG (the "reprogramming factors"), can reprogram somatic cells to induced pluripotent stem cells (19). Several reports have demonstrated that LIN28 binds to mRNAs, regulating their stability and translation (4,16,17). In addition, LIN28 can bind to the terminal loops of the precursor of the miRNA let-7, thereby blocking the processing of let-7 into its mature form (7, 8, 20 -26). Importantly, expression of LIN28 is highly restricted to ES 2 cells as well as dev...

Lin-28 Homologue A (LIN28A) Promotes Cell Cycle Progression via Regulation of Cyclin-dependent Kinase 2 (CDK2), Cyclin D1 (CCND1), and Cell Division Cycle 25 Homolog A (CDC25A) Expression in Cancer

“…Studies have excluded the possibility that LIN28A overexpression in tumors is due to an alteration of the LIN28A DNA copy number (42). Based on recent studies (42,67), there are at least two mechanisms that can lead to the repression of LIN28A expression in adult tissues that may be partially lost in human tumors: epigenetic silencing (42) and miRNA suppression (67). In fact, we have previously identified four LIN28A regulatory miRNAs that were globally down-regulated in human tumors (67).…”

“…Based on recent studies (42,67), there are at least two mechanisms that can lead to the repression of LIN28A expression in adult tissues that may be partially lost in human tumors: epigenetic silencing (42) and miRNA suppression (67). In fact, we have previously identified four LIN28A regulatory miRNAs that were globally down-regulated in human tumors (67). In addition, overexpression of LIN28 activators, such as the Myc oncogene (47,54,68), may serve as another mechanism to deregulate LIN28A expression in epithelial tumors.…”

Conserved microRNA pathway regulates developmental timing of retinal neurogenesis

“…Several previous studies have shown that Lin28b is a target of the three LP-miRNAs (29,30); however, no study to date has analyzed regulation of Prtg by miRNAs. Therefore, to directly test whether Prtg is a direct target of miR-125, miR-9, and let-7, we used a plasmid containing the 3′ UTR of Prtg cloned downstream of a luciferase reporter.…”

MicroRNA-30c promotes human adipocyte differentiation and co-repressesPAI-1andALK2

“…In line with this, studies have either proposed or validated functions of miR-30 family members in numerous types of cancer, [40][41][42][43][44][45][46] but also in several other biological contexts such as myocardial matrix remodelling, 47 apoptosis 48 and kidney Figure 5. Analysis of miR-30c, ALK2 and PAI-1 expression in white adipose tissue of murine obesity models.…”