let-7 Overexpression Leads to an Increased Fraction of Cells in G2/M, Direct Down-regulation of Cdc34, and Stabilization of Wee1 Kinase in Primary Fibroblasts

Abstract

microRNAs play a critically important role in a wide array of biological processes including those implicated in cancer, neurodegenerative and metabolic disorders, and viral infection. Although we have begun to understand microRNA biogenesis and function, experimental demonstration of their functional effects and the molecular mechanisms by which they function remains a challenge. Members of the let-7/miR-98 family play a critical role in cell cycle control with respect to differentiation and tumorigenesis. In this study, we show that exogenous addition of pre-let-7 in primary human fibroblasts results in a decrease in cell number and an increased fraction of cells in the G 2 /M cell cycle phase. Combining microarray techniques with DNA sequence analysis to identify potential let-7 targets, we discovered 838 genes with a let-7 binding site in their 3-untranslated region that were down-regulated upon overexpression of let-7b. Among these genes is cdc34, the ubiquitin-conjugating enzyme of the Skp1/cullin/F-box (SCF) complex. Cdc34 protein levels are strongly down-regulated by let-7 overexpression. Reporter assays demonstrated direct regulation of the cdc34 3-untranslated region by let-7. We hypothesized that low Cdc34 levels would result in decreased SCF activity, stabilization of the SCF target Wee1, and G 2 /M accumulation. Consistent with this hypothesis, small interfering RNA-mediated down-regulation of Wee1 reversed the G 2 /M phenotype induced by let-7 overexpression. We conclude that Cdc34 is a functional target of let-7 and that let-7 induces down-regulation of Cdc34, stabilization of the Wee1 kinase, and an increased fraction of cells in G 2 /M in primary fibroblasts. miRNAs 2 are non-coding, single-stranded, conserved RNAs of ϳ22 nucleotides that function as gene regulators (1). miRNAs have emerged as central post-transcriptional negative regulators and have been implicated in a wide array of biological processes including cell cycle control. In metazoans, individual miRNAs can down-regulate hundreds of mRNA targets by interacting with partially complementary sequences within their 3Ј-untranslated region (3Ј-UTR) (2, 3).The let-7 miRNA was originally discovered in Caenorhabditis elegans as a switch gene induced as cells exit the cell cycle when C. elegans reach their adult stage (4). In humans and mouse, like C. elegans, the expression of let-7 is barely detectable in embryonic developmental stages but increases after differentiation and in mature tissue (5). let-7 family members have been implicated as tumor suppressors. Some of the 12 members of the let-7 family map onto genomic regions altered or deleted in human tumors (6, 7). Further, members of the let-7 family of miRNAs are consistently down-regulated in lung and colon cancer (8 -10). In lung cancers, low levels of let-7 correlated with shorter survival after resection (9). Decreased let-7 levels in tumors are associated with elevated levels of Ras, which contains several let-7 binding sites within its 3Ј-UTR (8). let-7 expression is reduced in m...

Let-7b and microRNA-199a inhibit the proliferation of B16F10 melanoma cells

“…In vitro overexpression of let-7b in melanoma cells downregulated the expression of cyclins D1, D3, A and cyclin-dependent kinase (Cdk) 4 (21). Let-7b overexpression leads to an increased fraction of cells in G2/M, direct downregulation of Cdc34, and stabilization of Wee-1 kinase in primary human fibroblasts (22). microRNAs may also desensitize stem cells to external signals, leading to cell cycle arrest in G1/S transition (23,24).…”

MicroRNA dysregulation in gastric cancer: a new player enters the game

“…The expression of Wee1 is regulated by the Skp1/cullin/F-box complex that polyubiquitinates Wee1 for proteasome-dependent degradation (Watanabe et al, 2004). let-7, an miRNA frequently downregulated in gastric cancer (Zhang et al, 2007a), has been shown to target CDC34, the ubiquitinconjugating enzyme of the Skp1/cullin/F-box complex in primary fibroblasts, and thereby promoting the half-life and expression of Wee1 protein (Legesse-Miller et al, 2009). Whether a similar regulatory mechanism exists in gastric cancer cells, however, requires further elucidation.…”

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

“…It has been reported that LIN28A can bind to the terminal loops of the let-7 miRNA precursor, thereby blocking the processing of let-7 into its mature form (10, 11, 30 -39). In addition, several let-7 target genes, such as CCND1 (64), CCND2 (65), CCND3 (65), CCNA2 (64), CDK4 (64,65), CDC25A (65), and CDC34 (66), have been shown to function as regulators of the cell cycle. Therefore, we explored whether LIN28A could regulate the cell cycle in a let-7-dependent manner.…”