Atherosclerosis-associated endothelial cell apoptosis by miRNA let7-b-mediated downregulation of HAS-2

MicroRNAs (miRNAs) play essential roles in the regulation and pathophysiology of various types of human diseases including atherosclerosis. Increasing numbers of miRNAs have been identified to be important regulators in the progression of atherosclerosis by regulating gene expression. However, functional miRNAs and the underlying mechanisms involved in atherosclerosis need fully elucidation. In the present study, the function of miRNA let-7b was investigated in human aortic endothelial cells (HAECs). The results showed that downregulation of let-7b in the high-fat diet mice and HAECs was inversely correlated with the expression level of HAS-2. upregulation of let-7b significantly reduced apoptosis of HAECs. The results also revealed that HAS-2 was a target gene of let-7b and HAS-2 reduction reversed the antiapoptotic effect of let-7b through regulation of the P13K/Akt pathway. These results together suggest the potential of regulating the let-7b expression and endothelial apoptosis against development and progression of atherosclerosis.     

Cellular microRNA let-7c inhibits M1 protein expression of the H1N1 influenza A virus in infected human lung epithelial cells

The influenza virus (IV) triggers a series of signalling events inside host cells and induces complex cellular responses. Studies have suggested that host factors play an essential role in IV replication. MicroRNAs (miRNAs) represent a class of small non-coding RNAs that target mRNAs, triggering either translation repression or RNA degradation. Emerging research suggests that host-derived cellular miRNAs are involved in mediating the host-IV interaction. Using miRNA microarrays, we identified several miRNAs aberrantly expressed in IV-infected human lung epithelial cells (A549). Specifically, miR-let-7c was highly up-regulated in IV-infected A549 cells. PITA and miRanda database screening indicated that the let-7c seed sequence is a perfect complementary sequence match to the 3' untranslated region (UTR) of viral gene M1 (+) cRNA, but not to PB2 and PA. As detected by a luciferase reporter system, let-7c directly targeted the 3'-UTR of M1 (+) cRNA, but not PB2 and PA. To experimentally identify the function of cellular let-7c, precursor let-7c was transfected into A549 cells. Let-7c down-regulated IV M1 expression at both the (+) cRNA and protein levels. Furthermore, transfection with a let-7c inhibitor enhanced the expression of M1. Therefore, let-7c may reduce IV replication by degrading M1 (+) cRNA. This is the first report indicating that cellular miRNA regulates IV replication through the degradation of viral gene (+) cRNA by matching the 3'-UTR of the viral cRNA. These findings suggest that let-7c plays a role in protecting host cells from the virus in addition to its known cellular functions.     

Systematic gene regulation involving miRNAs during neuronal differentiation of mouse P19 embryonic carcinoma cell

MicroRNAs (miRNAs) are small noncoding RNA and play an essential role in gene regulation. In this study, we investigated regulation of gene expression during neuronal differentiation of mouse P19 embryonic carcinoma cells and described a systematic pathway of gene regulation involving miRNAs. In the pathway, downregulation of Lin28 involved in blocking the let-7 maturation and upregulation of let-7 occur following induction of the differentiation, thereby triggering suppression of the downstream High Mobility Group A2 (Hmga2) gene expression via activation of gene silencing mediated by let-7. Our data further suggest that miR-9, as well as miR-125b, participate in the reduction of the Lin28 expression. The gene regulation involving miRNAs likely contributes to a rapid and programmed change in gene expression in neuronal differentiation of P19 cells.     

A whole-genome RNAi Screen for C. elegans miRNA pathway genes

BACKGROUND: miRNAs are an abundant class of small, endogenous regulatory RNAs. Although it is now appreciated that miRNAs are involved in a broad range of biological processes, relatively little is known about the actual mechanism by which miRNAs downregulate target gene expression. An exploration of which protein cofactors are necessary for a miRNA to downregulate a target gene should reveal more fully the molecular mechanisms by which miRNAs are processed, trafficked, and regulate their target genes. RESULTS: A weak allele of the C. elegans miRNA gene let-7 was used as a sensitized genetic background for a whole-genome RNAi screen to detect miRNA pathway genes, and 213 candidate miRNA pathway genes were identified. About 2/3 of the 61 candidates with the strongest phenotype were validated through genetic tests examining the dependence of the let-7 phenotype on target genes known to function in the let-7 pathway. Biochemical tests for let-7 miRNA production place the function of nearly all of these new miRNA pathway genes downstream of let-7 expression and processing. By monitoring the downregulation of the protein product of the lin-14 mRNA, which is the target of the lin-4 miRNA, we have identified 19 general miRNA pathway genes. CONCLUSIONS: The 213 candidate miRNA pathway genes identified could act at steps that produce and traffic miRNAs or in downstream steps that detect miRNA::mRNA duplexes to regulate mRNA translation. The 19 validated general miRNA pathway genes are good candidates for genes that may define protein cofactors for sorting or targeting miRNA::mRNA duplexes, or for recognizing the miRNA base-paired to the target mRNA to downregulate translation.     

Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation

MicroRNAs (miRNAs) are approximately 22 nucleotide RNAs that negatively regulate the expression of protein-coding genes. In a present model of miRNA function in animals, miRNAs that form imperfect duplexes with their targets inhibit protein expression without affecting mRNA levels. Here, we report that in C. elegans, regulation by the let-7 miRNA results in degradation of its lin-41 target mRNA, despite the fact that its 3'UTR regulatory sequences can only partially base-pair with the miRNA. Furthermore, lin-14 and lin-28 are targets of the lin-4 miRNA, and we show that the mRNA levels for these protein-coding genes significantly decrease in response to lin-4 expression. This study reveals that mRNAs containing partial miRNA complementary sites can be targeted for degradation in vivo, raising the possibility that regulation at the level of mRNA stability may be more common than previously appreciated for the miRNA pathway.     

Mammalian DIS3L2 exoribonuclease targets the uridylated precursors of let-7 miRNAs

The mechanisms of gene expression regulation by miRNAs have been extensively studied. However, the regulation of miRNA function and decay has long remained enigmatic. Only recently, 3′ uridylation via LIN28A-TUT4/7 has been recognized as an essential component controlling the biogenesis of let-7 miRNAs in stem cells. Although uridylation has been generally implicated in miRNA degradation, the nuclease responsible has remained unknown. Here, we identify the Perlman syndrome-associated protein DIS3L2 as an oligo(U)-binding and processing exoribonuclease that specifically targets uridylated pre-let-7 in vivo. This study establishes DIS3L2 as the missing component of the LIN28-TUT4/7-DIS3L2 pathway required for the repression of let-7 in pluripotent cells.     

let-7 microRNAs induce tamoxifen sensitivity by downregulation of estrogen receptor α signaling in breast cancer

MicroRNAs (miRNAs) play an important regulatory role in breast tumorigenesis. Previously, we found that let-7 miRNAs were downregulated significantly in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. In this study, we further found that endogenous levels of let-7b and let-7i miRNAs are inversely correlated with levels of estrogen receptor (ER)-a36, a new variant of ER-α66, in the FFPE tissue set. Bioinformatic analysis suggested that ER-α36 may be another target of let-7 miRNAs. To test this hypothesis, cotransfection of let-7 mimics or inhibitors together with full-length or a fragment of ER-α36 3'UTR luciferase construct was performed, and we found that let-7b and let-7i mimics suppressed the activity of reporter gene significantly, which was enhanced remarkably by let-7b and let-7i inhibitors. Both mRNA and protein expression of ER-α36 were inhibited by let-7 mimics and enhanced by let-7 inhibitors. Furthermore, ER-α36 mediated nongenomic MAPK and Akt pathways were weakened by let-7b and let-7i mimics in triple negative breast cancer cell line MDA-MB-231. The reverse correlation between let-7 miRNAs and ER-α36 also exists in Tamoxifen (Tam)-resistant MCF7 cell line. Transfection of let-7 mimics to Tam-resistant MCF7 cells downregulated ER-α36 expression and enhanced the sensitivity of MCF7 cells to Tam in estrogen-free medium, which could be restored by overexpression of ER-α36 constructs without 3'UTR. Our results suggested a novel regulatory mechanism of let-7 miRNAs on ER-α36 mediated nongenomic estrogen signal pathways and Tam resistance.     

Tethering of human Ago proteins to mRNA mimics the miRNA-mediated repression of protein synthesis

MicroRNAs (miRNAs) are approximately 21-nt-long RNAs involved in regulating development, differentiation, and other processes in eukaryotes. In metazoa, nearly all miRNAs control gene expression by imperfectly base-pairing with the 3'-untranslated region (3'-UTR) of target mRNAs and repressing protein synthesis by an unknown mechanism. It is also unknown whether miRNA-mRNA duplexes containing mismatches and bulges provide specific features that are recognized by factors mediating the repression. miRNAs form part of ribonucleoprotein complexes, miRNPs, that contain Argonaute (Ago) and other proteins. Here we demonstrate that effects of miRNAs on translation can be mimicked in human HeLa cells by the miRNA-independent tethering of Ago proteins to the 3'-UTR of a reporter mRNA. Inhibition of protein synthesis occurred without a change in the reporter mRNA level and was dependent on the number, but not the position, of the hairpins tethering hAgo2 to the 3'-UTR. These findings indicate that a primary function of miRNAs is to guide their associated proteins to the mRNA.