Regulation of the let-7a-3 Promoter by NF-κB

Abstract

Changes in microRNA expression have been linked to a wide array of pathological states. However, little is known about the regulation of microRNA expression. The let-7 microRNA is a tumor suppressor that inhibits cellular proliferation and promotes differentiation, and is frequently lost in tumors. We investigated the transcriptional regulation of two let-7 family members, let-7a-3 and let-7b, which form a microRNA cluster and are located 864 bp apart on chromosome 22q13.31. Previous reports present conflicting data on the role of the NF-κB transcription factor in regulating let-7. We cloned three fragments upstream of the let-7a-3/let-7b miRNA genomic region into a plasmid containing a luciferase reporter gene. Ectopic expression of subunits of NF-κB (p50 or p65/RelA) significantly increased luciferase activity in HeLa, 293, 293T and 3T3 cells, indicating that the let-7a-3/let-7b promoter is highly responsive to NF-κB. Mutation of a putative NF-κB binding site at bp −833 reduced basal promoter activity and decreased promoter activity in the presence of p50 or p65 overexpression. Mutation of a second putative binding site, at bp −947 also decreased promoter activity basally and in response to p65 induction, indicating that both sites contribute to NF-κB responsiveness. While the levels of the endogenous primary let-7a and let-7b transcript were induced in response to NF-κB overexpression in 293T cells, the levels of fully processed, mature let-7a and let-7b miRNAs did not increase. Instead, levels of Lin-28B, a protein that blocks let-7 maturation, were induced by NF-κB. Increased Lin-28B levels could contribute to the lack of an increase in mature let-7a and let-7b. Our results suggest that the final biological outcome of NF-κB activation on let-7 expression may vary depending upon the cellular context. We discuss our results in the context of NF-κB activity in repressing self-renewal and promoting differentiation.

miR-20b, miR-98, miR-125b-1*, and let-7e* as new potential diagnostic biomarkers in ulcerative colitis

“…Wu et al [36] found let-7f differentially expressed in the intestinal tissue of active UC patients when compared to controls, and Zahm et al [68] found higher concentrations of let-7b miRNA in sera of pediatric CD patients vs controls. It has been reported that there is a regulatory interaction between NF-κB activation and expression of let-7 members [69][70][71][72] , which suggests a significant importance for the pathogenesis of IBD. Let-7 miRNAs has also been demonstrated to be repressed in inflammation, which result in increased expression of pro-inflammatory cytokines and enhanced inflammatory responses [72] .…”

Whole-genome microRNA screening identifies let-7 and mir-18 as regulators of germ layer formation during early embryogenesis

“…In mice, 14 different genomic loci encode for different let-7 family members (Roush and Slack 2008), which allows for a very diverse repertoire of transcriptional regulation. Surprisingly, only a few reports have identified transcriptional regulators of let-7 family members' primary transcripts, including Myc, which negatively regulates let-7a, let-7d, and let-7g in human P493-6 cells (Chang et al 2008), and NF-kB, which activates let-7a-3 and let-7b (Wang et al 2012). However, Figure 7.…”

The Hippo Pathway Effectors TAZ/YAP Regulate Dicer Expression and MicroRNA Biogenesis through Let-7

“…Transcriptional Reporter Assays-HEK293 cells were transfected with a transcriptional reporter that drives firefly luciferase expression from either a 1-kb or a 1.5-kb fragment of the Let-7 promoter (13). A pCMV-␤-galactosidase plasmid was cotransfected in these cells, and the levels of luciferase activity were normalized against the levels of ␤-galactosidase activity, as we have previously done for other reporters (14).…”