Tumour-suppressor microRNAs let-7 and mir-101 target the proto-oncogene MYCN and inhibit cell proliferation in MYCN-amplified neuroblastoma

Abstract

Background:MicroRNAs (miRNAs) regulate expression of many cancer-related genes through posttranscriptional repression of their mRNAs. In this study we investigate the proto-oncogene MYCN as a target for miRNA regulation.Methods:A luciferase reporter assay was used to investigate software-predicted miRNA target sites in the 3′-untranslated region (3′UTR) of MYCN. The miRNAs were overexpressed in cell lines by transfection of miRNA mimics or miRNA-expressing plasmids. Mutation of the target sites was used to validate MYCN 3′UTR as a direct target of several miRNAs. To measure miRNA-mediated suppression of endogenous N-myc protein, inhibition of proliferation and inhibition of clonogenic growth, miRNAs were overexpressed in a MYCN-amplified neuroblastoma cell line.Results:The results from this study show that MYCN is targeted by several miRNAs. In addition to the previously shown mir-34a/c, we experimentally validate mir-449, mir-19a/b, mir-29a/b/c, mir-101 and let-7e/mir-202 as direct MYCN-targeting miRNAs. These miRNAs were able to suppress endogenous N-myc protein in a MYCN-amplified neuroblastoma cell line. The let-7e and mir-202 were strong negative regulators of MYCN expression. The mir-101 and the let-7 family miRNAs let-7e and mir-202 inhibited proliferation and clonogenic growth when overexpressed in Kelly cells.Conclusion:The tumour-suppressor miRNAs let-7 and mir-101 target MYCN and inhibit proliferation and clonogenic growth of MYCN-amplified neuroblastoma cells.

MicroRNA-101 Inhibits Cell Proliferation, Invasion, and Promotes Apoptosis by Regulating Cyclooxygenase-2 in Hela Cervical Carcinoma Cells

“…cell proliferation, invasion, and angiogenesis (Semaan et al, 2011). MiR-101-1 has been found in the genomic fragile regions that are associated with abnormal deletion or amplification in cancer (Buechner et al, 2011). In recent years, several published studies have shown that miR-101 is decreased expressed in several cancer types including breast, lung, prostate, ovarian, colon, and liver cancers, and emerging evidence suggests a tumour suppressive role for this miRNA (Zhang et al, 2012;Schwarzenbacher et al, 2013;Zhang et al, 2013).…”

The N-myc Oncogene: Maximizing its Targets, Regulation, and Therapeutic Potential

“…This is a mechanism that is distinct from H-Ras/MAPK regulation of C-myc, which is primarily through protein stabilization. Various microRNAs (mir-34a, mir-101, and let-7) have also been documented to regulate N-myc by targeting mRNA, resulting in reduced N-myc expression and decreased proliferation of MYCN-amplified neuroblastoma cells (75,76).…”

Macro-management of microRNAs in cell cycle progression of tumor cells and its implications in anti-cancer therapy

“…In addition to regulating the miRNAs, myc is also directly targeted by miRNAs. In neuroblastoma, let-7e and miR-101 directly target the 3'-UTR, repress the expression of myc and inhibit the growth of tumor cells [92] . In non-small cell lung cancer, miR-145 inhibits proliferation and the G 1 /S transition by directly targeting c-myc [93] .…”