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. 2008 Apr 15;105(15):5874-8.
doi: 10.1073/pnas.0801130105. Epub 2008 Apr 4.

MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNAs encoding extracellular matrix proteins

Srikumar Sengupta  1 , Johan A den Boon, I-How Chen, Michael A Newton, Stephen A Stanhope, Yu-Juen Cheng, Chien-Jen Chen, Allan Hildesheim, Bill Sugden, Paul Ahlquist
Affiliations

MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNAs encoding extracellular matrix proteins

Srikumar Sengupta et al. Proc Natl Acad Sci U S A. .

Abstract

Using highly sensitive microarray-based procedures, we identified eight microRNAs (miRNAs) showing robust differential expression between 31 laser-capture-microdissected nasopharyngeal carcinomas (NPCs) and 10 normal healthy nasopharyngeal epithelial samples. In particular, miRNA mir-29c was expressed at one-fifth the levels in tumors as in normal epithelium. In NPC tumors, the lower mir-29c levels correlated with higher levels of multiple mRNAs whose 3' UTRs can bind mir-29c at target sequences conserved across many vertebrates. In cultured cells, introduction of mir-29c down-regulated these genes at the level of mRNA and inhibited expression of luciferase encoded by vectors having the 3' UTRs of these genes. Moreover, for each of several genes tested, mutating the mir-29c target sites in the 3' UTR abrogated mir-29c-induced inhibition of luciferase expression. Most of the mir-29c-targeted genes identified encode extracellular matrix proteins, including multiple collagens and laminin gamma1, that are associated with tumor cell invasiveness and metastatic potential, prominent characteristics of NPC. Thus, we identify eight miRNAs differentially expressed in NPC and demonstrate the involvement of one in regulating genes involved in metastasis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
mir-29c down-regulates accumulation of its target mRNAs. HeLa and HepG2 cells transfected with mir-29c precursor show lower levels of target mRNAs than untransfected cells as measured by quantitative real-time PCR normalized to GAPDH (Table S4). This down-regulation is more pronounced in HepG2 cells, possibly because of the lower basal level of these mRNAs in these cells, resulting in higher a miRNA:mRNA ratio posttransfection and attendant increased efficiency of mRNA down-regulation.
Fig. 2.
Fig. 2.
mir-29c inhibits expression of luciferase with 3′ UTRs derived from mir-29c's target genes. 3′ UTRs of target genes containing mir-29c-binding sites were cloned into vectors containing firefly luciferase that were transfected into HeLa cells. These cells were subsequently transfected with mir-29c precursor RNAs or mock-transfected. Compared with cells that were mock-transfected (set to 100%), mir-29c precursor-transfected cells show down-regulation in luciferase activity.
Fig. 3.
Fig. 3.
Mutations disrupting the binding of mir-29c to the 3′ UTRs of three target genes block mir-29c mediated inhibition of the expression of these genes. (A) Black-boxed nucleotides in the mRNA sequence indicate the extent of basepairing with mir-29c, and in particular how the mutations disrupt basepairing with the mir-29c seed sequence. (B) The wild-type or mutated 3′ UTRs of target mRNAs were cloned into vectors containing firefly luciferase for expression in HeLa cells, which were subsequently transfected with precursor mir-29c RNA or mock-transfected. Luciferase activity was no longer affected by mir-29c in cells transfected with constructs containing the mutated target sequence.
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