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. 2007 Oct 2;104(40):15805-10.
doi: 10.1073/pnas.0707628104. Epub 2007 Sep 21.

MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B

Muller Fabbri  1 , Ramiro Garzon, Amelia Cimmino, Zhongfa Liu, Nicola Zanesi, Elisa Callegari, Shujun Liu, Hansjuerg Alder, Stefan Costinean, Cecilia Fernandez-Cymering, Stefano Volinia, Gulnur Guler, Carl D Morrison, Kenneth K Chan, Guido Marcucci, George A Calin, Kay Huebner, Carlo M Croce
Affiliations

MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B

Muller Fabbri et al. Proc Natl Acad Sci U S A. .

Abstract

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate expression of many genes. Recent studies suggest roles of miRNAs in carcinogenesis. We and others have shown that expression profiles of miRNAs are different in lung cancer vs. normal lung, although the significance of this aberrant expression is poorly understood. Among the reported down-regulated miRNAs in lung cancer, the miRNA (miR)-29 family (29a, 29b, and 29c) has intriguing complementarities to the 3'-UTRs of DNA methyltransferase (DNMT)3A and -3B (de novo methyltransferases), two key enzymes involved in DNA methylation, that are frequently up-regulated in lung cancer and associated with poor prognosis. We investigated whether miR-29s could target DNMT3A and -B and whether restoration of miR-29s could normalize aberrant patterns of methylation in non-small-cell lung cancer. Here we show that expression of miR-29s is inversely correlated to DNMT3A and -3B in lung cancer tissues, and that miR-29s directly target both DNMT3A and -3B. The enforced expression of miR-29s in lung cancer cell lines restores normal patterns of DNA methylation, induces reexpression of methylation-silenced tumor suppressor genes, such as FHIT and WWOX, and inhibits tumorigenicity in vitro and in vivo. These findings support a role of miR-29s in epigenetic normalization of NSCLC, providing a rationale for the development of miRNA-based strategies for the treatment of lung cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Dnmt3A protein expression level in NSCLCs is inversely associated with overall survival. Kaplan–Meier curve showing survival of 172 NSCLC patients with different levels of Dnmt3A expression in tumors relative to adjacent normal lung. Patients with higher expression of Dnmt3A had shorter overall survival (P = 0.029).
Fig. 2.
Fig. 2.
Complementarity sites for miR-29s in the 3′-UTR region of DNMT3A and -3B. The capital and bold letters identify perfect base matches, according to the TARGETSCAN 3.1 software. The PicTar software identifies two additional match regions between miR-29a and DNMT3B, indicated with an asterisk.
Fig. 3.
Fig. 3.
MiR-29s directly target DNMT3A and -B. (a) Results of the luciferase assay for DNMT3s expression after transfection with miR-29s in A549 cells. (b) (Upper) Assessment of expression of DNMT3A and DNMT3B mRNAs by qRT-PCR, after transfection of A549 cells with miR-29s or a negative control. (Lower) Silencing of miR-29s with antisense molecules (AS) induces increased expression of DNMT3A and DNMT3B mRNA. (c) Western blot of proteins extracted from A549 cells that were cotransfected with the GFP repression vectors for the DNMT3A and -B−3′-UTR plus miR-29s or scrambled (Scr) oligonucleotides. (d) miR-29b acts as an endogenous primer to retrotranscribe its predicted DNMT3B mRNA target. Black, DNMT3B cDNA (GenBank accession no. NM_175848); blue, cloned and sequenced cDNAs experimentally obtained (eight clones analyzed); red, deduced RNA sequences and corresponding miR-29b. The upper underlined black and blue nucleotides have no homology between target and experimental cDNAs. The lower underlined red nucleotides represent RNA sequence complementary to cDNAs that lack homology to the miR-29b sequence. Nucleotides in bold represent the PicTar-predicted match site.
Fig. 4.
Fig. 4.
Correlation of endogenous miR-29 levels with DNMT3A/B mRNA levels. Inverse correlation between endogenous mRNA levels of DNMT3A and DNMT3B and endogenous levels of miR-29s determined by qRT-PCR in 14 NSCLCs. R, regression coefficient; □, regression line; ♦, actual sample correlations.
Fig. 5.
Fig. 5.
Effect of restoration of miR-29s on the cancer cell epigenome. (a) Global DNA methylation changes induced by miR-29s on A549 cells harvested 48 and 72 h after transfection. The results are compared with nontransfected (mock) cells and cells transfected with a scrambled oligonucleotide. (b) Determination of FHIT and WWOX mRNA levels in A549 and H1299 cells 48 h after transfection with miR-29s or a negative control by qRT PCR; miR-29s induced reexpression of FHIT and WWOX mRNAs. (c) Immunoblot of Fhit and Wwox proteins in A549 and H1299 cells 72 h after transfection with miR-29s or negative control; by 72 h, miR-29s induce increased expression of Fhit and Wwox proteins. The numbers above the immunoblot images represent the intensity of the bands relative to the GAPDH gene (upper row, Fhit; lower row, Wwox). (d) Graphical representation of the quantitative DNA methylation data for FHIT and WWOX promoter region by using the MassARRAY system. Each square represents a single CpG or a group of CpGs analyzed, and each arrow represents a sample. Methylation frequencies are displayed for each experiment in a color code that extends from light green (lower methylation frequencies) to bright red (higher methylation frequencies).
Fig. 6.
Fig. 6.
Effects of miR-29s on tumorigenicity of A549 cells. (a) Growth curve of A549 cells transfected in vitro with miR-29s or scrambled oligonucleotide or mock-transfected. (b) Percentages of live cells were measured in A549 cells transfected with scrambled oligonucleotide or with miR-29s oligonucleotides (100 nM final concentration). (c) Growth curve of engrafted tumors in nude mice injected with A549 cells pretransfected (48 h before injection) with miR-29s, scrambled oligonucleotides, or mock transfected. (d) Comparison of tumor engraftment sizes of mock-, scrambled-, and miR-29s-transfected A549 cells 21 days after injection in nude mice. The images show average-sized tumors from among five of each category. (e) Tumor weights ± SD in nude mice.
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