A combined array-based comparative genomic hybridization and functional library screening approach identifies mir-30d as an oncomir in cancer - PubMed (original) (raw)

. 2012 Jan 1;72(1):154-64.

doi: 10.1158/0008-5472.CAN-11-2484. Epub 2011 Nov 4.

Sippy Kaur, Joel Greshock, Heini Lassus, Xiaomin Zhong, Yanling Wang, Arto Leminen, Zhongjun Shao, Xiaowen Hu, Shun Liang, Dionyssios Katsaros, Qihong Huang, Ralf Bützow, Barbara L Weber, George Coukos, Lin Zhang

Affiliations

A combined array-based comparative genomic hybridization and functional library screening approach identifies mir-30d as an oncomir in cancer

Ning Li et al. Cancer Res. 2012.

Abstract

Oncomirs are microRNAs (miRNA) that acts as oncogenes or tumor suppressor genes. Efficient identification of oncomirs remains a challenge. Here we report a novel, clinically guided genetic screening approach for the identification of oncomirs, identifying mir-30d through this strategy. mir-30d regulates tumor cell proliferation, apoptosis, senescence, and migration. The chromosomal locus harboring mir-30d was amplified in more than 30% of multiple types of human solid tumors (n = 1,283). Importantly, higher levels of mir-30d expression were associated significantly with poor clinical outcomes in ovarian cancer patients (n = 330, P = 0.0016). Mechanistic investigations suggested that mir-30d regulates a large number of cancer-associated genes, including the apoptotic caspase CASP3. The guided genetic screening approach validated by this study offers a powerful tool to identify oncomirs that may have utility as biomarkers or targets for drug development.

©2011 AACR.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1

Figure 1

A clinically guided genetic screening approach to identify oncomirs A, aCGH frequency plots of ovarian (n = 109) and breast (n = 73) cancer specimens. Columns represent miRNAs which are ordered on the basis of location in the genome (from chromosome 1 to 22). B, Venn diagrams of miRNA genes with copy number gain shared by ovarian and breast cancers. C, a clinically guided genetic screening approach to identify oncomirs in vitro.

Figure 2

Figure 2

mir-30d regulates cancer cell proliferation and senescence in vitro. A, growth rates of the mir-30d mimic and control transfected tumor cells, measured using the MTT assay. B, growth rates of the mir-30d miRZips inhibitor and control miRZips expressed in tumor cells, measured by cell counts. C, cell-cycle analysis of the mir-30d miRZips inhibitor and control miRZips expressed in tumor cells using flow cytometry. D, β-Gal staining in control and mir-30d inhibitor transfected cells. E, percentage of β-Gal-positive cells after control and mir-30d inhibitor lentiviral transduction. OD, optical density. *P < 0.05.

Figure 3

Figure 3

mir-30d regulates cancer cell apoptosis in vitro. Tumor cells were transfected with mir-30d mimic or control mimic using Lipofectamine RNAiMAX transfection reagent. A, caspase-3 activity after control or camptothecin treatment, measured using the caspase-3 assay. B, total and cleaved caspase-3 after control and camptothecin treatment, detected by Western blots. C, percentage of apoptotic cells measured by flow cytometry using an Annexin V assay. D, total and cleaved PARP protein after control and camptothecin treatment, detected by Western blots. *P < 0.05.

Figure 4

Figure 4

Inhibition of mir-30d blocks xenograft tumor growth in vivo. A and B, the control and mir-30d knockdown tumors (MDA-MB-231) were injected subcutaneously (without Matrigel) into female nude mice (n = 5). Tumor growth rates were measured and followed up for 38 days after tumor cell injection. C, the control and mir-30d knockdown tumors (MDA-MB-231) were injected subcutaneously (with Matrigel) into female nude mice (n = 5). Tumor growth rates were measured and followed up for 38 days after tumor cell injection. D, the xenograft tumors (with Matrigel) were monitored using a bioluminescence optical imaging system at day 38 postinjection. Right: control; left: mir-30d inhibitor. E, quantitative data of the optical imaging shown as photons (p) per second. *P < 0.05.

Figure 5

Figure 5

mir-30d genomic amplification and clinical significance in cancer patients. A, correlation analysis between mir-30d DNA copy number alteration and miRNA expression in cell lines. DNA copy number was analyzed by aCGH. Mature mir-30d expression was quantified by real-time RT-PCR. B, mir-30d exhibited genomic amplifications at high frequencies in multiple human solid tumors by aCGH (left) and SNP array (right). C, SNP array validation in human breast, colon, and lung cancer cell lines. D, mir-30d expression in ovarian cancer was detected by in situ hybridization. E, overall survival in patients with ovarian cancer (n = 330) according to mir-30d staining scores.

Figure 6

Figure 6

mir-30d regulates a large number of transcripts/genes in cancer cells. A and C, the genome-wide transcriptional effect of mir-30d mimic transfection compared with control mimic transfection was analyzed using an Affymetrix Human Gene 1.0 ST oligonucleotide array in ovarian cancer cell line 2008 (A) and breast cancer cell line MDA-MB-231 (C). Light gray spots indicate downregulated transcripts. B and D, histograms of negative natural logarithms of P values derived from a 1-tailed Wilcoxon rank sum test applied to the distributions of hexamers in the 3′-UTRs of all downregulated versus unchanged transcripts. E, Venn diagrams of transcript numbers shared by downregulated transcripts in mir-30d mimic transfections in 2008 and MDA-MB-231 cells and predicted targets of mir-30d by TargetScan. F, the microarray results were validated by real-time RT-PCR in 4 tumor cells. Real-time RT-PCR validation of transcripts that were downregulated in both 2008 and MDA-MB-231 cells after transfection with the mir-30d mimic and that were also predicted mir-30d targets by TargetScan (E). Validations were done in 2008 and MDA-MB-231 cells, as well as in 2 independent cell lines, HCT116and A2780. *, P < 0.05; **, P < 0.01.

Figure 7

Figure 7

mir-30d regulates CASP3 in cancer cells. A, the schematic diagram of the mir-30d binding site in the CASP3 3′-UTR, which was broadly conserved among different species. Hsa, Human; Ptr, chimpanzee; Mml, Rhesus; Mmu, mouse; Rno, Rat; Cpo, Pig; Sar, Shrew; Cfa, Dog; Fca: Cat; Eca, Horse; Bta, Cow; Dno, armadillo; Laf, elephant. B, the mir-30d mimics and control mimic were transiently transfected into 4 cancer cell lines. At 48 hours posttransfection, total RNA were collected, and the endogenous CASP3 expression was examined by real-time RT-PCR. C, reporter assays using wild type and mir-30d binding site mutant CASP-3 3′-UTR. The mir-30d mimic transfection significantly reduced luciferase activity in the wild type but not the binding site mutant CASP3 3′-UTR reporters. WT, wild type. *P < 0.05; **P < 0.01.

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