Abstract
Micro-RNAs are approximately 21-25-nucleotide-long noncoding RNAs that regulate gene expression primarily at the post-transcriptional level in animals. Here, we report that micro-RNA-1 (miR-1), abundant in the cardiac and smooth muscles, is expressed in the lung and is down-regulated in human primary lung cancer tissues and cell lines. In situ hybridization demonstrated localization of miR-1 in bronchial epithelial cells. The tumor suppressor C/EBPalpha, frequently suppressed in lung cancer, reactivated miR-1 expression in the lung cancer cells. Repressed miR-1 was also activated in lung cancer cells upon treatment with a histone deacetylase inhibitor. These observations led us to examine the antitumorigenic potential of miR-1 in lung cancer cells. Expression of miR-1 in nonexpressing A549 and H1299 cells reversed their tumorigenic properties, such as growth, replication potential, motility/migration, clonogenic survival, and tumor formation in nude mice. Exogenous miR-1 significantly reduced expression of oncogenic targets, such as MET, a receptor tyrosine kinase, and Pim-1, a Ser/Thr kinase, frequently up-regulated in lung cancer. Similarly, the levels of two additional targets, FoxP1, a transcription factor with oncogeneic property, and HDAC4 that represses differentiation-promoting genes, were reduced in miR-1-expressing cells. Conversely, depletion of miR-1 facilitated N417 cell growth with concomitant elevation of these targets. Further, ectopic miR-1 induced apoptosis in A549 cells in response to the potent anticancer drug doxorubicin. Enhanced activation of caspases 3 and 7, cleavage of their substrate PARP-1, and depletion of anti-apoptotic Mcl-1 contributed to the sensitivity of miR-1-expressing cells to doxorubicin. Thus, miR-1 has potential therapeutic application against lung cancers.
Highlights
1), abundant in the cardiac and smooth muscles, is expressed in cancers are classified histopathologically as non-small cell lung the lung and is down-regulated in human primary lung cancer cancers
Ubiquitously expressed serum response factor (SRF) in concert with muscle-specific transcription factors activates these genes by binding to the upstream enhancer elements
Potential of Lung Cancer Cells— we examined the antitumorigenic function of miR-1 in lung cancer cells
Summary
Cell Culture and Tissue Procurement—Human lung cancer cell lines were obtained from ATCC. Human bronchial epithelial (BEAS-2B) cells and the lung cancer cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum, cells were harvested for RNA isolation, and whole cell extracts were subjected to Western blot analysis. TaqMan RT-PCR for Quantification of miR-1—DNasetreated total RNA was isolated from lung cancer tissues and cell lines, and mature miR-1 and miR-191 were measured with the Applied Biosystems TaqMan micro-RNA assay protocol, as described [31]. Cells were transfected with rat C/EBP␣ expression vector, and the miR-1 level was measured after 60 h by real time RT-PCR analysis. A549 cells (2 ϫ 106 in phosphate-buffered saline containing 50% Matrigel) expressing miR-1 or control micro-RNA were injected subcutaneously to flanks of nude mice. Western Blot Analysis—Proteins extracted from cells or tumor tissues were immunoblotted with different antibodies following a published protocol [31]. Statistical Analysis—Statistical significance of differences between groups was analyzed by unpaired Student’s t test, and p Յ 0.05 was considered to be statistically significant
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