The let-7 microRNA represses cell proliferation pathways in human cells.

MicroRNAs play important roles in animal development, cell differentiation, and metabolism and have been implicated in human cancer. The let-7 microRNA controls the timing of cell cycle exit and terminal differentiation in Caenorhabditis elegans and is poorly expressed or deleted in human lung tumors. Here, we show that let-7 is highly expressed in normal lung tissue, and that inhibiting let-7 function leads to increased cell division in A549 lung cancer cells. Overexpression of let-7 in cancer cell lines alters cell cycle progression and reduces cell division, providing evidence that let-7 functions as a tumor suppressor in lung cells. let-7 was previously shown to regulate the expression of the RAS lung cancer oncogenes, and our work now shows that multiple genes involved in cell cycle and cell division functions are also directly or indirectly repressed by let-7. This work reveals the let-7 microRNA to be a master regulator of cell proliferation pathways.

Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis.

Of the over 200 identified mammalian microRNAs (miRNAs), only a few have known biological activity. To gain a better understanding of the role that miRNAs play in specific cellular pathways, we utilized antisense molecules to inhibit miRNA activity. We used miRNA inhibitors targeting miR-23, 21, 15a, 16 and 19a to test efficacy of antisense molecules in reducing miRNA activity on reporter genes bearing miRNA-binding sites. The miRNA inhibitors de-repressed reporter gene activity when a miRNA-binding site was cloned into its 3'-untranslated region. We employed a library of miRNA inhibitors to screen for miRNA involved in cell growth and apoptosis. In HeLa cells, we found that inhibition of miR-95, 124, 125, 133, 134, 144, 150, 152, 187, 190, 191, 192, 193, 204, 211, 218, 220, 296 and 299 caused a decrease in cell growth and that inhibition of miR-21 and miR-24 had a profound increase in cell growth. On the other hand, inhibition of miR-7, 19a, 23, 24, 134, 140, 150, 192 and 193 down-regulated cell growth, and miR-107, 132, 155, 181, 191, 194, 203, 215 and 301 increased cell growth in lung carcinoma cells, A549. We also identified miRNA that when inhibited increased the level of apoptosis (miR-1d, 7, 148, 204, 210, 216 and 296) and one miRNA that decreased apoptosis (miR-214) in HeLa cells. From these screens, we conclude that miRNA-mediated regulation has a complexity of cellular outcomes and that miRNAs can be mediators of regulation of cell growth and apoptosis pathways.

A novel mean-centering method for normalizing microRNA expression from high-throughput RT-qPCR data.

BACKGROUND: Normalization is critical for accurate gene expression analysis. A significant challenge in the quantitation of gene expression from biofluids samples is the inability to quantify RNA concentration prior to analysis, underscoring the need for robust normalization tools for this sample type. In this investigation, we evaluated various methods of normalization to determine the optimal approach for quantifying microRNA (miRNA) expression from biofluids and tissue samples when using the TaqMan® Megaplex™ high-throughput RT-qPCR platform with low RNA inputs. FINDINGS: We compared seven normalization methods in the analysis of variation of miRNA expression from biofluid and tissue samples. We developed a novel variant of the common mean-centering normalization strategy, herein referred to as mean-centering restricted (MCR) normalization, which is adapted to the TaqMan Megaplex RT-qPCR platform, but is likely applicable to other high-throughput RT-qPCR-based platforms. Our results indicate that MCR normalization performs comparable to or better than both standard mean-centering and other normalization methods. We also propose an extension of this method to be used when migrating biomarker signatures from Megaplex to singleplex RT-qPCR platforms, based on the identification of a small number of normalizer miRNAs that closely track the mean of expressed miRNAs. CONCLUSIONS: We developed the MCR method for normalizing miRNA expression from biofluids samples when using the TaqMan Megaplex RT-qPCR platform. Our results suggest that normalization based on the mean of all fully observed (fully detected) miRNAs minimizes technical variance in normalized expression values, and that a small number of normalizer miRNAs can be selected when migrating from Megaplex to singleplex assays. In our study, we find that normalization methods that focus on a restricted set of miRNAs tend to perform better than methods that focus on all miRNAs, including those with non-determined (missing) values. This methodology will likely be most relevant for studies in which a significant number of miRNAs are not detected.

An optimized isolation and labeling platform for accurate microRNA expression profiling.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression in both plants and animals. miRNA genes have been implicated in a variety of important biological processes, including development, differentiation, apoptosis, fat metabolism, viral infection, and cancer. Similar to protein-coding messenger RNAs, miRNA expression varies between tissues and developmental states. To acquire a better understanding of global miRNA expression in tissues and cells, we have developed isolation, labeling, and array procedures to measure the relative abundance of all of the known human mature miRNAs. The method relies on rapid isolation of RNA species smaller than ~40 nucleotides (nt), direct and homogenous enzymatic labeling of the mature miRNAs with amine modified ribonucleotides, and hybridization to antisense DNA oligonucleotide probes. A thorough performance study showed that this miRNA microarray system can detect subfemtomole amounts of individual miRNAs from <1 mug of total RNA, with 98% correlation between independent replicates. The system has been applied to compare the global miRNA expression profiles in 26 different normal human tissues. This comprehensive analysis identified miRNAs that are preferentially expressed in one or a few related tissues and revealed that human adult tissues have unique miRNA profiles. This implicates miRNAs as important components of tissue development and differentiation. Taken together, these results emphasize the immense potential of microarrays for sensitive and high-throughput analysis of miRNA expression in normal and disease states.