Development of 5-FU-modified tumor suppressor microRNAs as a platform for novel microRNA-based cancer therapeutics.

MicroRNA (miRNAs) are pleiotropic post-transcriptional modulators of gene expression. Their inherently pleiotropic nature makes miRNAs strong candidates for the development of cancer therapeutics, yet despite their potential, there remains a challenge to deliver nucleic acid-based therapies into cancer cells. We developed a novel approach to modify miRNAs by replacing the uracil bases with 5-fluorouracil (5-FU) in the guide strand of tumor suppressor miRNAs, thereby combining the therapeutic effect of 5-FU with tumor-suppressive effect of miRNAs to create a potent, multi-targeted therapeutic molecule without altering its native RNAi function. To demonstrate the general applicability of this approach to other tumor-suppressive miRNAs, we screened a panel of 12 novel miRNA mimetics in several cancer types, including leukemia, breast, gastric, lung, and pancreatic cancer. Our results show that 5-FU-modified miRNA mimetics have increased potency (low nanomolar range) in inhibiting cancer cell proliferation and that these mimetics can be delivered into cancer cells without delivery vehicle both in vitro and in vivo, thus representing significant advancements in the development of therapeutic miRNAs for cancer. This work demonstrates the potential of fluoropyrimidine modifications that can be broadly applicable and may serve as a platform technology for future miRNA and nucleic acid-based therapeutics.

Functional and Potential Therapeutic Implication of MicroRNAs in Pancreatic Cancer.

The alarmingly low five-year survival rate for pancreatic cancer presents a global health challenge, contributing to about 7% of all cancer-related deaths. Late-stage diagnosis and high heterogeneity are the biggest hurdles in treating pancreatic cancer. Thus, there is a pressing need to discover novel biomarkers that could help in early detection as well as improve therapeutic strategies. MicroRNAs (miRNAs), a class of short non-coding RNA, have emerged as promising candidates with regard to both diagnostics and therapeutics. Dysregulated miRNAs play pivotal roles in accelerating tumor growth and metastasis, orchestrating tumor microenvironment, and conferring chemoresistance in pancreatic cancer. The differential expression profiles of miRNAs in pancreatic cancer could be utilized to explore novel therapeutic strategies. In this review, we also covered studies on recent advancements in various miRNA-based therapeutics such as restoring miRNAs with a tumor-suppressive function, suppressing miRNA with an oncogenic function, and combination with chemotherapeutic drugs. Despite several challenges in terms of specificity and targeted delivery, miRNA-based therapies hold the potential to revolutionize the treatment of pancreatic cancer by simultaneously targeting multiple signaling pathways.

The Role of MicroRNAs in Pancreatitis Development and Progression.

Pancreatitis (acute and chronic) is an inflammatory disease associated with significant morbidity, including a high rate of hospitalization and mortality. MicroRNAs (miRs) are essential post-transcriptional modulators of gene expression. They are crucial in many diseases' development and progression. Recent studies have demonstrated aberrant miRs expression patterns in pancreatic tissues obtained from patients experiencing acute and chronic pancreatitis compared to tissues from unaffected individuals. Increasing evidence showed that miRs regulate multiple aspects of pancreatic acinar biology, such as autophagy, mitophagy, and migration, impact local and systemic inflammation and, thus, are involved in the disease development and progression. Notably, multiple miRs act on pancreatic acinar cells and regulate the transduction of signals between pancreatic acinar cells, pancreatic stellate cells, and immune cells, and provide a complex interaction network between these cells. Importantly, recent studies from various animal models and patients' data combined with advanced detection techniques support their importance in diagnosing and treating pancreatitis. In this review, we plan to provide an up-to-date summary of the role of miRs in the development and progression of pancreatitis.

5-FU-miR-15a Inhibits Activation of Pancreatic Stellate Cells by Reducing YAP1 and BCL-2 Levels In Vitro.

Chronic pancreatitis is characterized by chronic inflammation and fibrosis, processes heightened by activated pancreatic stellate cells (PSCs). Recent publications have demonstrated that miR-15a, which targets YAP1 and BCL-2, is significantly downregulated in patients with chronic pancreatitis compared to healthy controls. We have utilized a miRNA modification strategy to enhance the therapeutic efficacy of miR-15a by replacing uracil with 5-fluorouracil (5-FU). We demonstrated increased levels of YAP1 and BCL-2 (both targets of miR-15a) in pancreatic tissues obtained from Ptf1aCreERTM and Ptf1aCreERTM;LSL-KrasG12D mice after chronic pancreatitis induction as compared to controls. In vitro studies showed that delivery of 5-FU-miR-15a significantly decreased viability, proliferation, and migration of PSCs over six days compared to 5-FU, TGFß1, control miR, and miR-15a. In addition, treatment of PSCs with 5-FU-miR-15a in the context of TGFß1 treatment exerted a more substantial effect than TGFß1 alone or when combined with other miRs. Conditioned medium obtained from PSC cells treated with 5-FU-miR-15a significantly inhibits the invasion of pancreatic cancer cells compared to controls. Importantly, we demonstrated that treatment with 5-FU-miR-15a reduced the levels of YAP1 and BCL-2 observed in PSCs. Our results strongly suggest that ectopic delivery of miR mimetics is a promising therapeutic approach for pancreatic fibrosis and that 5-FU-miR-15a shows specific promise.

Roles of microRNAs in Gastrointestinal Cancer Stem Cell Resistance and Therapeutic Development.

Resistance to cancer treatment is one of the major challenges currently faced when treating gastrointestinal (GI) cancers. A major contributing factor to this resistance is the presence of cancer stem cells (CSCs) in GI cancers (e.g., colorectal, pancreatic, gastric, liver cancer). Non-coding RNAs, such as microRNAs (miRNAs), have been found to regulate several key targets that are responsible for cancer stemness, and function as oncogenic miRNAs (oncomiRs) or tumor suppressor miRNAs. As a result, several miRNAs have been found to alter, or be altered by, the expression of CSC-defining markers and their related pathways. These miRNAs can be utilized to affect stemness in multiple ways, including directly targeting CSCs and enhancing the efficacy of cancer therapeutics. This review highlights current studies regarding the roles of miRNAs in GI CSCs, and efforts towards the development of cancer therapeutics.

Transcriptomic changes associated with DKK4 overexpression in pancreatic cancer cells detected by RNA-Seq.

The promotion of tumor development by Dickkopf 4 (DKK4) is receiving increased attention. However, the association between DKK4 and pancreatic cancer remains unclear. DKK4 expression was measured in pancreatic ductal adenocarcinoma tissues using qRT-PCR and immunohistochemistry. A DKK4-overexpressing pancreatic cancer cell line was established, and the differentially expressed genes (DEGs) that were induced by DKK4 were identified using transcriptome sequencing. The association between the identified DEGs and pancreatic cancer was assessed using gene ontology (GO), pathway analysis, pathway interaction networks, differentially expressed gene interaction network analysis, and co-expression gene networks. Finally, the accuracy of the analyses was validated using serial paraffin and frozen sections of clinical samples. DKK4 is highly expressed in pancreatic cancer tissues. DEGs of overexpression DKK4 of PANC-1 are mostly upregulated. GO and pathway analysis showed that DKK4 are associated with tumor and organ development and immune inflammation. The mitogen-activated protein kinase (MAPK) signaling pathway was the main signal transduction pathway that showed significant enrichment in overexpression DKK4 of PANC-1. The results of GO, pathway analyses, and differentially expressed gene interaction network identified genes that are closely associated with tumor development, including MAPK3, PIK3R3, VAV3, JAG1, and Notch3. The immunohistochemistry and immunofluorescence results suggested that DKK4 is co-expressed with MAPK3 and VAV3 in pancreatic cancer tissues. The results presented here show for the first time that DKK4 is highly expressed in pancreatic cancer tissues. Bioinformatics analysis of a DKK4-overexpressing of PANC-1 identified several oncogenes that are closely associated with tumors, and the MAPK signaling pathway is the core signal transduction pathway. DKK4 can be co-expressed with MAPK3 and VAV3 in pancreatic ductal adenocarcinoma tissues. Thus, DKK4 may have function on the development and progression of pancreatic cancer.

Genetic variant rs16430 6bp > 0bp at the microRNA-binding site in TYMS and risk of sporadic breast cancer risk in non-Hispanic white women aged ≤ 55 years.

Thymidylate synthase (TYMS) is involved in the folate metabolism and provision of nucleotides needed for DNA synthesis and repair. Thus, functional genetic variants in TYMS may alter cancer risk. In the study, we evaluated associations of three germline variants (rs2790 A > G, rs16430 6 bp > 0 bp, and rs1059394 C > T) in the predicted miRNA-binding sites of TYMS with risk of sporadic breast cancer in non-Hispanic white women aged ≤ 55. We found that carriers of the rs16430 0 bp variant allele had an increased risk of breast cancer [adjusted odd ratio (OR) = 1.37, 95% confidence interval (CI): 1.08-1.73; P = 0.010], compared with carriers of the 6 bp/6 bp genotype. This increased risk was more evident in older subjects (OR = 1.47, 95% CI = 1.06-2.03, P = 0.022), never smokers (OR = 1.67, 95% CI = 1.23-2.25, P < 0.001), never drinkers (OR = 1.44, 95% CI = 1.01-2.05, P = 0.043), and estrogen receptor-positive patients (OR = 1.46, 95% CI = 1.11-1.92, P = 0.006), regardless of tumor stages. The results are consistent with the functional analyses of rs16430 as previously reported, which showed that the 0 bp allele had a decrease in both luciferase activity by ∼ 70% and mRNA levels by ∼ 50% compared with the 6bp allele. Additionally, the rs16430 variant was predicted to influence the binding activity of miR-561. Taken together, these findings indicate that the TYMS rs16430 may contribute to the etiology of sporadic breast cancer in non-Hispanic white women aged ≤ 55 yr. Further validation in large population-based or cohort studies is needed.

Identification of KLF17 as a novel epithelial to mesenchymal transition inducer via direct activation of TWIST1 in endometrioid endometrial cancer.

Krüppel-like factor 17 (KLF17), a member of the KLF transcription factor family, has been shown to inhibit the epithelial-mesenchymal transition (EMT) and tumor growth. However, the expression, the cellular function and the mechanism of KLF17 in endometrioid endometrial cancer (EEC; a dominant type of endometrial cancer) remain elusive. Here, we report that among the KLF family members, KLF17 was consistently upregulated in EEC cell lines compared with immortalized endometrial epithelial cells. Overexpression of KLF17 in EEC cell lines induced EMT and promoted cell invasion and drug resistance, resulting in increased expression of TWIST1. In contrast, KLF17 suppression reversed EMT, diminished cell invasion, restored drug sensitivity and suppressed TWIST1 expression. Luciferase assays, site-directed mutagenesis and transcription factor DNA-binding analysis demonstrated that KLF17 transactivates TWIST1 expression by directly binding to the TWIST1 promoter. Knockdown of TWIST1 prevented KLF17-induced EMT. Consistent with these results, both KLF17 and TWIST1 levels were found to be elevated in EECs compared with normal tissues. KLF17 expression positively correlated with tumor grade but inversely correlated with estrogen and progesterone receptor expression. Thus, KLF17 may have an oncogenic role during EEC progression via initiating EMT through the regulation of TWIST1.

Systematic analysis of microRNA fingerprints in thrombocythemic platelets using integrated platforms.

Posttranscriptional and translational controls mediated by microRNAs (miRNA) regulate diverse biologic processes. We dissected regulatory effects of miRNAs relevant to megakaryocytopoiesis and platelet biology by analyzing expression patterns in 79 subjects with thrombocytosis and controls, and integrated data with transcriptomic and proteomic platforms. We validated a unique 21-miRNA genetic fingerprint associated with thrombocytosis, and demonstrated that a 3-member subset defines essential thrombocythemia (ET). The genetic signature includes functional guide and passenger strands of the previously uncharacterized miR 490 (5p and 3p), which displayed restricted, low-level expression in megakaryocytes/platelets (compared with leukocytes), and aberrant expression during thrombocytosis, most profound in ET. Overexpression of miR 490 in a bilineage differentiation model of megakaryocyte/erythroid progenitor formation was insufficient for hematopoietic colony differentiation and/or lineage specification. Integration of transcriptomic and mass spectrometric datasets with functional reporter assays identified dishevelled associated activator of morphogenesis 1 (DAAM1) as a miR 490 5p protein target demonstrating decreased expression in ET platelets, putatively by translational control (and not by mRNA target degradation). Our data define a dysregulated miRNA fingerprint in thrombocytosis and support a developmentally restricted function of miR 490 (and its putative DAAM1 target) to conditions associated with exaggerated megakaryocytopoiesis and/or proplatelet formation.

Development of gemcitabine-modified miRNA mimics as cancer therapeutics for pancreatic ductal adenocarcinoma.

Despite the recent advancement in diagnosis and therapy, pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, is still the most lethal cancer with a low five-year survival rate. There is an urgent need to develop new therapies to address this issue. In this study, we developed a treatment strategy by modifying tumor suppressor miRNAs, miR-15a and miR-194, with the chemotherapeutic gemcitabine (Gem) to create Gem-modified mimics, Gem-miR-15a and Gem-miR-194, respectively. In a panel of PDAC cell lines, we found that Gem-miR-15a and Gem-miR-194 induce cell-cycle arrest and apoptosis, and these mimics are potent inhibitors with IC50 values up to several hundred fold less than their native counterparts or Gem alone. Furthermore, we found that Gem-miR-15a and Gem-miR-194 retained miRNA function by downregulating the expression of several key targets including WEE1, CHK1, BMI1, and YAP1 for Gem-miR-15a, and FOXA1 for Gem-miR-194. We also found that our Gem-modified miRNA mimics exhibit an enhanced efficacy compared to Gem in patient-derived PDAC organoids. Furthermore, we observed that Gem-miR-15a significantly inhibits PDAC tumor growth in vivo without observing any noticeable signs of toxicity. Overall, our results demonstrate the therapeutic potential of Gem-modified miRNAs as a treatment strategy for PDAC.

Correction: Soung et al. Therapeutic Potential of Chemically Modified MiR-489 in Triple-Negative Breast Cancers. Cancers 2020, 12, 2209.

In the original publication [...].

Analysis of MicroRNAs in Ferroptosis.

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that are involved in a wide range of biological processes, including development, differentiation, and disease. They function by binding to the 3' untranslated region (UTR) of target mRNAs, leading to mRNA degradation or translational repression. miRNAs are involved in the regulation of many cellular processes, including cell proliferation, apoptosis, and metabolism. MiRNAs have been shown to modulate ferroptosis in several ways. Some miRNAs have been shown to promote ferroptosis by increasing the expression of genes involved in lipid peroxidation. Other miRNAs have been shown to inhibit ferroptosis by decreasing the expression of genes involved in iron uptake. The role of miRNAs in ferroptosis is still being studied, but they play a significant role in this cell death pathway. miRNAs may be potential targets for therapeutic intervention in diseases associated with ferroptosis, such as cancer and neurodegenerative diseases. This chapter outlines several methods used to study the connection between miRNAs and ferroptosis through target discovery and validation.

The Sensitization of Triple-Negative Breast Cancers to Poly ADP Ribose Polymerase Inhibition Independent of BRCA1/2 Mutation Status by Chemically Modified microRNA-489.

Chemoresistance and inefficient therapeutic efficacies in triple-negative breast cancers (TNBCs) are among the major clinical problems in breast cancers. A potential new method to sensitize these tumors to current treatment options is, therefore, urgent and necessary. Our previous studies demonstrated that miR-489 serves as one of the top tumor-suppressing miRs and features downregulated expression in metastatic TNBCs and that the restoration of miR-489 expression in TNBCs effectively inhibits the metastatic potentials of TNBCs both in vitro and in vivo. The chemical modification of miR-489 (CMM489) through the replacement of uracil with 5-FU further enhances the therapeutic potential of miR-489. In the present study, we tested the effects of CMM489 in synergizing DNA damage response (DDR) inhibitors such as PARP inhibitors. CMM489 is particularly effective in sensitizing TNBC cell lines with inherent resistance to PARP inhibitors regardless of BRCA mutation status. One of the anti-cancer mechanisms through which CMM489 synergizes with PARP inhibitors is the blockade of homologous recombination (HR) in TNBC cells upon DNA damage. The results of this study highlight the potential use of CMM489 in combination treatments with PARP inhibitors in TNBCs.

Development of Gemcitabine-Modified miRNA Mimics as Cancer Therapeutics for Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer, including its most common subtype, pancreatic adenocarcinoma (PDAC), has the lowest five-year survival rate among patients with pancreatic cancer in the United States. Despite advancements in anticancer treatment, the overall median survival for patients with PDAC has not dramatically improved. Therefore, there is an urgent need to develop new strategies of treatment to address this issue. Non-coding RNAs, including microRNAs (miRNAs), have been found to have major roles in carcinogenesis and the subsequent treatment of various cancer types like PDAC. In this study, we developed a treatment strategy by modifying tumor suppressor miRNAs, hsa-miRNA-15a (miR-15a) and hsa-miRNA-194-1 (miR-194), with the nucleoside analog chemotherapeutic gemcitabine (Gem) to create Gem-modified mimics of miR-15a (Gem-miR-15a) and miR-194 (Gem-miR-194). In a panel of PDAC cell lines, we found that Gem-miR-15a and Gem-miR-194 induce cell cycle arrest and apoptosis, and these mimics are potent inhibitors with IC 50 values up to several hundred fold less than their native counterparts or Gem alone. Furthermore, we found that Gem-miR-15a and Gem-miR-194 retained miRNA function by downregulating the expression of several key targets including WEE1, CHK1, BMI1, and YAP1 for Gem-miR-15a, and FOXA1 for Gem-miR-194. We also found that our Gem-modified miRNA mimics exhibit an enhanced efficacy compared to Gem alone in patient-derived PDAC organoids. Furthermore, we observed that Gem-miR-15a significantly inhibits PDAC tumor growth in vivo without observing any noticeable signs of toxicity. Overall, our results demonstrate the therapeutic potential of Gem-modified miRNAs as a treatment strategy for PDAC. One Sentence Summary: Yuen and Hwang et. al. have developed a potent therapeutic strategy for patients with pancreatic cancer by modifying microRNAs with gemcitabine.

Development of a 5-FU modified miR-129 mimic as a therapeutic for non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related deaths in the United States with non-small cell lung cancer (NSCLC) accounting for most cases. Despite advances in cancer therapeutics, the 5-year survival rate has remained poor due to several contributing factors, including its resistance to therapeutics. Therefore, there is a pressing need to develop therapeutics that can overcome resistance. Non-coding RNAs, including microRNAs (miRNAs), have been found to contribute to cancer resistance and therapeutics by modulating the expression of several targets involving multiple key mechanisms. In this study, we investigated the therapeutic potential of miR-129 modified with 5-fluorouracil (5-FU) in NSCLC. Our results show that 5-FU modified miR-129 (5-FU-miR-129) inhibits proliferation, induces apoptosis, and retains function as an miRNA in NSCLC cell lines A549 and Calu-1. Notably, we observed that 5-FU-miR-129 was able to overcome resistance to tyrosine kinase inhibitors and chemotherapy in cell lines resistant to erlotinib or 5-FU. Furthermore, we observed that the inhibitory effect of 5-FU-miR-129 can also be achieved in NSCLC cells under vehicle-free conditions. Finally, 5-FU-miR-129 inhibited NSCLC tumor growth and extended survival in vivo without toxic side effects. Altogether, our results demonstrate the potential of 5-FU-miR-129 as a highly potent cancer therapeutic in NSCLC.

A frequent somatic mutation in CD274 3'-UTR leads to protein over-expression in gastric cancer by disrupting miR-570 binding.

Inhibitory costimulatory molecule CD274 expresses in various cancers and contributes to cancer immune evasion by inhibiting T cell activation and proliferation, yet the regulatory mechanisms for CD274 overexpression in cancers are poorly understood. In this study, we discovered a novel mechanism of CD274 expression regulated by miR-570. A guanine-to-cytosine mutation at the 3'-UTR of CD274 mRNA led to CD274 overexpression by disrupting the miR-570 binding. The mutations were widely observed in cancers by sequencing of 276 gastrointestinal cancers (esophageal, gastric, colorectal, hepatocellular, and pancreatic cancers). This mutation was significantly associated with CD274 overexpression in gastric cancer (P = 1.44×10(-10)) and with the pathological features including differentiation grade, depth of tumor invasion, lymph node metastasis, and tumor-node-metastases (TNM) stage. These findings suggest a novel regulatory mechanism for CD274 overexpression in gastric cancer mediated by miR-570 and a somatic mutation in CD274 3'-UTR, and provide a new insight to gastric carcinogenesis.

Translational control analysis by translationally active RNA capture/microarray analysis (TrIP-Chip).

We have developed a new approach to systematically study post-transcriptional regulation in a small number of cells. Actively translating mRNAs are associated with polysomes and the newly synthesized peptide chains are closely associated with molecular chaperones such as hsp70s, which assist in the proper folding of nascent polypeptides into higher ordered structures. These chaperones provide an anchor with which to separate actively translating mRNAs associated with polysomes from free mRNAs. Affinity capture beads were developed to capture hsp70 chaperones associated with the polysome complexes. The isolated actively translating mRNAs were used for high-throughput expression profiling analysis. Feasibility was demonstrated using an in vitro translation system with known translationally regulated mRNA transcript thymidylate synthase (TS). We further developed the approach using HCT-116 colon cancer cells with both TS and p53 as positive controls. The steady-state levels of TS and p53 mRNAs were unaltered after 5-fluorouracil treatment as assessed by real-time qRT-PCR analysis. In contrast, the protein expression and polysome-associated mRNA levels of both genes were increased. These differences in translational rate were revealed with our new approach from 500 cells. This technology has the potential to make investigation of translational control feasible with limited quantities of clinical specimens.

Retraction Notice to: A Novel Mechanism of Doxorubicin Resistance and Tumorigenesis Mediated by MicroRNA-501-5p-Suppressed BLID.

[This retracts the article DOI: 10.1016/j.omtn.2018.06.011.].

MicroRNA-194 inhibits epithelial to mesenchymal transition of endometrial cancer cells by targeting oncogene BMI-1.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is the key process driving cancer metastasis. Oncogene/self renewal factor BMI-1 has been shown to induce EMT in cancer cells. Recent studies have implied that noncoding microRNAs (miRNAs) act as crucial modulators for EMT. The aims of this study was to determine the roles of BMI-1 in inducing EMT of endometrial cancer (EC) cells and the possible role of miRNA in controlling BMI-1 expression. METHODS AND RESULTS: We evaluated the expression of BMI-1 gene in a panel of EC cell lines, and detected a strong association with invasive capability. Stable silencing of BMI-1 in invasive mesenchymal-type EC cells up-regulated the epithelial marker E-cadherin, down-regulated mesenchymal marker Vimentin, and significantly reduced cell invasion in vitro. Furthermore, we discovered that the expression of BMI-1 was suppressed by miR-194 via direct binding to the BMI-1 3'-untranslated region 3'-UTR). Ectopic expression of miR-194 in EC cells induced a mesenchymal to epithelial transition (MET) by restoring E-cadherin, reducing Vimentin expression, and inhibiting cell invasion in vitro. Moreover, BMI-1 knockdown inhibited in vitro EC cell proliferation and clone growth, correlated with either increased p16 expression or decreased expression of stem cell and chemoresistance markers (SOX-2, KLF4 and MRP-1). CONCLUSION: These findings demonstrate the novel mechanism for BMI-1 in contributing to EC cell invasion and that repression of BMI-1 by miR-194 could have a therapeutic potential to suppress EC metastasis.

A personalized microRNA microarray normalization method using a logistic regression model.

MOTIVATION: MicroRNA (miRNA) is a set of newly discovered non-coding small RNA molecules. Its significant effects have contributed to a number of critical biological events including cell proliferation, apoptosis development, as well as tumorigenesis. High-dimensional genomic discovery platforms (e.g. microarray) have been employed to evaluate the important roles of miRNAs by analyzing their expression profiling. However, because of the small total number of miRNAs and the absence of well-known endogenous controls, the traditional normalization methods for messenger RNA (mRNA) profiling analysis could not offer a suitable solution for miRNA analysis. The need for the establishment of new adaptive methods has come to the forefront. RESULTS: Locked nucleic acid (LNA)-based miRNA array was employed to profile miRNAs using colorectal cancer cell lines under different treatments. The expression pattern of overall miRNA profiling was pre-evaluated by a panel of miRNAs using Taqman-based quantitative real-time polymerase chain reaction (qRT-PCR) miRNA assays. A logistic regression model was built based on qRT-PCR results and then applied to the normalization of miRNA array data. The expression levels of 20 additional miRNAs selected from the normalized list were post-validated. Compared with other popularly used normalization methods, the logistic regression model efficiently calibrates the variance across arrays and improves miRNA microarray discovery accuracy. AVAILABILITY: Datasets and R package are available at http://gauss.usouthal.edu/publ/logit/.