Affymetrix miRNA Array Research Articles

Porphyromonas gingivalis LPS‐stimulated BMSC‐derived exosome promotes osteoclastogenesis via miR‐151‐3p/PAFAH1B1

AbstractObjectivesPorphyromonas gingivalis‐LPS regulated bone metabolism by triggering dysfunction of osteoblasts directly, and affecting activity of osteoclasts through intracellular communication. Exosome, as the mediator of intercellular communication, was important vesicle to regulate osteogenesis and osteoclastogenesis. This research was designed for investigating the mechanism of BMSCs‐EXO in modulating osteoclastic activity under the P. gingivalis‐LPS.Materials and methodsThe cytotoxicity and osteogenic effects of P. gingivalis‐LPS on BMSCs was evaluated, and then osteoclastic activity of RAW264.7 co‐cultured with exosomes was detected. Besides, Affymetrix miRNA array and luciferase reporter assay were used to identify the target exosomal miRNA signal pathway.ResultsBMSCs' osteogenic differentiation and proliferation were decreased under 1 and 10 μg/mL P. gingivalis‐LPS. Osteoclastic‐related genes and proteins levels were promoted by P. gingivalis‐LPS‐stimulated BMSCs‐EXO. Based on the miRNA microarray analysis, exosomal miR‐151‐3p was lessened in BMExo‐LPS group, which facilitated osteoclastic differentiation through miR‐151‐3p/PAFAH1B1.ConclusionsPorphyromonas gingivalis‐LPS could regulated bone metabolism by inhibiting proliferation and osteogenesis of BMSCs directly. Also, P. gingivalis‐LPS‐stimulated BMSCs‐EXO promoted osteoclastogenesis via activating miR‐151‐3p/PAFAH1B1 signal pathway.

EXTH-23. EFFECT OF EVOGLIPTIN AND TEMOZOLOMIDE COMBINATION THERAPY FOR THE TREATMENT OF GLIOBLASTOMA, IN VITRO AND IN VIVO

Abstract A recent study showed that high dipeptidyl-peptidase IV (DPP4) expression affects glioma progression. The purpose of this study is to investigate the synergistic effect of a DDP4 inhibitor, evogliptin and temzolomide (TMZ) combination therapy for glioblastoma (GBM) in vitro and in vivo. U87, U252, and A172 cells were treated with 50, 250, and 500 uM TMZ or with 5, 10, and 20 mM evogliptin for 24, 48 and 72 hrs. For in vivo experiment, GBM xenograft models were used. Three groups (n=5 mice) were treated with TMZ(15mg/kg/day), evogliptin (2mg/25g/day), and combination of TMZ and evogliptin via i.p. injection for 4 weeks. For evaluating the relevant mechanism, Affymetrix miRNA arrays are used. The MTT assay revealed that the combination treatment had higher cytotoxicity against U87, U251, and A172 cells than the monotherapy had after 48 and 72-hr treatment. mRNA DDP4 levels were much more decreased after combination administration. The median survival of each group was 43.6, 55.2, 53.2, 65.2, and 71.3 days in the control, evogliptin, TMZ, the first combination therapy with low-dose evogliptin (2mg/25g/day) and TMZ, and the second combination therapy with high-dose evogliptin (10mg/25g/day) and TMZ, respectively. In Affymetrix miRNA arrays, miR-1246, miR-149-3p, miR-3148, miR-4440, miR-4484, miR-4535, miR-6126, miR-6780b-5p, and miR-7110-5p were increased after evogliptin treatment. miR-149-5p, miR-23a-5p, miR-3195, miR-4330, miR-4497, miR-4669, miR-671-3p, and miR-744-5p were decreased. miR-4440 and miR-6780b-5p were also increased following combination therapy. In the TCGA dataset, the survival of glioma patients with low DPP4 expression is longer than that in patients with high DPP4. A DDP4 inhibitor, evoglipin regulates miRNA in GBM cell lines. The combination of evogliptin and TMZ had a moderate effect on the treatment of GBM in vitro and in vivo. miR-4440 and miR-6780b-5p could be associated with inhibiting the proliferation of GBM cells by targeting DPP4.

Dysregulation of the miRNome unveils a crosstalk between obesity and prostate cancer: miR-107 asa personalized diagnostic and therapeutic tool

Prostate-specific antigen (PSA) is the gold-standard marker to screen prostate cancer (PCa) nowadays. Unfortunately, its lack of specificity and sensitivity makes the identification of novel tools to diagnose PCa an urgent medical need. In this context, microRNAs (miRNAs) have emerged as potential sources of non-invasive diagnostic biomarkers in several pathologies. Therefore, this study was aimed at assessing for the first time the dysregulation of the whole plasma miRNome in PCa patients and its putative implication in PCa from a personalized perspective (i.e., obesity condition). Plasma miRNome from a discovery cohort (18 controls and 19 PCa patients) was determined using an Affymetrix-miRNA array, showing that the expression of 104 miRNAs was significantly altered, wherein six exhibited a significant receiver operating characteristic (ROC) curve to distinguish between control and PCa patients (area under the curve [AUC] = 1). Then, a systematic validation using an independent cohort (135 controls and 160 PCa patients) demonstrated that miR-107 was the most profoundly altered miRNA in PCa (AUC = 0.75). Moreover, miR-107 levels significantly outperformed the ability of PSA to distinguish between control and PCa patients and correlated with relevant clinical parameters (i.e., PSA). These differences were more pronounced when considering only obese patients (BMI > 30). Interestingly, miR-107 levels were reduced in PCa tissues versus non-tumor tissues (n = 84) and in PCa cell lines versus non-tumor cells. In vitro miR-107 overexpression altered key aggressiveness features in PCa cells (i.e., proliferation, migration, and tumorospheres formation) and modulated the expression of important genes involved in PCa pathophysiology (i.e., lipid metabolism [i.e., FASN] and splicing process). Altogether, miR-107 might represent a novel and useful personalized diagnostic and prognostic biomarker and a potential therapeutic tool in PCa, especially in obese patients.

GATA4-driven miR-206-3p signatures control orofacial bone development by regulating osteogenic and osteoclastic activity.

Growth disorders in the orofacial bone development process may lead to orofacial deformities. The balance between bone matrix formation by mesenchymal lineage osteoblasts and bone resorption by osteoclasts is vital for orofacial bone development. Although the mechanisms of orofacial mesenchymal stem cells (OMSCs) in orofacial bone development have been studied intensively, the communication between OMSCs and osteoclasts remains largely unclear.Methods: We used a neural crest cell-specific knockout mouse model to investigate orofacial bone development in GATA-binding protein 4 (GATA4) morphants. We investigated the underlying mechanisms of OMSCs-derived exosomes (OMExos) on osteoclastogenesis and bone resorption activity in vitro. miRNAs were extracted from OMExos, and differences in miRNA abundances were determined using an Affymetrix miRNA array. Luciferase reporter assays were used to validate the binding between GATA4 and miR-206-3p in OMSCs and to confirm the putative binding of miR-206-3p and its target genes in OMSCs and osteoclasts. The regulatory mechanism of the GATA4-miR-206-3p axis in OMSC osteogenic differentiation and osteoclastogenesis was examined in vitro and in vivo.Results: Wnt1-Cre;Gata4fl/fl mice (cKO) not only presented inhibited bone formation but also showed active bone resorption. Osteoclasts cocultured in vitro with cKO OMSCs presented an increased capacity for osteoclastogenesis, which was exosome-dependent. Affymetrix miRNA array analysis showed that miR-206-3p was downregulated in exosomes from shGATA4 OMSCs. Moreover, the transcriptional activity of miR-206-3p was directly regulated by GATA4 in OMSCs. We further demonstrated that miR-206-3p played a key role in the regulation of orofacial bone development by directly targeting bone morphogenetic protein-3 (Bmp3) and nuclear factor of activated T -cells, cytoplasmic 1 (NFATc1). OMExos and agomiR-206-3p enhanced bone mass in Wnt1-cre;Gata4fl/flmice by augmenting trabecular bone structure and decreasing osteoclast numbers.Conclusion: Our findings confirm that miR-206-3p is an important downstream factor of GATA4 that regulates the functions of OMSCs and osteoclasts. These results demonstrate the efficiency of OMExos and microRNA agomirs in promoting bone regeneration, which provide an ideal therapeutic tool for orofacial bone deformities in the future.

Influence of Obesity in the miRNome: miR-4454, a Key Regulator of Insulin Response Via Splicing Modulation in Prostate

Obesity is a major health problem associated with severe comorbidities, including type 2 diabetes and cancer, wherein microRNAs (miRNAs) might be useful as diagnostic/prognostic tools or therapeutic targets. To explore the differential expression pattern of miRNAs in obesity and their putative role in obesity-related comorbidities such as insulin resistance. An Affymetrix-miRNA array was performed in plasma samples from normoweight (n = 4/body mass index < 25) and obese subjects (n = 4/body mass index > 30). The main changes were validated in 2 independent cohorts (n = 221/n = 18). Additionally, in silico approaches were performed and in vitro assays applied in tissue samples and prostate (RWPE-1) and liver (HepG2) cell-lines. A total of 26 microRNAs were altered (P < 0.01) in plasma of obese subjects compared to controls using the Affymetrix-miRNA array. Validation in ampler cohorts revealed that miR-4454 levels were consistently higher in obesity, associated with insulin-resistance (Homeostatic Model Assessment of Insulin Resistance/insulin) and modulated by medical (metformin/statins) and surgical (bariatric surgery) strategies. miR-4454 was highly expressed in prostate and liver tissues and its expression was increased in prostate and liver cells by insulin. In vitro, overexpression of miR-4454 in prostate cells resulted in decreased expression levels of INSR, GLUT4, and phosphorylation of AMPK/AKT/ERK, as well as in altered expression of key spliceosome components (ESRP1/ESRP2/RBM45/RNU2) and insulin-receptor splicing variants. Obesity was associated to an alteration of the plasmatic miRNA landscape, wherein miR-4454 levels were higher, associated with insulin-resistance and modulated by obesity-controlling interventions. Insulin regulated miR-4454, which, in turn may impair the cellular response to insulin, in a cell type-dependent manner (i.e., prostate gland), by modulating the splicing process.

MicroRNA-154-5p regulates the HPV16 E7-pRb pathway in Cervical Carcinogenesis by targeting CUL2.

Cervical cancer, induced by persistent HPV infection, has a high mortality rate. The E3 ubiquitin ligase Cullin 2 (CUL2) is critical for HPV16 E7-mediated degradation of retinoblastoma protein (pRb). Dysregulation of microRNAs (miRNAs) is induced during tumorigenesis; however, the association between miRNA networks and CUL2, specific to cervical cancer, remains unknown. Herein, we determined miRNA profiles in cervical cancer tissues using an Affymetrix miRNA array. We found that miR-154-5p was downregulated during cancer progression using real-time quantitative reverse transcription PCR in 130 biopsy specimens. Bioinformatics analysis and dual-luciferase reporter assays indicated that miR-154-5p directly targets the CUL2 3'UTR. To determine the functional consequences of modulating miR-154-5p and CUL2 levels, HPV16-positive cervical cancer cell line (SiHa) was transfected with miR-154-5p mimic, miR-154-5p inhibitor, or CUL2 siRNA. The proliferation, migration, and invasion of transfected cells were evaluated using CCK8 cell counting kit, wound-healing assay, and Transwell invasion assay. Increased miR-154-5p expression promoted significantly reduced SiHa cell proliferation, migration, and invasion, whereas the miR-154-5p inhibitor had the opposite effect. CUL2 silencing had similar effects to those of the miR-154-5p mimic. Consistent with the inverse correlation between miR-154-5p and CUL2 levels, CUL2 silencing also increased pRb expression. To our knowledge, this is the first study to demonstrate that miR-154-5p regulates pRb expression by targeting CUL2 3'UTR, thereby playing a tumor-suppressive role in HPV16 E7-induced cervical carcinogenesis.

MiRNA profile of neuroprotection mechanism of echinomycin in Parkinson’s disease

Echinomycin was known as the inhibitor of Hif1a. Discovery of echinomycin make expect for anti-breast cancer drug, but it has only useful in a limited dose in tumor because toxicity was appeared even in normal cells. In here, we developed echinomycin for Parkinson’s disease drug. For evidence, we performed the miRNA array using affymetrix miRNA array, and through the miRNA profiling we predicted the effects of echinomycin in Parkinson’s disease and finally, predicted phenomenon was verified by experiments such as viability assay and signaling checkup. In conclusion, Notch signaling pathway was decelerated and b-catenin stabilization was increased.

MicroRNAs as outcome predictors in patients with metastatic colorectal cancer treated with bevacizumab in combination with FOLFOX

Bevacizumab is a humanized anti-vascular endothelial growth factor monoclonal antibody, used in combination with a oxaliplatin-based chemotherapy in the treatment of metastatic colorectal cancer (mCRC). The aim of the present study was to identify microRNA (miRNA)-based predictive biomarkers of therapy response in order to avoid unnecessary and costly therapy to non-responding patients. High-throughput miRNA microarray profiling (Affymetrix miRNA array) was performed on a discovery cohort of patients with mCRC. The discovery cohort was (n=20) divided into either responding (n=10) or non-responding (n=10) groups of bevacizumab/5-flourouracil, leucovorin, oxaliplatin (FOLFOX) treatment according to Response Evaluation Criteria in Solid Tumors criteria. Validation of candidate miRNAs was performed on an independent cohort of 41 patients with mCRC using quantitative reverse transcription polymerase chain reaction. Normalized data were subjected to receiver operating characteristic and Kaplan-Meier analyses. In total, 67 miRNAs were identified to be differentially expressed when miRNA expression was compared between responding and non-responding patients to bevacizumab/FOLFOX treatment (P<0.05). A total of 7 miRNAs were chosen for independent validation, which confirmed significantly higher expression of miR-92b-3p, miR-3156-5p, miR-10a-5p and miR-125a-5p (P<0.005) in tumor tissue of responding patients compared with non-reponding patients. Using the combination of miRNAs, the present study identified responders to the therapy with sensitivity 82% and specificity 64% (area under the curve = 0.8015). In conclusion, 4 predictive miRNAs associated with progression-free survival (PFS) were identified in patients with mCRC treated with bevacizumab/FOLFOX. Following further independent validations, detection of these miRNA may enable identification of patients with mCRC who may potentially benefit from the therapy.

Abstract 223: Brain-derived Neurotrophic Factor Promotes Regenerative Functions of Human Endothelial Progenitor Cells - Roles of miRNAs

Our previous study has demonstrated that brain-derived neurotrophic factor (BDNF) enhances anti-oxidant capacity of endothelial progenitor cells (EPCs). Since BDNF is produced in cerebrovascular endothelial cells, and functions as a vasoprotective molecule, we hypothesized that BDNF might enhance regenerative capacity of EPCs. Early EPCs (circulating angiogenic cells) were derived from healthy male subjects (48±1.5 years old, n=10). EPCs were treated with BDNF 100 ng/ml for 7 hours, and miRNA transcriptome was examined using Affymetrix miRNA array (version 3.0). BDNF significantly changed 31 miRNAs (n=4, P&lt;0.05, fold-change cut off=1.5). Notably, miR-181c level was increased by 1.9 fold (P&lt;0.05) after BDNF treatment. Since miR-181c is known to down-regulate TNF-α expression, BDNF may protect EPCs by promoting anti-inflammatory phenotype. We also found that BDNF decreased miR-433 level by 1.6 fold (P&lt;0.05). We further evaluated miR-433 function in human early EPCs. Knock-down of miR-433 in EPCs using siRNA significantly increased expression of guanylate binding protein 2 (GBP2) (n=6, P&lt;0.05), an established direct target for miR-433. In contrast, treatment with miR-433 mimic significantly reduced GBP2 protein expression in EPCs (n=4, P&lt;0.05). Since GBP2 has been shown to play an important role in the growth and colony formation of hematopoietic progenitors, BDNF/miR-433/GBP2 signaling may enhance angiogenic capacity of EPCs. In addition, neither miR-433-siRNA nor miR433 mimic significantly changed protein expressions of other targets such as growth factor receptor-binding protein2, cAMP response element-binding protein-1, cyclin-dependent kinase 6, and γ glutamyl cysteine synthetase (n=4-6, P&gt;0.05). In summary, changes in miRNA transcriptome induced by treatment with BDNF may promote anti-inflammatory and angiogenic phenotype of EPCs. These effects of BDNF may have important implications for therapeutic enhancement of regenerative capacity of EPCs.

Abstract 473: A tumor and immune related miRNA signature predicts progression-free survival of melanoma patients treated with ipilimumab

Abstract Background Patients with regionally advanced melanoma were treated with neoadjuvant ipilimumab in a previously reported study (Tarhini et al, PLOS One 2014). MicroRNA (miRNA) expression profiles of tumors of treated patients were investigated for their therapeutic predictive value. Methods Patients were treated with ipilimumab (10 mg/kg IV every 3 weeks x2 doses) bracketing surgery. Tumor specimens were obtained at baseline and following ipilimumab at definitive surgery (week 6-8). MiRNA expression profiling was performed on the tumor biopsies of 30 patients using Affymetrix miRNA array (v.4). Significance Analysis of Microarrays (SAM) was performed to test the association of each differentially expressed miRNA molecule with outcome. Targets of the selected miRNAs were obtained from miRTarBase (http://mirtarbase.mbc.nctu.edu.tw). Functional annotation analysis of the list of miRNA target genes were performed using DAVID (https://david.ncifcrf.gov). The FDR method was used to adjust for multiple testing in SAM and the functional analysis. Results An expression profile consisting of a 4-miRNA signature was associated with improved progression free survival. The signature consisted of miR-34c (previously reported to suppress cancer growth and invasion), miR-711 (reported as a prognostic marker in cutaneous T-cell lymphomas and to target and suppress Heat Shock Protein 70 highly expressed in melanoma), miR-641 (activates MAPK by targeting NF1 and cooperates with its host gene AKT2 in human cancer) and miR-22 (reported to function as a tumour suppressor). Functional annotation analysis of target genes for the 4-miRNA signature was statistically significantly enriched for various cancer-related pathways including regulation of cell proliferation (GO:0042147), regulation of apoptosis (GO:0042981), MAPK signaling pathway (hsa04010) and positive regulation of T cell activation (GO:0050870). Conclusions MiRNA expression profiling identified a 4-miRNA signature that is significantly associated with PFS in advanced melanoma patients treated with neoadjuvant ipilimumab. Preliminary results show that targets of these 4 miRNAs are biologically relevant and important. These findings warrant further investigation in relation to ipilimumab and other immunotherapeutics. Citation Format: Ahmad Tarhini, Priyanka Vallabhaneni, Theofanis Floros, William A. LaFramboise, Panayiotis V. Benos, Lucas Santana dos Santos. A tumor and immune related miRNA signature predicts progression-free survival of melanoma patients treated with ipilimumab. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 473.

Novel Association of miR-451 with the Incidence of TEVG Stenosis in a Murine Model.

The development of a tissue-engineered vascular graft (TEVG) holds great promise for advancing the field of cardiac surgery. Despite the successful translation of this technology, previous reports identify the primary mode of graft failure as stenosis secondary to intimal hyperplasia. MicroRNAs (miRNAs) regulate gene expression by interfering with mRNA function and recent research has suggested miRNA as a potential therapeutic target. The role of miRNAs in TEVGs during neotissue formation is currently unknown. In this study, we investigated if miRNAs regulate the inhibition of graft stenosis. Biodegradable PGA-P(LA/CL) scaffolds were implanted as inferior vena cava interposition grafts in a murine model (n = 14). Mice were sacrificed 14 days following implantation and TEVGs were harvested for histological analysis and miRNA profiling using Affymetrix miRNA arrays. Graft diameters were measured histologically, and the largest grafts (patent group) and smallest grafts (stenosed group) were profiled (n = 4 for each group). Cell population in each graft was analyzed with immunohistochemistry using antismooth muscle actin (SMA) and antimacrophage (F4/80) antibodies. The graft diameter was significantly greater in the patent group (0.63 ± 0.06 mm) than in the stenosed group (0.17 ± 0.06 mm) (p < 0.01). Cell proliferation was significantly greater in the stenosed grafts than in patent grafts (p < 0.01: SMA [187 ± 11 vs. 77 ± 8 cells] vs. p = 0.025: F4/80 [245 ± 23 vs. 187 ± 11 cells]). MiRNA array of 1416 genes showed that in stenosed grafts, mir-451, mir-338, and mir-466 were downregulated and mir-154 was upregulated. Mir-451 exhibited the greatest difference in expression between stenosed and patent grafts by -3.1-fold. Significant negative correlation was found between the expression of mir-451 and cell proliferation (SMA: r = -0.86, p = 0.003; F4/80: r = -0.89, p = 0.001). Our data, along with previous evidence that mir-451 regulates tumor suppressor genes, suggest that downregulation of mir-451 promotes acute proliferation of macrophages and smooth muscle cells, thereby inducing TEVG stenosis. Adequate expression of mir-451 may be critical for improving TEVG patency.

Circulating microRNA-196a as a candidate diagnostic biomarker for chronic hepatitis C.

Previous studies have demonstrated the inhibitory effect of microRNA (miR)-196a on hepatitis C virus (HCV) expression in human hepatocytes. However, the clinical implications of aberrant miR-196a expression and the application of circulating miR-196a in the diagnosis and management of chronic hepatitis C (CHC) require further investigation. The present study aimed to examine the possibility of using serum miR-196a as a biomarker for CHC. The Affymetrix miRNA array platform was used for miRNA expression profiling in adenovirus (Ad)-HCV core-infected (HepG2-HCV) and Ad-enhanced green fluorescence protein (EGFP)-infected HepG2 cells (HepG2-control). miR-196a downregulation and levels were analyzed using stem-loop reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of the sera of 43 patients with CHC and 22 healthy controls. A total of six miRNAs were identified as significantly different (≥1.5 fold; P≤0.05) between the two groups. Of note, significant miR-196a downregulation was observed in HepG2-HCV as compared with HepG2-EGFP. Furthermore, as compared with that of the healthy control group, serum miR-196a was demonstrated to be significantly lower in patients with CHC. In addition, analysis of the receiver operating characteristic (ROC) curve for serum miR-196a revealed an area under the ROC curve of 0.849 (95% confidence interval, 0.756–0.941; P<0.001) with 81.8% sensitivity and 76.7% specificity in discriminating chronic HCV infection from healthy controls at a cut-off value of 6.115×10‒5, demonstrating significant diagnostic value for CHC. However, no correlation was identified between serum miR-196a and alanine aminotransferase, aspartate aminotransferase or HCV-RNA. In conclusion, the present study identified circulating miR-196a as a specific and noninvasive candidate biomarker for the diagnosis of CHC.

MiR-222 contributes to sex-dimorphic cardiac eNOS expression via ets-1

It is well recognized that there is sex-dimorphic expression of mRNA and protein in the heart; however, the underlying mechanism is poorly understood. Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiac function, and the expression levels of eNOS differ between male and female hearts. The aim of this study was to examine whether expression of specific microRNA (miRNA, miR) in males and females contributes to changes in the expression of eNOS. miRNA was extracted from the myocardium of male and female C57BL/6 mice and subjected to an Affymetrix miRNA array. Decreased expression of miR-222 was discovered in females and confirmed by qRT-PCR from whole heart or isolated cardiomyocytes. The transcription factor V-ets erythroblastosis virus E26 oncogene homolog-1 (ets-1) was identified as a potential target of miR-222 using TargetScan, and fivefold increased ets-1 protein expression in females was confirmed by Western blot. Targeting of ets-1 by miR-222 was determined in HEK293 cells overexpressing luciferase under regulation of either the ets-1 3'-UTR, a null 3'-UTR control, or a scrambled ets-1 3'-UTR and treated with a small molecule miR-222 mimic or inhibitor. Additionally qRT-PCR confirmed that mRNA levels of the ets-1 transcriptional target, eNOS, were 25% higher in females. Compared with untreated myocyte controls, 50% inhibition of eNOS expression was achieved by treatment with a miR-222 mimic, compared with a 25% increase due to miR-222 inhibitor. Our findings indicate that sex-dependent miR-222 regulation alters the expression of the cardiac regulatory protein eNOS.

Abstract 3086: Modulation of microRNA profile in lung adenocarcinoma cell line following TIMP-1 downregulation.

Abstract Non-small cell carcinoma accounts for 85% of lung cancer cases and has a 5 year survival rate of only 17%. In recent years, tissue inhibitor of metalloproteinase-1 (TIMP-1) has emerged as an important prognostic marker. In several cancers including non-small cell lung cancer, high serum levels of TIMP-1 have been associated with poor prognosis. Previously, we have shown that overexpressing TIMP-1 in H2009, a lung adenocarcinoma cell line resulted in aggressive tumors when implanted in the mouse brain. In the present study, we have knocked down TIMP-1 expression in the non-small cell lung cancer cell line A549 using shRNA. In vitro assays using the TIMP-1 knockdown clones defined decreased migration in wound assays (p&amp;lt;0.01), as well as reduced invasion (p&amp;lt;0.05) using Boyden chamber assays. We investigated these functional alterations by examining microRNAs in these cell lines. MicroRNAs represent a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level and control crucial signaling and physiological processes functioning either as oncogenes or tumor-suppressors. The miRNA expression profile of A549 cell line and its TIMP1 knockdown clone were evaluated using the Affymetrix miRNA microarray platform on Gene-Chip miRNA 3.0 array in A549 cell line. Following hybridization and data acquisition, we used Partek Genomics Suite ® software for RNA normalization and to determine statistically significant differences in miRNA expression between experimental groups by ANOVA and pairwise comparisons (two-sidedα=0.05). Using the Affymetrix miRNA array we have identified altered miRNAs expression in the clones, with 72 differentially regulated miRNAs following TIMP-1 knockdown. Using quantitative real time PCR we have selectively validated several of the miRNAs that showed the maximal difference. We have confirmed a 2 to 4-fold down regulation of MiR-10b, miR-29a and miR-30 and a 3-fold upregulation of miR-125a-5p. Based on KEGG pathway analysis, this group of validated miRNAs are involved in the MAPK signaling pathway. Western blot analysis showed activation of the apoptosis inducing p38 MAPK pathway in the knockdown clones. Additional mechanistic dissection of these changes and involved pathways will contribute further to our understanding of the role of TIMP-1 in lung cancer. (This research was supported by a Georgia Distinguished Cancer Scholar award to AMR). Citation Format: Sampa Ghoshal-Gupta, Ammar Kutiyanawalla, Shahanawaz Jiwani, Juhi Ojha, Ravindra Kolhe, Amyn M. Rojiani, Mumtaz V. Rojiani. Modulation of microRNA profile in lung adenocarcinoma cell line following TIMP-1 downregulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3086. doi:10.1158/1538-7445.AM2013-3086

Abstract 5277: MicroRNA expression profile of astrocytoma in archived formalin-fixed paraffin-embedded brain tumor samples.

Abstract Astrocytomas, a tumor derived from astrocytic glial cells, are the most common type of adult primary central nervous system (CNS) neoplasms, representing around 76% of all primary brain tumors. The World Health Organization (WHO) classified astrocytomas into four histological grades spanning from grade I, a benign tumor, to grade IV, a highly malignant tumors. This classification is somewhat subjective and more reliable molecular markers for tumor grading are necessary. MicroRNAs (miRNAs) are small (≈22-nucleotide) noncoding RNAs that, in mammals, regulate genes at the translational level by binding to loosely complimentary sequences in the 3’-UTR of target mRNAs. There is increased in vitro and in vivo evidence that various miRNAs are deregulated in a variety of cancer including astrocytoma. The aim of this study was to assess miRNAs dysregulated in astrocytoma according to its grade using archived formalin-fixed paraffin-embedded (FFPE) samples. Total RNA was isolated, labeled, and hybridized to Affymetrix miRNA arrays. Images were analyzed using the PARTEK software. Eighty-five miRNAs were differentially regulated in grade II, 72 miRNAs in grade III and 118 miRNAs in GBM; compared with control samples. Several non-previously described miRNA in astrcoytoma were identified in this study. The most differentially expressed miRNAs in each tumor grade, were selected for further validation by TAqMan-based real-time PCR. Particularly, miR-92b was upregulated in all astrocytoma grades. Opposite, miR-548p, a miRNA regulating antiapoptotic molecules, was downregulated in GBM tumor tissues, and in well-established GBM cell lines. However, the role of these miRNAs in GBM cells has not been studied. We are currently performing in vitro studies to clarify the role of some of these microRNAs in the cell proliferation of GBM cells. Citation Format: Mónica Rivera-Díaz, Mario Quintero, Julio López, P E. Vivas-Mejia. MicroRNA expression profile of astrocytoma in archived formalin-fixed paraffin-embedded brain tumor samples. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5277. doi:10.1158/1538-7445.AM2013-5277

Analytical variables influencing the performance of a miRNA based laboratory assay for prediction of relapse in stage I non-small cell lung cancer (NSCLC)

BackgroundLaboratory assays are needed for early stage non-small lung cancer (NSCLC) that can link molecular and clinical heterogeneity to predict relapse after surgical resection. We technically validated two miRNA assays for prediction of relapse in NSCLC. Total RNA from seventy-five formalin-fixed and paraffin-embedded (FFPE) specimens was extracted, labeled and hybridized to Affymetrix miRNA arrays using different RNA input amounts, ATP-mix dilutions, array lots and RNA extraction- and labeling methods in a total of 166 hybridizations. Two combinations of RNA extraction- and labeling methods (assays I and II) were applied to a cohort of 68 early stage NSCLC patients.ResultsRNA input amount and RNA extraction- and labeling methods affected signal intensity and the number of detected probes and probe sets, and caused large variation, whereas different ATP-mix dilutions and array lots did not. Leave-one-out accuracies for prediction of relapse were 63% and 73% for the two assays. Prognosticator calls ("no recurrence" or "recurrence") were consistent, independent on RNA amount, ATP-mix dilution, array lots and RNA extraction method. The calls were not robust to changes in labeling method.ConclusionsIn this study, we demonstrate that some analytical conditions such as RNA extraction- and labeling methods are important for the variation in assay performance whereas others are not. Thus, careful optimization that address all analytical steps and variables can improve the accuracy of prediction and facilitate the introduction of microRNA arrays in the clinic for prediction of relapse in stage I non-small cell lung cancer (NSCLC).