Stem-loop RT Research Articles

Evaluation of the expression level of microRNA-21, microRNA-15a, microRNA-372 in human follicular fluid stem cells-derived oocyte-like cells (OLCs).

Today, researchers have succeeded in achieving oocyte-like cells through the in vitro differentiation of stem cells. MicroRNAs are key regulators of oocyte development. In this study we decided to evaluate the expression pattern of microRNA-21, microRNA-15a, and microRNA-372 in oocyte-like cells, to determine the maturation stage of oocyte-like cells. Human follicular fluid samples were collected and centrifuged, and their cells were divided into 3 groups; day 7 as control group, days 14 and 21. During this period, the cells were evaluated for their morphological appearance and viability by inverted microscopy. RNA isolation was performed and cDNA was reversely transcribed by specific stem-loop RT primers. Real-time RT-PCR was used to detect microRNA expression. The relative expression of microRNA-21 and microRNA-15a on day 21 was significantly down-regulated compared to the control group (day 7), but microRNA-372 did not show a significant difference. Also, on day 14 compared to the control group (day 7), microRNA-21 did not show a significant difference; but microRNA-15a and microRNA-372 were significantly down-regulated. MicroRNA-21 and microRNA-15a on day 21 compared to day 14 revealed down-regulated levels, but microRNA-372 revealed up-regulated levels. Our results showed significant decreases in the expression of microRNA-21 and microRNA-15a in oocyte-like cells, as well as in oocytes, which may lead to cytoplasmic maturation, germinal vesicle break down and the completion of meiosis І. In addition, down-regulation expression of microRNA-372 maybe a confirmation that mesenchymal stem cells have differentiated into germ cells, and these cells were differentiated into oocyte-like cells.

B-370 Clinical Utility of microRNA Quantitative Analysis Using Double Quencher Probes and Methylation Analysis by Methylation Sensitive Restriction Enzyme Polymerase Chain Reaction for Bladder Cancer

Abstract Background Histone modifications, DNA methylation, and microRNA (miRNA)-mediated gene silence are examples of epigenetic changes that regulate gene expression without causing sequence changes. The early phases of neoplastic transformation are characterized by the occurrence of these epigenetic changes, which are believed to be crucial for the development and progression of cancer. To increase the sensitivity and practicality of miRNA analysis, we developed a measuring technique using a ZEN double quencher into hydrolysis probe in reverse transcription quantitative polymerase chain reaction (RT-qPCR). Additionally, we developed a method for DNA methylation analysis employing methylation-sensitive restriction enzymes (MSRE) and methylation-insensitive restriction enzymes (MIRE) that can analyze a number of samples quickly. We examined the clinical usefulness of using these systems to understand the level of miRNA expression and methylation in bladder cancer. Methods In this study, 64 biopsied tissues from transurethral bladder tumor resection (TURBT) were used. For miRNA extraction, Cica geniusTM RNA Prep Kit (For Tissue) II (KANTO CHEMICAL CO.,INC.) and a solution that improves miRNA extraction efficiency were used. Using stem-loop RT primers, complementary DNA was created from the isolated miRNA solution. Quantitative analysis was performed by RT-qPCR using a ZEN double quencher probe and miR-21,30a, 200c, and U6 were used as target. Genomic DNA (gDNA) was extracted from the TURBT sample using High Pure PCR Template Preparation Kit (Roche). Restriction enzyme digestion was performed using HapII (Takara Bio) as MSRE and MspI (Takara Bio) as MIRE; the latter has the same recognition site as HapII but is not affected by the presence or absence of methylation. Real-time PCR was performed on the LightCycler 96 (Roche) using Universal ProbeLibrary probes (Roche) targeting ESM1, SFMBT2, and SEPTIN9. From the obtained Cp value, each methylation rate was calculated. The Fisher’s exact test was used to statistically analyze the relationship among miRNA, methylation data and other clinicopathological factors. Results For miR-30a, we observed a higher expression of miRNA was linked to more advanced T stage (P = 0.047). Additionally, ESM1 methylation exhibited a significant correlation with greater grade (P = 0.004) and T stage progress (P = 0.018). Data on miRNA abundance and methylation rate were combined in order to uncover a link with clinical data that was not apparent from either set of data separately. Samples with higher values for both miR-21 and ESM1 methylation rates were shown to have high grade (P = 0.007) and progress to the T stage (P = 0.006). Furthermore, samples with higher values for both miR-200c and SEPTIN9 methylation rates did not progress to the T stage (P = 0.013). Conclusion The combined miRNA and methylation data revealed relationships with clinical results that could not be discovered with either data separately, highlighting the clinical usefulness of the current study’s findings.

Evaluating the Differential Expression of miR-146a, miR-222, and miR-9 in Matched Serum and Follicular Fluid of Polycystic Ovary Syndrome Patients: Profiling and Predictive Value.

Polycystic ovary syndrome (PCOS) is the most prevalent endocrine disorder of women in reproductive age with significant effects on reproductive and metabolic functions. Many molecular players may be involved in PCOS pathology; however, miRNAs possess great ability in gene expression control in normal ovarian function and folliculogenesis. We appraised the relative expression of miR-146a, miR-222, miR-9, and miR-224 in serum and follicular fluid (FF) of PCOS patients compared to control subjects. PCOS (n = 35) and control (n = 30) subjects were recruited in the study during their enrolment in IVF cycles. Serum and FF of human subjects were collected and stored. Total RNA was isolated from samples and cDNA was synthesized using miRNA-specific stem-loop RT primers. Quantitative real-time PCR was used to evaluate the expression of miRNAs relative to U6 expression. The predictive value of miRNAs' expression for discrimination of PCOS patients from control subjects was evaluated by receiver-operating characteristic (ROC) curve analysis. miR-224 was not detected in serum and FF samples. Significantly, higher levels of miR-146a and miR-9 in serum of PCOS group were detected. In contrast, relative expression of miR-146a and miR-9 significantly decreased in FF. In PCOS group, relative expression of all detected miRNAs was elevated in serum in comparison to FF, whereas in control group no change was noticed. Combination of FF miRNAs showed improved predictive value with area under the ROC curve (AUC) of 0.84, 93.8% sensitivity, and 83.3% specificity. Contradicting alternations of miRNAs in serum and FF are indicative of different sources of miRNAs in body fluids. Presumptive target genes of studied miRNAs in signalling pathways may show the potential role of these miRNA in folliculogenesis.

Quantification of microRNAs in Cells and Tissues Using Stem-Loop RT PCR and qPCR.

MicroRNA s are small RNA molecules that regulate gene expression by binding to the 3' untranslated region of the mRNA of their target genes. MicroRNA expression is altered in medulloblastoma as compared to the normal brain and this alteration is often associated with the pathogenesis of this tumor. The quantification of microRNA expression is carried out using quantitative/real-time polymerase chain reaction (PCR). In this chapter, we describe the protocol for the quantification of microRNA s in medulloblastoma tissues and cultured cells. This is carried out in three steps: (1) Extraction of total RNA, (2) Stem-loop reverse-transcriptase PCR, and (3) quantitative PCR.

Identification and the potential involvement of miRNAs in the regulation of artemisinin biosynthesis in A. annua

Micro RNAs (miRNAs) play crucial regulatory roles in multiple biological processes. Recently they have garnered the attention for their strong influence on the secondary metabolite production in plants. Their role in the regulation of artemisinin (ART) biosynthesis is, however, not fully elucidated. ART is a potent anti-malarial compound recommended by WHO for the treatment of drug-resistant malaria. It is produced by Artemisia annua (A. annua). The lower in planta content of ART necessitates a deep understanding of regulatory mechanisms involved in the biosynthesis of this metabolite. In this study, using modern high throughput small RNA-sequencing by Illumina Nextseq 500 platform for identification and stem-loop RT PCR for validation, miRNAs were identified in the leaf sample of A. annua plant. Here, we report a total of 121 miRNAs from A. annua that target several important genes and transcription factors involved in the biosynthesis of ART. This study revealed the presence of some important conserved miRNA families, miR396, miR319, miR399, miR858, miR5083 and miR6111 not identified so far in A. annua. The expression patterns and correlation between miRNAs and their corresponding targets at different developmental stages of the plant using real-time PCR indicate that they may influence ART accumulation. These findings thus, open new possibilities for the rational engineering of the secondary metabolite pathways in general and ART biosynthesis in particular.

In-particle stem-loop RT-qPCR for specific and multiplex microRNA profiling

Here we report a novel method of microRNA (miRNA) profiling with particle-based multiplex quantitative reverse transcription polymerase chain reaction (RT-qPCR). To achieve target-specific reaction in a particle, the stem-loop RT primer and forward primer for each target miRNA were chemically immobilized to the particle. Target-specific cDNA synthesis proceeds with the stem-loop RT primer and then qPCR subsequently proceeds with the forward primer to rapidly achieve a quantitative result. High-fidelity multiplex assay was also accomplished in a single PCR process by loading multiple particles for each specific miRNA. The method for primer supply in the particles, involving confinement of the target-specific RT and PCR primers in the matrix of particles, led to the reduction of nonspecific reactions and improved the selectivity of the miRNA assay while minimizing labor in a multiple target assay. Specifically, this particle-based assay enabled the differentiation of mature miRNA from precursor with selectivity of 270:1 in terms of amplification speed. This advanced method also showed good discrimination among highly homologous let-7 family members, with cross-reaction rates of less than 5%. We demonstrated a very simple process of five-plex miRNA profiling in total RNA, and the measured changes in expression level were consistent with those from a conventional singleplex method.

Redox-sensitive, PEG-shielded carboxymethyl PEI nanogels silencing MicroRNA-21, sensitizes resistant ovarian cancer cells to cisplatin

A series of branched polyethylenimine (PEI) modifications including PEGylation (PEG2k-PEI) for steric shielding, redox-sensitive crosslinking for synthesis PEG2k-PEI-ss nanogels and subsequent carboxymethylation (PEG2k-CMPEI-ss) for modulation of the polymer pka have been introduced for cellular delivery of Anti-miR-21. The synthesis was characterized using 1H NMR, FTIR, TNBS, potentiometric titration, particle size and ζ potential. Loading of Anti-miR-21 at various N/P ratios was investigated by gel retardation, ethidium bromide dye exclusion, heparin sulfate competition and DNase I digestion experiments. The miR-21 silencing was measured by stem-loop RT PCR in A2780 ovarian cancer cell lines whether it is sensitive to resistant to cisplatin. It has been shown that PEG2k-CMPEI-ss was well suited for delivery of Anti-miR-21 in terms of nucleic acid loading, preservation against extracellular matrix and nucleases and sequence-specific silencing of miRNA-21 in vitro. Moreover, it has been demonstrated that pre-treating cells with Anti-miR-21 loaded nanogels can sensitize them to cis-Pt even at non-toxic concentraions. The results indicate that PEG2k-CMPEI-ss mediated microRNA delivery can be considered as a novel strategy for ovarian cancer therapy.

Expression of miR-15a, miR-145, and miR-182 in granulosa-lutein cells, follicular fluid, and serum of women with polycystic ovary syndrome (PCOS).

Polycystic ovary syndrome (PCOS) is one of the most common endocrinopathies that affects women in reproductive age. MicroRNAs (miRNAs) play crucial roles in normal function of female reproductive system and folliculogenesis. Deregulated expression of miRNAs in PCOS condition may be significantly implicated in the pathogenesis of PCOS. We determined relative expression of miR-15a, miR-145, and miR-182 in granulosa-lutein cells (GLCs), follicular fluid (FF), and serum of PCOS patients. Human subjects were divided into PCOS (n=20) and control (n=21) groups. GLCs, FF, and serum were isolated and stored. RNA isolation was performed and cDNA was reversely transcribed using specific stem-loop RT primers. Relative expression of miRNAs was calculated after normalization against U6 expression. Correlation of miRNAs' expression level with basic clinical features and predictive value of miRNAs in FF and serum were appraised. Relative expression of miR-145 and miR-182 in GLCs was significantly decreased in PCOS, but miR-182 in FF of PCOS patients revealed up-regulated levels. Significant correlations between level of miRNAs in FF and serum and hormonal profile of subjects were observed. MiR-182 in FF showed a significant predictive value with AUC of 0.73, 76.4% sensitivity, and 70.5% specificity which was improved after combination of miR-182 and miR-145. A significant dysregulation of miR-145 and miR-182 in GLCs of PCOS may indicate their involvement in pathogenesis of PCOS. Differential up-regulation of miR-182 in FF of PCOS patients with its promising predictive values for discrimination of PCOS reinforced the importance of studying miRNAs' profile in FF.

MP60-20 IDENTIFICATION OF MICRORNA REGULATING SUNITINIB RESISTANCE IN RENAL CELL CARCINOMA CELLS

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II1 Apr 2017MP60-20 IDENTIFICATION OF MICRORNA REGULATING SUNITINIB RESISTANCE IN RENAL CELL CARCINOMA CELLS Noriya Yamaguchi, Tetsuya Yumioka, Kunishige Onuma, Hideto Iwamoto, Toshihiko Masago, Shuichi Morizane, Masashi Honda, Mitsuhiko Osaki, Futoshi Okada, and Atsushi Takenaka Noriya YamaguchiNoriya Yamaguchi More articles by this author , Tetsuya YumiokaTetsuya Yumioka More articles by this author , Kunishige OnumaKunishige Onuma More articles by this author , Hideto IwamotoHideto Iwamoto More articles by this author , Toshihiko MasagoToshihiko Masago More articles by this author , Shuichi MorizaneShuichi Morizane More articles by this author , Masashi HondaMasashi Honda More articles by this author , Mitsuhiko OsakiMitsuhiko Osaki More articles by this author , Futoshi OkadaFutoshi Okada More articles by this author , and Atsushi TakenakaAtsushi Takenaka More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1857AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Sunitinib is indicated as first-line treatment of metastatic renal cell carcinoma (mRCC) and suppress angiogenesis and tumor cell proliferation through binding to vascular endothelial growth factor receptor and platelet-derived growth factor receptor. However, the clinical benefit in progression free survival is limited and almost patients have a relapse of disease due to acquired resistance. MicroRNA, which are non-protein-coding small RNAs, are involved in cancer progression and regulate gene expression at the post-transcriptional level by binding to the untranslated region (3‘UTR) of target mRNAs. The purposes of this study were to generate sunitinib resistant RCC cell lines and to detect candidate miRNAs for regulating sunitinib resistance. METHODS The renal cell carcinoma cell line ACHN and RCC23 were cultured in RPMI1640 with 10% Fetal Bovine Serum and 1% of Penicillin-Streptomycin. Sunitinib resistant cells were generated by continuous exposure for three months and gradually increasing doses of sunitinib up to IC50% (inhibitory concentration) of each cell. We used these cells as sunitinib resistant ACHN and RCC23 cell lines (SR-ACHN, SR-RCC23) for the subsequent verification. For microarray analysis, total RNA was labeled using a 3D-Gene miRNA labeling kit. Reverse transcription was performed with a stem-loop RT Megaplex Primer Pool and the Taqman MicroRNA Reverse Transcription Kit. RESULTS According to previous MTT assay, the IC50% of ACHN cell was 10μM and that of RCC23 cell was 14μM. SR-ACHN and SR-RCC23 cells exhibited significantly higher resistance to sunitinib treatment compared with that of these sunitinib sensitive cells. Microarray analysis was performed comparing ACHN vs SR-ACHN and RCC23 vs SR-RCC23, respectively, to evaluate the miRNA profiles of each cells. In SR-ACHN and SR-RCC23 cells as compared with ACHN and RCC23 cells, the CT values of miR-575, -642b-3p and -4430 have significantly increased, while the CT values of miR-18a-5p, -29b-1-5p, -431-3p and -4521 have significantly decreased with real-time RT-PCR. CONCLUSIONS We created sunitinib resistant cell lines SR-ACHN and SR-RCC23 and identified microRNAs considering related with sunitnib resistant by performing microarray. By regulating these microRNAs may contribute to the improvement of sunitinib resistance. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e797-e798 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Noriya Yamaguchi More articles by this author Tetsuya Yumioka More articles by this author Kunishige Onuma More articles by this author Hideto Iwamoto More articles by this author Toshihiko Masago More articles by this author Shuichi Morizane More articles by this author Masashi Honda More articles by this author Mitsuhiko Osaki More articles by this author Futoshi Okada More articles by this author Atsushi Takenaka More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Clinicopathological features associated to MiRNA-195 expression in patients with breast cancer: Evidence of a potential biomarker.

Background & Objective:MiRNAs are a systematic class of small non-coding RNAs with impending role as tumor biomarkers. Our objective was to identify the level of expression of Mir-195 in patients with breast cancer along with its correlation with clinicopathologic features.Methods:A total of 209 females in which 139 histologically diagnosed breast cancer (BC) cases and 70 healthy controls matched for age, their relative clinical and histopathological findings were recorded from their laboratory reports and hospital record of the patients. Plasma was used for extraction of total RNA and cDNA was prepared by using both miR-195 stem loop RT primers and gene specific antisense primers while U6 IT was used as control. Quantitative real-time PCR (qRT-PCR) for miR-195 expression status was performed and amplification (down regulation) was recorded.Results:Of 139 samples the expression of miR-195 was down regulated in 72.6% cases and the remaining 27.3% cases behaved same as 70 healthy or normal controls. Significant correlation of low miR-195 expression with higher differentiation grade, lymph node metastasis and clinical stage was found.Conclusion:Significant correlation between miR-195 expression and some clinicopathological features were recognized. MiR-195 could be used as potential non-invasive, molecular biomarker for early detection of breast cancer.

Stem-Loop qRT-PCR for the Detection of Plant microRNAs.

Plant microRNAs (miRNAs) play important roles in the posttranscriptional regulation of protein-coding genes, and they are essential for a normal development and survival. Mature miRNAs are cleaved from larger precursor RNAs and are typically 21-22 nt long.The small size, the lack of a common feature like a poly(A) tail, 3' end-modifications, and presence of a precursor-all these factors affect the detection and hinder the quantification of miRNAs. The stem-loop qRT-PCR method described here is designed to detect and quantify mature miRNAs in a fast, specific, accurate, and reliable manner. Firstly, a miRNA-specific stem-loop RT primer is hybridized to miRNA and then reverse transcribed. Next, the RT product is amplified and monitored in real time using a miRNA-specific forward primer and a universal reverse primer. This method enables miRNA expression profiling from as little as 10 pg of total RNA, and it is suitable for a relatively high-throughput analysis of miRNA expression.

Considerations for optimization of microRNA PCR assays for molecular diagnosis

ABSTRACTThe remarkable stability of microRNAs in biofluids underlies their potential as biomarkers, but their small size presents challenges for detection by RT-qPCR. The heterogeneity of microRNAs, with each one comprising a series of variants or ‘isomiRs’, adds additional complexity. Presented here are the key considerations for use of RT-qPCR to measure microRNAs and their isomiRs, with a focus on plasma. Modified nucleotides can be incorporated into primer sequences to enhance affinity and provide increased specificity and sensitivity for RT-qPCR assays. Approaches based upon polyA tailing and use of a common oligo(dT)-based reverse transcription oligonucleotide will detect most isomiRs. Conversely, stem-loop RT oligonucleotides and sequence specific probes can enable detection of specific isomiRs of interest. Next generation sequencing of all the products of a microRNA RT-PCR reaction is a promising new approach for both microRNA quantification and characterization.

Molecular cystoscopy: Micro-RNAs could be a marker for identifying genotypic changes for transitional cell carcinoma of the urinary bladder.

Introduction:Normal-looking mucosa may harbor genetic changes preceding a visible tumor. This study was aimed at exploring the role of the quantitative expression of micro-RNAs (miRNAs) in bladder cancer tissue in comparison with normal mucosa and healthy controls (HCs) as a molecular marker.Materials and Methods:Between October 2011 to December 2012, tissue from the bladder tumor of 21 patients (cases tumor, CT), normal mucosa (case control, CC) of the same patients (n-21) and normal bladder mucosa from 10 HCs were obtained. miRNAs of angiogenesis, endothelial mesenchymal transition and apoptosis were quantified using stem–loop RT Taq Man polymerase chain reaction. Statistical analysis was performed using the Chi square and independent sample T tests by using SPSS version 16.Results:The mean age of the patients and controls were 55.41 ± 11.03 and 52.14 ± 13.04 years. miR-21, miR-205, miR-126, miR-10b and miR-200a were highly expressed in CT (P < 0.027, <0.048, <0.025, <0.029 and < 0.005) as compared with HC. Expression of miR-21 and miR-129 were both correlated with grade and stage (P = 0.001 and < 0.009, respectively) and the level of expression was different in the same grade of non-muscle invasive tumors. The fold change of miR129, miR205 and miR200a was significantly higher in the normal-looking mucosa of bladder tumor patients than the HC (P < 0.005).Conclusion:Expression of miR129, miR205 and miR200a in the normal-looking mucosa of bladder cancer patients was significantly higher than the normal mucosa of a HC. This may help in predicting recurrence and formulating the follow-up strategy.

MicroRNA-34a induces a senescence-like change via the down-regulation of SIRT1 and up-regulation of p53 protein in human esophageal squamous cancer cells with a wild-type p53 gene background

MiR-34a has been reported as a non-coding RNA universally expressed in normal old cells and a probable suppressor of diverse cancer cells; however, this miRNA's expression and anti-tumor mechanism in esophageal squamous cancer cells (ESCC) remains unclear. We explored these questions in three human ESCC lines, KYSE-450, KYSE-410, and ECa-109, with wild-type p53 and mutant p53 backgrounds. Through a specific stem-loop RT primer for miR-34a, we examined the relevant expression level of miR-34a in these three cell lines using real-time reverse transcription PCR (qRT-PCR). We found that the expression level of miR-34a induced by the DNA damage agent adrmycin (ADR) was both p53- and time-dependent. Following incubation with miR-34a, cellular growth inhibition was exhibited differently in the three cell lines harbored with different p53 backgrounds. Furthermore, the MTT assay demonstrated an miR-34a-related cytotoxic effect in cell growth. Senescence-associated β-galactosidase (SA-β-Gal) staining was used to examine senescence-like phenotypes induced by miR-34a. Mechanistic investigation suggested that the down-regulation of Sirtuin1 (SIRT1) and up-regulation of p53/p21 contributed to the anti-tumor mechanism of miR-34a in wild-type p53 ECa-109 cells, while neither of the apoptosis-related proteins PARP and caspase-3 caused significant changes. In summary, our findings indicated that the intrinsic expression of miR-34a was relatively low and was expressed differently among different p53 backgrounds and ADR treatment times. The anti-tumor effect of miR-34a was primarily dependent on the regulation of SIRT1 and p53/p21 protein, not apoptosis-associated proteins.

Establishment and in-house validation of stem-loop RT PCR method for MicroRNA398 expression analysis

MicroRNAs (miRNAs) belong to the class of small non-coding RNAs which have important roles throughout development as well as in plant response to diverse environmental stresses. Some of plant miRNAs are essential for regulation and maintenance of nutritive homeostasis when nutrients are in excess or shortage comparing to optimal concentration for certain plant species. Better understanding of miRNAs functions implies development of efficient technology for profiling their gene expression. We set out to establish validate the methodology for miRNA gene expression analysis in cucumber grown under suboptimal mineral nutrient regimes, including iron deficiency. Reverse transcription by ?stem-loop? primers in combination with Real time PCR method is one of potential approaches for quantification of miRNA gene expression. In this paper we presented a method for ?stem loop? primer design specific for miR398, as well as reaction optimization and determination of Real time PCR efficiency. Proving the accuracy of this method was imperative as ?stem loop? RT which consider separate transcription of target and endogenous control. The method was verified by comparison of the obtained data with results of miR398 expression achieved using a commercial kit based on simultaneous conversion of all RNAs in cDNAs.

Small RNA existed in commercial reverse transcriptase: primary evidence of functional small RNAs.

MicroRNAs (miRNAs) are small RNA molecules (approximately 22 nucleotides long) that take part in the post-transcriptional regulation of gene expression (Chen and Rajewsky, 2007). Thousands of these RNAs have been reported since their discovery. These small molecules have been shown to play an important regulatory role in a wide range of biological and pathological processes (Brennecke et al., 2003; Cuellar and McManus, 2005; Lim et al., 2005). Recently, miRNAs have been found in serum, plasma, urine and other bodily fluids (Chen et al., 2008; Weber et al., 2010). Interestingly, the expression profiles of these extracellular miRNAs are correlated with various diseases; they may be used as biomarkers in diagnosing and monitoring human diseases (Lu et al., 2005). Various techniques have emerged to quantify miRNAs. MiRNA expression can be quantified by two-step RT-qPCR, which can provide relative quantification (Chen et al., 2005). MiRNAs can also be hybridized onmicroarrays, slides or chips with probes for hundreds of miRNA targets, and relative miRNA levels can be determined in different samples (Shingara et al., 2005).High-throughput sequencingmethodscanalsobe used to profile miRNAs (Buermans et al., 2010). RT-qPCR is usually performed as a standard for accuracy and sensitivity during the typical procedure to determine the miRNA expressionprofile. For this reason, the quality of theRT-qPCRsystem is closely related to the final determination and result. We were surprised to find that the background of certain miRNAs was very high in the RT-qPCR results, even in notemplate-controls during practice trials. By considering the high specificity of the stem-loop RT primers, we studied the origin of this high background in miRNA RT-qPCR and found that commercial avian myeloblastosis virus (AMV) reverse transcriptase may contain miRNAs. In practice, DEPC-treated water or ultrapure water (Invitrogen) served as no-template-controls for miRNA quantitation. However, certain miRNAs (for example, Let-7a, miR-16, miR-26a, and miR-191 in this study) exhibited high background signals in these no-template-controls when AMV reverse transcriptase was used (Fig. 1A). DNA sequencing showed that the PCR products were correct (data not shown). We tried two other commercial AMV reverse SMALL RNA IN REVERSE TRANSCRIPTASE: TOO SMALL TO BE FOUND

MiRNA-24 and miRNA-466i-5p controls inflammation in rat hepatocytes.

miRNA-24 and miRNA-466i-5p controls inflammation in rat hepatocytes.

Endothelial Cell MicroRNA Expression in Human Late-Onset Fuchs' Dystrophy

MicroRNAs (miRNAs) are a class of endogenous noncoding RNA and post transcriptionally modulate gene expression during development and disease. Our study investigated the differential miRNA expression in human Fuchs' endothelial corneal dystrophy (FECD) compared with normal endothelium to identify miRNA sequences that are involved in the pathogenesis of FECD. Comparative miRNA expression profiles of endothelial samples obtained from FECD patients during lamellar corneal transplant surgery and from normal donor globes were generated using OpenArray plate technology. Differential expression of individual miRNAs was validated in the original and in independent samples using stem-loop RT qPCR assays. Expression of miRNA target genes was assessed using qPCR and tissue microarray (TMA) immunolabeling. Our results demonstrate downregulation of 87 miRNAs in FECD compared with normal endothelium (>3-fold change; P < 0.01). Correspondingly, DICER1, (encoding an endoribonuclease critical to miRNA biogenesis) showed a moderate but significant decrease in FECD samples (P < 0.05). Significant repression of three miR-29 family members (miR-29a-3p, miR-29b-2-5p, and miR-29c-5p) was paralleled by upregulation of their extracellular matrix associated mRNA targets collagen I and collagen IV. Tissue microarray immunolabeling showed histologically verifiable subendothelial collagen I and collagen IV deposition and increased endothelial laminin protein expression in FECD samples. The present study provides the first miRNA profile in FECD and normal endothelial cells and demonstrates widespread miRNA downregulation in FECD. Decreased endothelial expression of miR-29 family members may be associated with increased subendothelial extracellular matrix accumulation in FECD.

Differential expression of microRNAs induced by trastuzumab emtansine (T-DM1) during megakaryocytopoiesis.

645 Background: Treatment with the HER2-targeted antibody–drug conjugate T-DM1 resulted in significantly longer PFS and OS vs lapatinib + capecitabine in patients previously treated with trastuzumab and a taxane in the phase 3 study EMILIA. Thrombocytopenia (TCP) was the dose-limiting toxicity for patients treated with T-DM1, although platelets do not express HER2. In EMILIA, grade 3/4 TCP was observed in 12.9% of T-DM1-treated patients. We have previously shown that T-DM1 inhibits megakaryocyte (Mk) production and differentiation. Here, we investigated the effect of T-DM1 on microRNAs (miRNAs) associated with megakaryocytopoiesis. Methods: Human stem cells (HSCs; CD133+/CD34+) from 8 donors were differentiated into Mks in the presence of T-DM1, trastuzumab, or vehicle. Total RNA was extracted using the miRNeasy MiniKit. cDNA was prepared using the Taqman miRNA RT Kit, FAM-MGB probes, and stem-loop RT primer pool set. miRNA expression was measured using the 96.96 Dynamic Array Chip on the Biomark HD Reader. Data were analyzed using Fluidigm real-time analysis software Spotfire 5, and SAS 9.2. hsa−let−7g and hsa−miR−671−3p were chosen as reference miRNAs due to their low variation between treatments and time points. Median normalization was also applied. Results: A total of 526 miRNA RT-qPCR assays were used to map miRNA expression during differentiation of HSCs from 8 separate donors to Mks in vitro over 30 days. Several miRNAs demonstrated temporal changes in their expression profiles during maturation, suggesting these miRNAs are potential drivers of Mk differentiation. T-DM1 treatment inhibited Mk production and differentiation. Concomitant modifications in the expression of specific miRNAs were observed. These modifications were not present in trastuzumab- or vehicle-treated cells, suggesting these miRNAs may be involved in the development of T-DM1–induced TCP. Conclusions: These results suggest that the miRNAs have the potential to be used as biomarkers for TCP in patients treated with T-DM1 and possibly other DM1 conjugates. Specific miRNA alterations related to T-DM1 treatment will be discussed following investigations using clinical samples to validate these preliminary data.

MicroRNA-31 Activates the RAS Pathway and Functions as an Oncogenic MicroRNA in Human Colorectal Cancer by Repressing RAS p21 GTPase Activating Protein 1 (RASA1)

MicroRNAs (miRNAs) are known to play a vital role in colorectal cancer. We found a widespread disruption in miRNA expression during colorectal tumorigenesis using microarray and quantitative RT-PCR analysis; of the 161 miRNAs altered in colorectal cancer compared with normal adjacent tissue samples, miR-31 was the most significantly dysregulated. We identified candidate targets of miR-31 using bioinformatics approaches and validated RAS p21 GTPase activating protein 1 (RASA1) as a direct target. First, we found an inverse correlation between miR-31 and RASA1 protein levels in vivo. Second, in vitro evidence demonstrated that RASA1 expression was significantly decreased by treatment with pre-miR-31-LV, whereas anti-miR-31-LV treatment increased RASA1 protein levels. Third, a luciferase reporter assay confirmed that miR-31 directly recognizes a specific location within the 3'-untranslated region of RASA1 transcripts. Furthermore, the biological consequences of miR-31 targeting RASA1 were examined by the cell proliferation assay in vitro and by the immunodeficient mouse xenograft tumor model in vivo. Taken together, our results demonstrate for the first time that miR-31 plays a significant role in activating the RAS signaling pathway through the inhibition of RASA1 translation, thereby improving colorectal cancer cell growth and stimulating tumorigenesis.