Stem-loop RT-PCR Research Articles

Dynamic Expression of MicroRNA-127 During Porcine Prenatal and Postnatal Skeletal Muscle Development

MicroRNAs (miRNAs), evolutionarily conserved non-coding RNAs in length 21-24 bp, play a critical role in skeletal muscle development. In this study, to explore the function of mircoRNA-127 in porcine skeletal muscle development, eight tissue samples from adult pigs and longissimus muscle samples at 26 developmental stages were collected from Tongcheng and Landrace pigs. The spatial-temporal expression profiles of miRNA-127 were carried out using step-loop quantitative real-time PCR (stem-loop RT-PCR). To explore the molecular functions of miRNA-127, we predicted its target genes and performed functional annotation using bioinformatics methods. Results suggested that miRNA-127 was abundantly expressed in heart, ovary, uterus and spleen tissues and was weakly expressed in liver, lung, kidney and small intestine in both Tongcheng and Landrace pigs. And miRNA-127 showed significant expression differences in heart, ovary, spleen and uterus tissues between these two breeds. miRNA-127 basically kept at a relatively stable high level in middle and later embryonic stages and a low expression level in early embryonic stages and postnatal stages, but the expression levels of miRNA-127 were higher in Tongcheng pigs than in Landrace at most developmental stages. miRNA-127 potentially regulated 240 candidate genes. Results of Gene Ontology and KEGG pathway analysis indicated that these genes could be involved in many molecular functions and mechanisms, such as regulation of the force of heart contraction, regulation of transcription, regulation of T cell differentiation, MAPK signaling pathway and GnRH signaling pathway. Many significantly enriched GO terms and KEGG pathways were related to skeletal muscle development. This study will be helpful to understand the biological function for miRNA-127 and identify candidate gene associated with meat production traits in pigs.

MicroRNA-182 targets special AT-rich sequence-binding protein 2 to promote colorectal cancer proliferation and metastasis.

BackgroundIncreasing evidence has revealed that microRNAs (miRNA) played a pivotal role in regulating cancer cell proliferation and metastasis. The deregulation of miR-182 has been identified in colorectal cancer (CRC). However, the role and mechanism of miR-182 in CRC have not been completely understood yet.MethodsThe expression levels of miR-182 in CRC tissues and CRC cell lines were examined by performing stem-loop quantitative RT-PCR. The stable over-expression miR-182 cell lines and control cell lines were constructed by lentivirus infection. Subsequently, CCK-8 assay, plate colony formation assay, cell migration, invasion assay and experimental animal models were performed to detect the biological functions of miR-182 in vitro and in vivo. A luciferase reporter assay was conducted to confirm target associations. Western blot and immunohistochemical analysis were performed to examine the expression changes of molecular markers that are regulated by miR-182.ResultsWe found that miR-182 expression is increased in CRC cells that originated from metastatic foci and human primary CRC tissues with lymph node metastases. The ectopic expression of miR-182 enhanced cell proliferation, invasion, and migration in vitro. Stable overexpression of miR-182 also facilitated tumor growth and metastasis in vivo too. Further research showed that miR-182 could directly target the 3’untranslated region (3’UTR) of SATB2 mRNA and subsequently repress both the mRNA and protein expressions of SATB2, which we identified in previous studies as a CRC metastasis-associated protein. Restoring SATB2 expression could reverse the effects of miR-182 on CRC cell proliferation and migration. Investigations of possible mechanisms underlying these behaviors induced by miR-182 revealed that miR-182 induced epithelial-mesenchymal transition (EMT) by modulating the expression of key cellular molecules in EMT.ConclusionsOur results illustrated that the up-regulation of miR-182 played a pivotal role in CRC tumorigenesis and metastasis, which suggesting a potential implication of miR-182 in the molecular therapy for CRC.

Rapid and efficient isolation of high-quality small RNAs from recalcitrant plant species rich in polyphenols and polysaccharides.

Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are important regulators of plant development and gene expression. The acquisition of high-quality small RNAs is the first step in the study of its expression and function analysis, yet the extraction method of small RNAs in recalcitrant plant tissues with various secondary metabolites is not well established, especially for tropical and subtropical plant species rich in polysaccharides and polyphenols. Here, we developed a simple and efficient method for high quality small RNAs extraction from recalcitrant plant species. Prior to RNA isolation, a precursory step with a CTAB-PVPP buffer system could efficiently remove compounds and secondary metabolites interfering with RNAs from homogenized lysates. Then, total RNAs were extracted by Trizol reagents followed by a differential precipitation of high-molecular-weight (HMW) RNAs using polyethylene glycol (PEG) 8000. Finally, small RNAs could be easily recovered from supernatant by ethanol precipitation without extra elimination steps. The isolated small RNAs from papaya showed high quality through a clear background on gel and a distinct northern blotting signal with miR159a probe, compared with other published protocols. Additionally, the small RNAs extracted from papaya were successfully used for validation of both predicted miRNAs and the putative conserved tasiARFs. Furthermore, the extraction method described here was also tested with several other subtropical and tropical plant tissues. The purity of the isolated small RNAs was sufficient for such applications as end-point stem-loop RT-PCR and northern blotting analysis, respectively. The simple and feasible extraction method reported here is expected to have excellent potential for isolation of small RNAs from recalcitrant plant tissues rich in polyphenols and polysaccharides.

Higher miR-21 expression in invasive breast carcinomas is associated with positive estrogen and progesterone receptor status in patients from Serbia.

MicroRNAs play essential role in breast carcinoma progression and invasion. Our principal goals were to assess clinicopathological and prognostic correlations of microRNA-21 (miR-21) expression levels in a group of 39 Serbian breast cancer patients with invasive lobular (ILC), ductal (IDC), or mixed (ILC-IDC) breast carcinomas and in order to discover the role of miR-21 in potential novel form of stratification of the patients with different estrogen receptor (ER) and progesterone receptor (PR) status. MiR-21 expression levels were measured by stem-loop real-time RT-PCR using TaqMan technology. ER, PR, human epidermal growth factor 2 receptor (Her-2), and proliferative index (Ki-67) were evaluated by immunohistochemistry. MiR-21 levels do not vary among ILC, IDC, and ILC-IDC subgroups. MiR-21 expression levels varied significantly in the age, tumor size, Ki-67, and different grade (p = 0.030, p = 0.036, p = 0.027 and p = 0.032, respectively) subgroups. ER+ and PR+ showed higher miR-21 levels than their negative receptor status paired groups ER- and PR- with p = 0.012 and p = 0.018, respectively. MiR-21 positively correlated with ER and PR status (p = 0.018, ρ = 0.379 and p = 0.034, ρ = 0.345, respectively). Our findings suggest that miR-21 emulates transitional form of expression and that the levels of expression might be useful for stratification of the patients with different receptor status with the purpose to seek for new therapy approaches especially for the patients with the lack of response to conventional endocrine therapy.

The association between miR‐499a polymorphism and oral squamous cell carcinoma progression

To investigate the association of miR-499a genetic polymorphism with the risk of oral leukoplakia, oral submucous fibrosis (OSF), oral squamous cell carcinoma (OSCC), and clinicopathological outcomes of OSCC. The genotyping of miR-499a T>C (rs3746444) using TagMan assay was conducted in two case-control studies of 1549 subjects. miR-499a-5p and miR-499a-3p were assayed using stem-loop RT-PCR for 63 paired OSCC and adjacent normal tissues. T/C+C/C genotypes [adjusted odds ratio (AOR) 1.84, P=0.032] and C allelic type (AOR 1.91, P=0.007) at miR-499a T>C were associated with an increased risk of BQ-related OSF as compared to those with T/T genotype or T allelic type, respectively. Conversely, T/C+C/C genotypes and C allelic type decreased the risk of OSCC, especially for non-BQ-related OSCC (for genotype: AOR 0.49, P=0.010; for allelic type: AOR 0.50, P=0.007). Additionally, downregulation of miR-499a-5p was found in OSCC tissues (P=0.001) and correlated with the TT genotype (P=0.001). The T/C+C/C genotypes of MiR-499a may contribute to an increased risk of BQ-related OSF, but a decreased risk of OSCC. miR-499a T>C influences the expression levels of miR-499a-5p during the tumorigenesis of OSCC.

Overexpression of miRNA‐195 induced by hyperthyroidism is attenuated by the AT1 receptor blockage (868.6)

Introduction: miRNA‐195 is increased during cardiac hypertrophy, and it´s overexpression is sufficient to drive cardiac growth. We demonstrated that the AT1 receptor (AT1R) participates of the thyroid hormone (TH)‐induced cardiac hypertrophy. However, the potential modulation of the miRNA‐195 in TH‐mediated cardiac growth, as well as the possible contribution of the AT1R in this event is unknown.Objective: The aim of this study was to investigate the cardiac miRNA‐195 levels in TH‐induced cardiac hypertrophy, as well as the potential role of the AT1R in this response.Methods: The hyperthyroidism was induced by injections of T4 for 14 days. The T4 group was subdivided into two groups: T4 and T4 plus Losartan (an AT1R blocker). Cardiac hypertrophy was assessed by heart weight (HW) to tibia length (TL) ratio and by the ANF and skeletal‐alpha actin gene expression. The analysis of the miRNA levels was assessed by miRNA Stem Loop RT‐PCR. Data are presented as mean±SD (n=5).Results: The hyperthyroid group presented cardiac hypertrophy, as evaluated by the higher HW to TL length ratio, as well as by the increased ANF and skeletal‐alpha actin mRNA levels. However, the AT1R blocker prevented the cardiac hypertrophy induced by T4. In addition, the cardiac growth in response to TH was accompanied by higher cardiac levels of miRNA‐195 (4.9±0.7, P<0.01 vs control), which were attenuated by the use of the AT1R blocker (2.1±0.5, P<0.01 vs T4).Conclusions: These results evidence for the first time that T4‐induced cardiac hypertrophy is accompanied by a cardiac overexpression of miRNA‐195, and that the AT1R participates at least in part of this response.Grant Funding Source: Supported by FAPESP (Foundation for the Support of Research in the State of Sao Paulo) and by CNPq.

MP23-02 THE CLUSTERED MICRORNA-23B/27B FUNCTION AS TUMOR SUPPRESSORS AND USEFUL PROGNOSTIC MARKERS IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 2014MP23-02 THE CLUSTERED MICRORNA-23B/27B FUNCTION AS TUMOR SUPPRESSORS AND USEFUL PROGNOSTIC MARKERS IN RENAL CELL CARCINOMA Tomoaki Ishihara, Takeshi Chiyomaru, Satoru Inoguchi, Hideki Enokida, Naohiko Seki, and Masayuki Nakagawa Tomoaki IshiharaTomoaki Ishihara More articles by this author , Takeshi ChiyomaruTakeshi Chiyomaru More articles by this author , Satoru InoguchiSatoru Inoguchi More articles by this author , Hideki EnokidaHideki Enokida More articles by this author , Naohiko SekiNaohiko Seki More articles by this author , and Masayuki NakagawaMasayuki Nakagawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.870AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES A growing body of evidence suggests that microRNAs (miRNAs) play an important role in cancer diagnosis and therapy. MicroRNA-23b (miR-23b) and miR-27b are closely located on chromosome 9q22.32, so called clustered miRNAs. MiR-23b/27b cluster is down-regulated in several human malignancies. Our recent miRNA expression signature of renal cell carcinoma (RCC) revealed that miR-23b and miR-27b expression were reduced in RCC, suggesting that miR-23b/27b cluster is a candidate tumor suppressor. The aim of this study is to investigate the functional significance of miR-23b/27b cluster, and to identify its target genes in RCC. METHODS We evaluated miR-23b and miR-27b expressions in 27 RCC clinical specimens and 27 normal kidney tissues by stem-loop RT-PCR. We analyzed their correlation with clinicopathological features. Furthermore, overall survival (OS) of 27 RCC patients was evaluated using the Kaplan-Meier method. We performed gain-of-function studies (cell migration and invasion assays) by mature miRNAs transfection into RCC cell lines (A498 and 786-O). To identify the targets potentially regulated by the miR-23b/27b cluster, we applied genome-wide gene expression analysis and in silico study. RESULTS The expression of miR-23b and miR-27b was significantly reduced in the RCC specimens (respectively, P<0.0001 and P=0.0002). MiR-23b and miR-27b expression level were significantly lower in recurrence group of patients compared to no-recurrence group (P=0.0147 and P=0.0313). MiR-23b and miR-27b expression level was significantly lower in high T stage (over pT3) compared to low T stage (under pT2) (P=0.0257 and P=0.0146). MiR-23b/27b expression level was significantly lower in high grade group compared to low grade group (P=0.0019). Kaplan-Meier analysis showed that the low miR-23b/27b group had lower OS probability compared to the high miR-23b/27b group (P=0.0664). Significant inhibitions of cell migration and invasion were observed in the miR-23b and miR-27b transfectants. In silico analysis exhibited 22 putative target oncogenes of miR-23b/27b cluster. CONCLUSIONS Our data indicated that miR-23b/27b cluster functioned as pivotal suppressor of cell migration and invasion in RCC and might be a prognostic cancer biomarker. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e243 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Tomoaki Ishihara More articles by this author Takeshi Chiyomaru More articles by this author Satoru Inoguchi More articles by this author Hideki Enokida More articles by this author Naohiko Seki More articles by this author Masayuki Nakagawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP23-14 FUNCTIONAL SIGNIFICANCE OF TUMOR SUPPRESSIVE MICRORNA-143/145 CLUSTER IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 2014MP23-14 FUNCTIONAL SIGNIFICANCE OF TUMOR SUPPRESSIVE MICRORNA-143/145 CLUSTER IN RENAL CELL CARCINOMA Hideki Enokida, Hirofumi Yoshino, Takeshi Chiyomaru, Toshihiko Itesako, Naohiko Seki, and Masayuki Nakagawa Hideki EnokidaHideki Enokida More articles by this author , Hirofumi YoshinoHirofumi Yoshino More articles by this author , Takeshi ChiyomaruTakeshi Chiyomaru More articles by this author , Toshihiko ItesakoToshihiko Itesako More articles by this author , Naohiko SekiNaohiko Seki More articles by this author , and Masayuki NakagawaMasayuki Nakagawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.882AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Our recent studies of microRNA (miRNA) expression signatures have demonstrated that the miRNA-143/145 cluster is significantly downregulated in several types of cancer and represents a putative tumor-suppressive miRNA in human cancers. The aim of this study was to investigate the functional significance of the miR-143/145 cluster in cancer cells and to identify novel molecular targets of the miR-143/145 cluster in renal cell carcinoma (RCC). METHODS We evaluated miR-143 and miR-145 expressions in 18 pair of clear cell RCC and adjacent noncancerous tissues by stem-loop RT-PCR. We performed gain-of-function studies (cell migration and invasion assays) by mature miRNAs transfection into RCC cell lines (786-O and A498). To identify the biological processes or pathways potentially regulated by the miR-143/145 cluster, we applied genome-wide gene expression analysis and in silico study; TargetScan database and GENECODIS software, which assigned a number of the putative miRNA targets to known pathways in KEGG. Furthermore, we performed gene expression analyses of all candidate genes involved in each of the pathways by using microarray expression data containing 53 clear cell RCCs and 23 noncancerous kidney tisuues, and constructed heat map diagrams. RESULTS The expression levels of miR-143 and miR-145 were significantly downregulated in RCC tissues compared with adjacent noncancerous tissues. A significant positive correlation was recognized between miR-143 and miR-145 expression. Restoration of mature miR-143 or miR-145 in 786-O and A498 RCC cells revealed that both mature miRNAs significantly inhibited cancer cell proliferation and invasion, suggesting that the miR-143/145 cluster functioned as a tumor suppressor in RCC. Gene expression data and in silico database analysis implied several promising target genes of the cluster, including hexokinase-2 (HK2) gene, which encodes a glycolytic enzyme crucial for the Warburg effect in cancer cells. Luciferase reporter assays showed that both miR-143 and miR-145 directly regulated HK2. In clinical RCC specimens, the expression of HK2 was significantly higher in cancer tissues than in noncancerous tissues. Silencing HK2 RCC enhanced cell proliferation and invasion, suggesting that HK2 has oncogenic functions in RCC. CONCLUSIONS Thus, our data demonstrated that loss of the tumor-suppressive miR-143/145 cluster enhanced RCC cell proliferation and invasion through targeting HK2 that is crucial for the Warburg effect. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e248 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Hideki Enokida More articles by this author Hirofumi Yoshino More articles by this author Takeshi Chiyomaru More articles by this author Toshihiko Itesako More articles by this author Naohiko Seki More articles by this author Masayuki Nakagawa More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Identification and Analysis of Differential miRNAs in PK-15 Cells after Foot-and-Mouth Disease Virus Infection

The alterations of MicroRNAs(miRNAs) in host cell after foot-and-mouth disease virus (FMDV) infection is still obscure. To increase our understanding of the pathogenesis of FMDV at the post-transcriptional regulation level, Solexa high-throu MicroRNAs (miRNAs) play an important role both in the post-transcriptional regulation of gene expression and host-virus interactions. Despite investigations of miRNA expression ghput sequencing and bioinformatic tools were used to identify differentially expressed miRNAs and analyze their functions during FMDV infection of PK-15cells. Results indicated that 9,165,674 and 9,230,378 clean reads were obtained, with 172 known and 72 novel miRNAs differently expressed in infected and uninfected groups respectively. Some of differently expressed miRNAs were validated using stem-loop real-time quantitative RT-PCR. The GO annotation and KEGG pathway analysis for target genes revealed that differently expressed miRNAs were involved in immune response and cell death pathways.

Comprehensive Analysis of Human Cytomegalovirus MicroRNA Expression during Lytic and Quiescent Infection

BackgroundHuman cytomegalovirus (HCMV) encodes microRNAs (miRNAs) that function as post-transcriptional regulators of gene expression during lytic infection in permissive cells. Some miRNAs have been shown to suppress virus replication, which could help HCMV to establish or maintain latent infection. However, HCMV miRNA expression has not been comprehensively examined and compared using cell culture systems representing permissive (lytic) and semi-permissive vs. non-permissive (latent-like) infection.MethodsViral miRNAs levels and expression kinetics during HCMV infection were determined by miRNA-specific stem-loop RT-PCR. HCMV infected THP-1 (non-permissive), differentiated THP-1 (d-THP-1, semi-permissive) and human embryo lung fibroblasts (HELs, fully-permissive) were examined. The impact of selected miRNAs on HCMV infection (gene expression, genome replication and virus release) was determined by Western blotting, RT-PCR, qPCR, and plaque assay.ResultsAbundant expression of 15 HCMV miRNAs was observed during lytic infection in HELs; highest peak inductions (11- to 1502-fold) occurred at 48 hpi. In d-THP-1s, fourteen mRNAs were detected with moderate induction (3- to 288-fold), but kinetics of expression was generally delayed for 24 h relative to HELs. In contrast, only three miRNAs were induced to low levels (3- to 4-fold) during quiescent infection in THP-1s. Interestingly, miR-UL70-3p was poorly induced in HEL (1.5-fold), moderately in THP-1s (4-fold), and strongly (58-fold) in d-THP-1s, suggesting a potentially specific role for miR-UL70-3p in THP-1s and d-THP-1s. MiR-US33, -UL22A and -UL70 were further evaluated for their impact on HCMV replication in HELs. Ectopic expression of miR-UL22A and miR-UL70 did not affect HCMV replication in HELs, whereas miR-US33 inhibited HCMV replication and reduced levels of HCMV US29 mRNA, confirming that US29 is a target of miR-US33.ConclusionsViral miRNA expression kinetics differs between permissive, semi-permissive and quiescent infections, and miR-US33 down-regulates HCMV replication. These results suggest that miR-US33 may function to impair entry into lytic replication and hence promote establishment of latency.

The difference in miR-21 expression levels between invasive and non-invasive breast cancers emphasizes its role in breast cancer invasion

MicroRNA-21 (miR-21) overexpression is characteristic for various types of tumors, but it is still unknown whether its expression levels differ between invasive and non-invasive breast carcinomas. The main goal of the study was to determine the difference in miR-21 expression among normal tissue, non-invasive, invasive with non-invasive component, and pure invasive breast cancer samples, to explain its potential role and significance in breast cancer invasiveness. The second goal was to propose miR-21 as molecular marker of breast cancer invasiveness and potential target for future anti-miR therapies for the prevention of invasion and metastasis. In order to reveal the role of miR-21 in breast cancer invasiveness, we measured miR-21 expression levels in 44 breast cancer and four normal samples by stem-loop real-time RT-PCR using TaqMan technology. Relative expression levels of miR-21 were significantly higher in invasive than in other groups (P=0.002) and significantly higher in invasive compared with invasive with non-invasive component group in histological (P=0.043) and nuclear grade 2 (P=0.036), estrogen-receptor-positive (ER+) (P=0.006), progesterone-receptor-positive (PR+) (P=0.008), ER+PR+ (P=0.007), and proliferation index (Ki-67)≤20% (P=0.036) tumors. Our findings suggest that miR-21 could be independent molecular marker of breast cancer invasiveness and potential target for future anti-miR therapies for the prevention of invasion and metastasis.

HCV Core Protein-Induced Down-Regulation of microRNA-152 Promoted Aberrant Proliferation by Regulating Wnt1 in HepG2 Cells

BackgroundHepatitis C virus (HCV) has been reported to regulate cellular microRNAs (miRNAs). The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma (HCV-HCC), but HCV core-regulated miRNAs are largely unknown. Our preliminary experiments revealed significant down-regulation of microRNA-152 (miR-152) by HCV core protein in HepG2 cells. Through target gene prediction softwares, Wnt1 was predicted to be a potential target of miR-152. The present study was initiated to investigate whether miR-152 is aberrantly regulated by the HCV core protein, and involved in the regulation of the aberrant proliferation of HCV-HCC cells.MethodsMiR-152 levels were examined by stem-loop real-time RT-PCR (SLqRT-PCR). Cell proliferation was analyzed by MTT and colony formation assay. Cell cycle analysis was performed by flow cytometry. Luciferase reporter assay was conducted to confirm miRNA-target association. Wnt1 expression was determined by real-time qPCR and Western blotting.ResultsHCV core protein significantly suppressed miR-152 expression, and led to significant Wnt1 up-regulation with a concomitant aberrantly promoted proliferation. Moreover, we validated that miR-152 inhibition promoted, while miR-152 mimics inhibited cell proliferation. Using, qRT–PCR and western blot, Wnt1 was demonstrated to be regulated by miR-152. Luciferase activity assay showed that while miR-152 mimics significantly reduced the luciferase activity by 83.76% (P<0.0001), miR-152 inhibitor showed no effect on luciferase reporter. Most notably, salvage expression of miR-152 after Ad-HCV core infection for 24 h almost totally reversed the proliferation-promoting effect of the HCV core protein, and meanwhile, reduced the expression of both Wnt1 mRNA and protein to basal levels.ConclusionThese findings provide important evidence that the reduced miR-152 expression by HCV core protein can indirectly lose an inhibitory effect on Wnt1, which might, at least partially lead to cell proliferation of liver cancer cells. MiR-152 may have a therapeutic potential to suppress liver cancer proliferation.

Frequent co-expression of miRNA-5p and -3p species and cross-targeting in induced pluripotent stem cells.

Background: A miRNA precursor generally gives rise to one major miRNA species derived from the 5' arm, and are called miRNA-5p. However, more recent studies have shown co-expression of miRNA-5p and -3p, albeit in different concentrations, in cancer cells targeting different sets of transcripts. Co-expression and regulation of the -5p and -3p miRNA species in stem cells, particularly in the reprogramming process, have not been studied.Methods: In this work, we investigated co-expression and regulation of miRNA-5p and -3p species in human induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and embryonic stem cells (ESC) using a nanoliter-scale real-time PCR microarray platform that included 1,036 miRNAs.Results: In comparing iPSC and ESC, only 32 miRNAs were found to be differentially expressed, in agreement of the ESC-like nature of iPSC. In the analysis of reprogramming process in iPSCs, 261 miRNAs were found to be differentially expressed compared with the parental MSC and pre-adipose tissue, indicating significant miRNA alternations in the reprogramming process. In iPSC reprogrammed from MSC, there were 88 miRNAs (33.7%), or 44 co-expressed 5p/3p pairs, clearly indicating frequent co-expression of both miRNA species on reprogramming. Of these, 40 pairs were either co-up- or co-downregulated indicating concerted 5p/3p regulation. The 5p/3p species of only 4 pairs were regulated in reverse directions. Furthermore, some 5p/3p species of the same miRNAs were found to target the same transcript and the same miRNA may cross-target different transcripts of proteins of the G1/S transition of the cell cycle; 5p/3p co-targeting was confirmed in stem-loop RT-PCR.Conclusion: The observed cross- and co-regulation by paired miRNA species suggests a fail-proof scheme of miRNA regulation in iPSC, which may be important to iPSC pluripotency.

Genome-wide characterization of microRNA in foxtail millet (Setaria italica)

BackgroundMicroRNAs (miRNAs) are a class of short non-coding, endogenous RNAs that play key roles in many biological processes in both animals and plants. Although many miRNAs have been identified in a large number of organisms, the miRNAs in foxtail millet (Setaria italica) have, until now, been poorly understood.ResultsIn this study, two replicate small RNA libraries from foxtail millet shoots were sequenced, and 40 million reads representing over 10 million unique sequences were generated. We identified 43 known miRNAs, 172 novel miRNAs and 2 mirtron precursor candidates in foxtail millet. Some miRNA*s of the known and novel miRNAs were detected as well. Further, eight novel miRNAs were validated by stem-loop RT-PCR. Potential targets of the foxtail millet miRNAs were predicted based on our strict criteria. Of the predicted target genes, 79% (351) had functional annotations in InterPro and GO analyses, indicating the targets of the miRNAs were involved in a wide range of regulatory functions and some specific biological processes. A total of 69 pairs of syntenic miRNA precursors that were conserved between foxtail millet and sorghum were found. Additionally, stem-loop RT-PCR was conducted to confirm the tissue-specific expression of some miRNAs in the four tissues identified by deep-sequencing.ConclusionsWe predicted, for the first time, 215 miRNAs and 447 miRNA targets in foxtail millet at a genome-wide level. The precursors, expression levels, miRNA* sequences, target functions, conservation, and evolution of miRNAs we identified were investigated. Some of the novel foxtail millet miRNAs and miRNA targets were validated experimentally.

Identification and characterization of microRNAs involved in growth of blunt snout bream (Megalobrama amblycephala) by Solexa sequencing

BackgroundBlunt snout bream (Megalobrama amblycephala) is an economically important fish species in the Chinese freshwater polyculture system for its delicacy and high economic value. MicroRNAs (miRNAs) play important roles in regulation of almost all biological processes in eukaryotes. Although previous studies have identified thousands of miRNAs from many species, little information is known for miRNAs of M. amblycephala. To investigate functions of miRNAs associated with growth of M. amblycephala, we adopted the Solexa sequencing technology to sequence two small RNA libraries prepared from four growth related tissues (brain, pituitary, liver and muscle) of M. amblycephala using individuals with relatively high and low growth rates.ResultsIn this study, we have identified 347 conserved miRNAs (belonging to 123 families) and 22 novel miRNAs in M. amblycephala. Moreover, we observed sequence variants and seed edits of the miRNAs. Of the 5,166 single nucleotide substitutions observed in two libraries, the most abundant were G-to-U (15.9%), followed by U-to-C (12.1%), G-to-A (11.2%), and A to G (11.2%). Subsequently, we compared the expression patterns of miRNAs in the two libraries (big-size group with high growth rate versus small-size group with low growth rate). Results indicated that 27 miRNAs displayed significant differential expressions between the two libraries (p < 0.05). Of these, 16 were significantly up-regulated and 11 were significantly down-regulated in the big-size group compared to the small-size group. Furthermore, stem-loop RT-PCR was applied to validate and profile the expression of the differentially expressed miRNAs in ten tissues, and the result revealed that the conserved miRNAs expressed at higher levels than the novel miRNAs, especially in brain, liver and muscle. Also, targets prediction of differentially expressed miRNAs and KEGG pathway analysis suggested that differentially expressed miRNAs are involved in growth and metabolism, signal transduction, cell cycle, neural development and functions.ConclusionsThe present study provides the first large-scale characterization of miRNAs in M. amblycephala and miRNA profile related to different growth performances. The discovery of miRNA resource from this study is expected to contribute to a better understanding of the miRNAs roles playing in regulating the growth biological processes and the study of miRNA function and phenotype-associated miRNA identification in fish.

Genome-wide identification and characterization of miRNAs in the hypocotyl and cotyledon of cauliflower (Brassica oleracea L. var. botrytis) seedlings

MicroRNAs (miRNAs) are a class of small endogenous, non-coding RNAs that have key regulatory functions in plant growth, development, and other biological processes. Hypocotyl and cotyledon are the two major tissues of cauliflower (Brassica oleracea L. var. botrytis) seedlings. Tissue culture experiments have indicated that the regenerative abilities of these two tissues are significantly different. However, the characterization of miRNAs and their roles in regulating organ development in cauliflower remain unexplored. In the present study, two small RNA libraries were sequenced by Solexa sequencing technology. 99 known miRNAs belonging to 28 miRNA families were identified, in which 6 miRNA families were detected only in Brassicaceae. A total of 162 new miRNA sequences with single nucleotide substitutions corresponding to the known miRNAs, and 32 potentially novel miRNAs were also first discovered. Comparative analysis indicated that 42 of 99 known miRNAs and 17 of 32 novel miRNAs exhibited significantly differential expression between hypocotyl and cotyledon, and the differential expression of several miRNAs was further validated by stem-loop RT-PCR. In addition, 235 targets for 89 known miRNAs and 198 targets for 24 novel miRNAs were predicted, and their functions were further discussed. The expression patterns of several representative targets were also confirmed by qRT-PCR analysis. The results identified that the transcriptional expression patterns of miRNAs were negatively correlated with their targets. These findings gave new insights into the characteristics of miRNAs in cauliflower, and provided important clues to elucidate the roles of miRNAs in the tissue differentiation and development of cauliflower.

Down-Regulation of PTEN Expression Modulated by Dysregulated miR-21 Contributes to the Progression of Esophageal Cancer

miR-21, a putative tumor oncomiR, is a frequently overexpressed miRNA in a variety of tumors. Because it targets tumor-suppressor genes it has been linked to tumor progression. In this study we investigated the role of miR-21 in esophageal squamous cell carcinoma (ESCC), and its possible mechanism. Expression of miR-21 was detected by stem-loop RT-PCR in tissue from 76 invasive ESCC at stage I-IV and in their corresponding para-cancerous histological normal tissues (PCHNT). Thirty endoscopic esophageal mucosal biopsy specimens from non-tumor patients were used as controls. Expression of PTEN in 76 paired ESCC and PCHNT was investigated by real-time RT-PCR and an immunohistochemical method, respectively. Paired tumor and PCHNT specimens of 20 ESCC cases were randomly selected for western blot analysis. The effect of miR-21 on PTEN expression was assessed in the ESCC cell line with an miR-21 inhibitor to reduce miR-21 expression. Furthermore, the roles of miR-21 in cell biology were analyzed by use of miR-21 inhibitor-transfected cells. Stem-loop RT-PCR revealed miR-21 was significantly overexpressed in ESCC tissues and cell lines. Overexpression of miR-21 correlated with tumor status, lymph node metastasis, and clinical stage. We demonstrated that knockdown of miR-21 significantly increased expression of PTEN protein. Consequent PTEN expression reduced cell proliferation, invasion, and migration. Our findings suggest that miR-21 could be a potential oncomiR, probably by regulation of PTEN, and a novel prognostic factor for ESCC patients.

Cloning, expression and target gene prediction of let-7 in Paralichthys olivaceus metamorphosis

The let-7 gene is a critical regulator of developmental timing events of the larval-to-adult transition.Here,Paralichthys olivaceus let-7 was identified by a stem-loop RT-PCR.The precursor of let-7 was cloned and was proven to be highly conserved among different species.Quantitative analysis revealed that the expression levels of let-7 were generally variably enhanced during the first five metamorphic stages and peaked during the late metamorphic stages(36 dph).At the end of P.olivaceus metamorphosis(41 dph),expression of let-7 was re-duced.To study the effects of triiodothyronine(T3) on the expression of let-7,six different concentrations(0 nmol?L?1,50 nmol?L?1,75 nmol?L?1,100 nmol?L?1,200 nmol?L?1) were used to treat P.olivaceus kidney cells in vitro.Quantitative PCR showed that let-7 levels increased after T3 treatment,especially at 75 nmol?L?1,indicating that T3 regulated the expression of let-7.Further investigation of target genes demonstrated that the 3′-untranslated regions(3?-UTRs) of 29 mRNAs were possible target sites matched with let-7.These mRNA targets played key roles in diverse biological processes: development,cell proliferation,signal transduction,virus immune response,and signaling pathways.These re sults suggested that let-7 played an essential role in regulating the developmental timing of P.olivaceus metamorphosis.

Quantification of Circulating miRNAs in Plasma: Effect of Preanalytical and Analytical Parameters on Their Isolation and Stability

Circulating miRNAs are intensively evaluated as promising blood-based biomarkers. This growing interest in developing assays for circulating miRNAs necessitates careful consideration of the effects of preanalytical and analytical parameters on the isolation, stability, and quantification of circulating miRNAs. By using quantitative stem-loop RT-PCR, we compared the relative efficiencies of four miRNA isolation systems and different storage conditions. The effect of the data normalization procedure on the quantification of circulating miRNA levels in plasma from 30 healthy individuals and 30 patients with non-small cell lung carcinoma was estimated by measuring endogenous hsa-miR-21 and hsa-miR-16 and exogenous cel-miR-39 that was spiked in all samples at the same concentration. Silica column-based RNA extraction methods are more effective and reliable with respect to TRIzol LS. Endogenous circulating miRNA levels are unstable when plasma is stored at 4°C, and samples should be kept at -70°C, where the extracted miRNAs remain stable for up to 1 year. When normalization is based on combined endogenous and exogenous control miRNAs, differences in miRNA recovery and differences in cDNA synthesis between samples are compensated. Using this normalization procedure and hsa-miR-21 as a biomarker, we could clearly discriminate healthy individuals from patients with cancer. Experimental handling and the use of exogenous and endogenous controls for normalization are critical for the reliable quantification of circulating miRNA levels in plasma.

High-throughput sequencing of small RNAs from pollen and silk and characterization of miRNAs as candidate factors involved in pollen-silk interactions in maize.

In angiosperms, successful pollen-pistil interactions are the prerequisite and guarantee of subsequent fertilization and seed production. Recent profile analyses have helped elucidate molecular mechanisms underlying these processes at both transcriptomic and proteomic levels, but the involvement of miRNAs in pollen-pistil interactions is still speculative. In this study, we sequenced four small RNA libraries derived from mature pollen, in vitro germinated pollen, mature silks, and pollinated silks of maize (Zea mays L.). We identified 161 known miRNAs belonging to 27 families and 82 novel miRNAs. Of these, 40 conserved and 16 novel miRNAs showed different expression levels between mature and germinated pollen, and 30 conserved and eight novel miRNAs were differentially expressed between mature and pollinated silks. As candidates for factors associated with pollen-silk (pistil) interactions, expression patterns of the two sets of differentially expressed miRNAs were confirmed by stem-loop real-time RT-PCR. Transcript levels of 22 predicted target genes were also validated using real-time RT-PCR; most of these exhibited expression patterns contrasting with those of their corresponding miRNAs. In addition, GO analysis of target genes of differentially expressed miRNAs revealed that functional categories related to auxin signal transduction and gene expression regulation were overrepresented. These results suggest that miRNA-mediated auxin signal transduction and transcriptional regulation have roles in pollen-silk interactions. The results of our study provide novel information for understanding miRNA regulatory roles in pollen-pistil interactions.