MicroRNA miR-7 is preferentially expressed in endocrine cells of the developing and adult human pancreas

MicroRNAs (miRNA) are small non-coding RNAs that inhibit gene expression through binding to complementary messenger RNA sequences. miRNAs have been predicted to target genes important for pancreas development, proper endocrine cell function and metabolism. We previously described that miRNA-7 (miR-7) was the most abundant and differentially expressed islet miRNA, with 200-fold higher expression in mature human islets than in acinar tissue. Here we have analyzed the temporal and spatial expression of miR-7 in human fetal pancreas from 8 to 22 weeks of gestational age (wga). Human fetal (8–22 wga) and adult pancreases were processed for immunohistochemistry, in situ hybridization, and quantitative RT-PCR of miRNA and mRNA. miR-7 was expressed in the human developing pancreas from around 9 wga and reached its maximum expression levels between 14 and 18 wga, coinciding with the exponential increase of the pancreatic endocrine hormones. Throughout development miR-7 expression was preferentially localized to endocrine cells and its expression persisted in the adult pancreas. The present study provides a detailed analysis of the spatiotemporal expression of miR-7 in developing human pancreas. The specific localization of miR-7 expression to fetal and adult endocrine cells indicates a potential role for miR-7 in endocrine cell differentiation and/or function. Future functional studies of a potential role for miR-7 function in islet cell differentiation and physiology are likely to identify novel targets for the treatment of diabetes and will lead to the development of improved protocols for generating insulin-producing cells for cell replacement therapy.     

Genome-wide identification of cold-responsive and new microRNAs in Populus tomentosa by high-throughput sequencing

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate the expression of target mRNAs in plant growth, development, abiotic stress responses, and pathogen responses. Cold stress is one of the most common abiotic factors affecting plants, and it adversely affects plant growth, development, and spatial distribution. To understand the roles of miRNAs under cold stress in Populus tomentosa, we constructed two small RNA libraries from plantlets treated or not with cold conditions (4 °C for 8 h). High-throughput sequencing of the two libraries identified 144 conserved miRNAs belonging to 33 miRNA families and 29 new miRNAs (as well as their corresponding miRNA¹s) belonging to 23 miRNA families. Differential expression analysis showed that 21 miRNAs were down-regulated and nine miRNAs were up-regulated in response to cold stress. Among them, 19 cold-responsive miRNAs, two new miRNAs and their corresponding miRNA¹s were validated by qRT-PCR. A total of 101 target genes of the new miRNAs were predicted using a bioinformatics approach. These target genes are involved in growth and resistance to various stresses. The results demonstrated that Populus miRNAs play critical roles in the cold stress response.     

Identification of endogenous microRNA references in porcine serum for quantitative real-time PCR normalization

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that regulate the expression of genes, and they affect important biological and physiological states. Circulating miRNAs in blood are useful markers of metabolism and economic traits. Expression levels of circulating miRNAs have been estimated using quantitative real-time PCR (qPCR). Proper normalization is critical for accurate miRNA expression analysis. However, there is no study which systematically presented endogenous reference genes for evaluating circulating miRNA expression in pigs. In this study, ten porcine miRNAs (let-7a, miR-16, miR-17, miR-23a, miR-26a, miR-93, miR-103, miR-107, miR-127 and miR-191), based on the literature, were chosen as candidate reference miRNAs in serum. We evaluated the expression stability value of these miRNAs in Berkshire, Duroc, Landrace and Yorkshire pigs using geNorm and NormFinder. We determined the optimal combination of reference miRNAs for qPCR experiments: miR-127 and miR-17 in Berkshire pigs; miR-127 and miR-93 in Duroc and Landrace pigs; miR-127 and miR-16 in Yorkshire pigs. miR-127 was the best reference gene in pigs, regardless of the breed. Our study is crucial for the discovery of novel biomarkers in pigs. The reference miRNAs presented in this study could be used as appropriate reference genes for the measurement of circulating miRNA levels in studies of physiological blood metabolites.

     

Comparison of Whole Blood RNA Preservation Tubes and Novel Generation RNA Extraction Kits for Analysis of mRNA and MiRNA Profiles

Background

Whole blood expression profiling is frequently performed using PAXgene (Qiagen) or Tempus (Life Technologies) tubes. Here, we compare 6 novel generation RNA isolation protocols with respect to RNA quantity, quality and recovery of mRNA and miRNA.

Methods

3 PAXgene and 3 Tempus Tubes were collected from participants of the LIFE study with (n = 12) and without (n = 35) acute myocardial infarction (AMI). RNA was extracted with 4 manual protocols from Qiagen (PAXgene Blood miRNA Kit), Life Technologies (MagMAX for Stabilized Blood Tubes RNA Isolation Kit), and Norgen Biotek (Norgen Preserved Blood RNA Purification Kit I and Kit II), and 2 (semi-)automated protocols on the QIAsymphony (Qiagen) and MagMAX Express-96 Magnetic Particle Processor (Life Technologies). RNA quantity and quality was determined. For biological validation, RNA from 12 representative probands, extracted with all 6 kits (n = 72), was reverse transcribed and mRNAs (matrix metalloproteinase 9, arginase 1) and miRNAs (miR133a, miR1), shown to be altered by AMI, were analyzed.

Results

RNA yields were highest using the Norgen Kit I with Tempus Tubes and lowest using the Norgen Kit II with PAXgene. The disease status was the second major determinant of RNA yields (LIFE-AMI 11.2 vs. LIFE 6.7 µg, p<0.001) followed by the choice of blood collection tube. (Semi-)automation reduced overall RNA extraction time but did not generally reduce hands-on-time. RNA yields and quality were comparable between manual and automated extraction protocols. mRNA expression was not affected by collection tubes and RNA extraction kits but by RT/qPCR reagents with exception of the Norgen Kit II, which led to mRNA depletion. For miRNAs, expression differences related to collection tubes (miR30b), RNA isolation (Norgen Kit II), and RT/qRT reagents (miR133a) were observed.

Conclusion

We demonstrate that novel generation RNA isolation kits significantly differed with respect to RNA recovery and affected miRNA but not mRNA expression profiles.     

Role of Deregulated microRNAs in Breast Cancer Progression Using FFPE Tissue

MicroRNAs (miRNAs) contribute to cancer initiation and progression by silencing the expression of their target genes, causing either mRNA molecule degradation or translational inhibition. Intraductal epithelial proliferations of the breast are histologically and clinically classified into normal, atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). To better understand the progression of ductal breast cancer development, we attempt to identify deregulated miRNAs in this process using Formalin-Fixed, Paraffin-Embedded (FFPE) tissues from breast cancer patients. Following tissue microdissection, we obtained 8 normal, 4 ADH, 6 DCIS and 7 IDC samples, which were subject to RNA isolation and miRNA expression profiling analysis. We found that miR-21, miR-200b/c, miR-141, and miR-183 were consistently up-regulated in ADH, DCIS and IDC compared to normal, while miR-557 was uniquely down-regulated in DCIS. Interestingly, the most significant miRNA deregulations occurred during the transition from normal to ADH. However, the data did not reveal a step-wise miRNA alteration among discrete steps along tumor progression, which is in accordance with previous reports of mRNA profiling of different stages of breast cancer. Furthermore, the expression of MSH2 and SMAD7, two important molecules involving TGF-β pathway, was restored following miR-21 knockdown in both MCF-7 and Hs578T breast cancer cells. In this study, we have not only identified a number of potential candidate miRNAs for breast cancer, but also found that deregulation of miRNA expression during breast tumorigenesis might be an early event since it occurred significantly during normal to ADH transition. Consequently, we have demonstrated the feasibility of miRNA expression profiling analysis using archived FFPE tissues, typically with rich clinical information, as a means of miRNA biomarker discovery.     

MicroRNA expression changes during human leukemic HL-60 cell differentiation induced by 4-hydroxynonenal,a product of lipid peroxidation

4-Hydroxynonenal (HNE) is one of several lipid oxidation products that may have an impact on human pathophysiology. It is an important second messenger involved in the regulation of various cellular processes and exhibits antiproliferative and differentiative properties in various tumor cell lines. The mechanisms by which HNE affects cell growth and differentiation are only partially clarified. Because microRNAs (miRNAs) have the ability to regulate several cellular processes, we hypothesized that HNE, in addition to other mechanisms, could affect miRNA expression. Here, we present the results of a genome-wide miRNA expression profiling of HNE-treated HL-60 leukemic cells. Among 470 human miRNAs, 10 were found to be differentially expressed between control and HNE-treated cells (at p < 0.05). Six miRNAs were down-regulated (miR-181a*, miR-199b, miR-202, miR-378, miR-454-3p, miR-575) and 4 were up-regulated (miR-125a, miR-339, miR-663, miR-660). Three of these regulated miRNAs (miR-202, miR-339, miR-378) were further assayed and validated by quantitative real-time RT-PCR. Moreover, consistent with the down-regulation of miR-378, HNE also induced the expression of the SUFU protein, a tumor suppressor recently identified as a target of miR-378. The finding that HNE could regulate the expression of miRNAs and their targets opens new perspectives on the understanding of HNE-controlled pathways. A functional analysis of 191 putative gene targets of miRNAs modulated by HNE is discussed.     

Serum microRNA-29a is a promising novel marker for early detection of colorectal liver metastasis

Background: Colorectal cancer (CRC) metastasis occurs in various organs, most frequently in liver. Serological examination including tumor and biochemical markers for liver function evaluation is routinely performed, though its accuracy is not high. MicroRNAs (miRNAs) have been implicated in a variety of human diseases including cancer, and have many characteristics of an ideal biomarker most notably their inherent stability and resilience. Recently, several studies have indicated that circulating miRNAs hold much potential as novel noninvasive biomarkers for cancer and other disease processes. The objective of this study was to investigate the potential of serum miRNAs as novel biomarkers for CRC with liver metastasis. Methods: This study was divided into three phases: (I) 3 candidate serum miRNAs were detected by using real-time RT-PCR, corresponding 38 CRC patients with liver metastasis and 36 CRC patients without metastasis. (II) Marker validation by real-time RT-PCR on a similar cohort of age- and sex-matched CRC patients without (n = 20) and with liver metastasis (n = 20). (III) We examined the correlation between the expressions of candidate serum miRNAs with clinical parameters of CRC patients. Results: Serum miR-29a was significantly higher in colorectal liver metastasis (CRLM) patients than in CRC patients. This marker yielded a receiver operating characteristic curve area of 80.3%. At a cutoff value of 0.155, the sensitivity was 75% and the specificity was 75% in discriminating metastatic from non-metastatic patients. In addition, increased levels of miR-29a expression were also observed in colorectal tumors from CRLM patients compared with CRC patients. No significant difference was observed in the levels of serum miR-92a between metastatic and non-metastatic patients. Conclusions: These findings suggest that serum miR-29a has strong potential as a novel noninvasive biomarker for early detection of CRC with liver metastasis.     

Metabolism of the synthetic progestogen norethynodrel by human ketosteroid reductases of the aldo-keto reductase superfamily

Menopause is associated with changes in lipid levels resulting in increased risk of atherosclerosis and cardiovascular events. Hormone therapy (HT) and atorvastatin have been used to improve lipid profile in postmenopausal women.Effects of HT, atorvastatin and APOE polymorphisms on serum lipids and APOE and LXRA expression were evaluated in 87 hypercholesterolemic postmenopausal women, randomly selected for treatment with atorvastatin (AT, n = 17), estrogen or estrogen plus progestagen (HT, n = 34) and estrogen or estrogen plus progestagen associated with atorvastatin (HT + AT, n = 36). RNA was extracted from peripheral blood mononuclear cells (PBMC) and mRNA expression was measured by TaqMan^® PCR. APOE ?2/?3/?4 genotyping was performed using PCR-RFLP.Total cholesterol (TC), LDL-c and apoB were reduced after each treatment (p < 0.001). Triglycerides, VLDL-c and apoAI were reduced only after atorvastatin (p < 0.05), whereas triglycerides and VLDL-c were increased after HT (p = 0.01). HT women had lower reduction on TC, LDL-c and apoB than AT and HT + AT groups (p < 0.05). APOE mRNA expression was reduced after atorvastatin treatment (p = 0.03). Although LXRA gene expression was not modified by atorvastatin, it was correlated with APOE mRNA before and after treatments. Basal APOE mRNA expression was not influenced by gene polymorphisms, however the reduction on APOE expression was more pronounced in ?3?3 than in ?3?4 carriers.Atorvastatin down-regulates APOE mRNA expression and it is modified by APOE genotypes in PBMC from postmenopausal women.     

Serum MicroRNAs Related with Chemoradiotherapy Resistance in Advanced-Stage Cervical Squamous Cell Carcinoma

OBJECTIVE: To investigate the serum microRNAs as biomarkers in predicting chemoradiotherapy resistance in advanced-stage cervical squamous cell carcinoma (ACSCC) patients. METHODS: Serum samples were collected from International Federation of Gynecology and Obstetrics (FIGO) stage IIB to IIIB cervical squamous cell carcinoma patients treated with platinum based Concomitant Chemoradiotherapy (CCRT) in our hospital during September 2013 to November 2015. Twenty well-matched samples (10 resistant and 10 sensitive) were chosen to screen the miRNA expression profile using serum samples pooled with microarrays. miRNAs expressed significantly different between two groups were further verified in 131 patients (29 resistant and 102 sensitive) serum samples with TaqMan Real-time PCR. The AUC was used to evaluate the accuracy of the biomarkers for prediction. RESULTS: MiR-136-5, miR-152-3p and miR-206 were expressed significantly different between sensitive and resistant groups. Results of 131 patients verification showed that the levels of miR-206 in sensitive samples and resistant samples were 2.715 ± 0.2115 and 14.64 ± 1.184, respectively, which was significantly different (P < .0001), while miR-136-5p and miR-152-3p could not be tested without pre-amplification reactions. Univariate analysis revealed that miR-206 expression was significantly associated with patients' DFS. Multivariate analysis demonstrated that miR-206 expression, tumor differentiation and pelvic lymph nodes metastasis were the independent prognostic factors associated with DFS in this cohort (P = .008, 0.000, 0.000, respectively). The probability of the prognostic accuracy of miR-206 expression in predicting chemoradiotherapy sensitivity of ACSCC patients was 91.3% (79.3% sensitivity and 92.2% specificity). CONCLUSION: Serum miR-206 is a powerful tool in predicting chemoradiotherapy sensitivity in ACSCC patients.     

Identification of microRNAs from Plutella xylostella larvae associated with parasitization by Diadegma semiclausum

MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signaling and immune response. Small RNA deep sequencing technology provided an opportunity for a thorough survey of miRNAs in a global key pest Plutella xylostella as well as comparative analysis of miRNA expression profile of the insect in association with parasitization by Diadegma semiclausum. Combining the deep sequencing data and bioinformatics, 235 miRNAs were identified from P. xylostella. Differential expression of host cellular miRNAs in response to parasitism was examined by making small RNA libraries from parasitized and naive second instar larvae of P. xylostella. Bantam, miR-276*, miR-10, miR-31 and miR-184 were detected as five most abundant miRNAs in both libraries and 96 miRNAs were identified that were differentially expressed after parasitization. Bantam*, miR-184 and miR-281* were significantly down-regulated and two miRNAs miR-279b and miR-2944b* were highly induced in parasitized larvae. Interestingly, high copy numbers and differential expression of several miRNA passenger strands (miRNA*) suggest their potential roles in host-parasitoid interaction. In conclusion, expression profiling of miRNAs provided insights into their possible involvement in insect immune response to parasitism and offer an important resource for further studies.     

Isothermal sensitive detection of microRNA using an autonomous DNA machine recycling output as input

An autonomous DNA machine recycling the output as the input for isothermal, sensitive, and specific detection of miRNAs has been developed. This machine shows considerably high signal amplification efficiency (～1000-fold) and thus a low detection limit (～20 amol). The machine also shows high specificity, discriminating 50 amol of synthetic miRNA from 100-fold larger amounts of its family member and from 100 ng of unrelated total RNAs. Moreover, it is available for practically detecting natural miRNAs in total RNAs.     

Assessment of RNA in human breast tissue sampled by random periareolar fine needle aspiration and ductal lavage and processed as fixed or frozen specimens

Ductal lavage (DL) and random periareolar fine needle aspiration (RPFNA) have both been proposed as minimally invasive techniques to sample breast tissue during breast cancer prevention trials. Laser capture microdissection (LCM), linear RNA amplification and quantitative real-time polymerase chain reaction (qPCR) theoretically overcome the limitations of small specimen size obtained with DL and RPFNA. In order to test the yield, relative stability and amplifiability of RNA from fixed and archived RPFNA and DL specimens, breast tissue was sampled from individual high risk women (n = 9) by both DL and RPFNA. RPFNA samples showed good RNA/cDNA yield and amplification while only 2 of 9 of the paired DL specimens had cDNA of adequate quality for subsequent PCR. One and two rounds of linear amplification provided approximately a 200- and 20,000-fold enrichment of RNA, respectively. PCR analysis consistently detected ER and COX-1 mRNA in the majority of RPFNA samples examined while pS2, PCNA, VEGF and survivin expression varied with subject. RNA yield and/or stability was greater for fixed and archived RPFNA than DL specimens of breast tissue. In a subsequent study examining an expanded biomarker gene panel in fixed vs. frozen RPFNA samples, mRNA profiles and ranked relative mRNA abundance were similar (r = 0.89) for frozen and fixed RPFNA specimens. In summary, frozen RPFNA samples may be optimal for RNA endpoints in human breast cancer prevention trials but fixed RPFNA specimens allow similar analyses with greater convenience.     

Towards Clinical Applications of Blood-Borne miRNA Signatures: The Influence of the Anticoagulant EDTA on miRNA Abundance

Background

Circulating microRNAs (miRNAs) from blood are increasingly recognized as biomarker candidates for human diseases. Clinical routine settings frequently include blood sampling in tubes with EDTA as anticoagulant without considering the influence of phlebotomy on the overall miRNA expression pattern. We collected blood samples from six healthy individuals each in an EDTA blood collection tube. Subsequently, the blood was transferred into PAXgene^TM tubes at three different time points, i.e. directly (0 min), 10 min, and 2 h after phlebotomy. As control blood was also directly collected in PAXgene^TM blood RNA tubes that contain a reagent to directly lyse blood cells and stabilize their content. For all six blood donors at the four conditions (24 samples) we analyzed the abundance of 1,205 miRNAs by human Agilent miRNA V16 microarrays.

Results

While we found generally a homogenous pattern of the miRNA abundance in all 24 samples, the duration of the EDTA treatment appears to influence the miRNA abundance of specific miRNAs. The most significant changes are observed after longer EDTA exposition. Overall, the impact of the different blood sample conditions on the miRNA pattern was substantially lower than intra-individual variations. While samples belonging to one of the six individuals mostly cluster together, there was no comparable clustering for any of the four tested blood sampling conditions. The most affected miRNA was miR-769-3p that was not detected in any of the six PAXgene blood samples, but in all EDTA 2h samples. Accordingly, hsa-miR-769-3p was also the only miRNA that showed a significantly different abundance between the 4 blood sample conditions by an ANOVA analysis (Benjamini-Hochberg adjusted p-value of 0.003). Validation by qRT-PCR confirmed this finding.

Conclusion

The pattern of blood-borne miRNA abundance is rather homogenous between the four tested blood sample conditions of six blood donors. There was a clustering between the miRNA profiles that belong to a specific blood donor, but not between any of the four tested blood sampling conditions. The results show a limited overall impact of the blood sampling conditions on the miRNA pattern. Notwithstanding, the abundance of single miRNAs can be significantly altered by different blood sampling conditions.     

MicroRNA Expression Differences in Human Hematopoietic Cell Lineages Enable Regulated Transgene Expression

Blood microRNA (miRNA) levels have been associated with and shown to participate in disease pathophysiology. However, the hematopoietic cell of origin of blood miRNAs and the individual blood cell miRNA profiles are poorly understood. We report the miRNA content of highly purified normal hematopoietic cells from the same individuals. Although T-cells, B-cells and granulocytes had the highest miRNA content per cell, erythrocytes contributed more cellular miRNA to the blood, followed by granulocytes and platelets. miRNA profiling revealed different patterns and different expression levels of miRNA specific for each lineage. miR-30c-5p was determined to be an appropriate reference normalizer for cross-cell qRT-PCR comparisons. miRNA profiling of 5 hematopoietic cell lines revealed differential expression of miR-125a-5p. We demonstrated endogenous levels of miR-125a-5p regulate reporter gene expression in Meg-01 and Jurkat cells by (1) constructs containing binding sites for miR-125a-5p or (2) over-expressing or inhibiting miR-125a-5p. This quantitative analysis of the miRNA profiles of peripheral blood cells identifies the circulating hematopoietic cellular miRNAs, supports the use of miRNA profiles for distinguishing different hematopoietic lineages and suggests that endogenously expressed miRNAs can be exploited to regulate transgene expression in a cell-specific manner.     

Methane output of rabbits (Oryctolagus cuniculus) and guinea pigs (Cavia porcellus) fed a hay-only diet: implications for the scaling of methane production with body mass in non-ruminant mammalian herbivores

It is assumed that small herbivores produce negligible amounts of methane, but it is unclear whether this is a physiological peculiarity or simply a scaling effect. A respiratory chamber experiment was conducted with six rabbits (Oryctolagus cuniculus, 1.57 ± 0.31 kg body mass) and six guinea pigs (Cavia porcellus, 0.79 ± 0.07 kg) offered grass hay ad libitum. Daily dry matter (DM) intake and DM digestibility were 50 ± 6 g kg^? 0.75 d^? 1 and 55 ± 6% in rabbits and 59 ± 11 g kg^? 0.75 d^? 1 and 61 ± 3% in guinea pigs, respectively. Methane production was similar for both species (0.20 ± 0.10 L d^? 1 and 0.22 ± 0.08 L d^? 1) and represented 0.69 ± 0.32 and 1.03 ± 0.29% of gross energy intake in rabbits and guinea pigs, respectively. In relation to body mass (BM) guinea pigs produced significantly more methane. The data on methane per unit of BM obtained in this study and from the literature on the methane output of elephant, wallabies and hyraxes all lay close to a regression line derived from roughage-fed horses, showing an increase in methane output with BM. The regression, including all data, was nearly identical to that based on the horse data only (methane production in horses [L d^? 1] = 0.18 BM [kg]^{0.97 (95%CI 0.92–1.02)}) and indicates linear scaling. Because feed intake typically scales to BM^0.75, linear scaling of methane output translates into increasing energetic losses at increasing BM. Accordingly, the data collection indicates that an increasing proportion of ingested gross energy is lost because relative methane production increases with BM. Different from ruminants, such losses (1%–2% of gross energy) appear too small in non-ruminant herbivores to represent a physiologic constraint on body size. Nevertheless, this relationship may represent a physiological disadvantage with increasing herbivore body size.