Search

Glucose metabolic reprogramming is a hallmark of cancer. Cancer cells rapidly adjust their energy source from oxidative phosphorylation to glycolytic metabolism in order to efficiently proliferate in a hypoxic environment, but the mechanism underlying this switch is still incompletely understood. Here, we report that hypoxia potently induces the RNA-binding protein HuR to specifically bind primary miR-199a transcript to block miR-199a maturation in hepatocellular carcinoma (HCC) cells. We demonstrate that this hypoxia-suppressed miR-199a plays a decisive role in limiting glycolysis in HCC cells by targeting hexokinase-2 (Hk2) and pyruvate kinase-M2 (Pkm2). Furthermore, systemically delivered cholesterol-modified agomiR-199a inhibits [(18)F]-fluorodeoxyglucose uptake and attenuates tumor growth in HCC tumor-bearing mice. These data reveal a novel mechanism of reprogramming of cancer energy metabolism in which HuR suppresses miR-199a maturation to link hypoxia to the Warburg effect and suggest a promising therapeutic strategy that targets miR-199a to interrupt cancerous aerobic glycolysis.

Extracellular matrix protein 1 (ECM1) is related to strong invasiveness and poor prognosis in major malignancies, but the underlying mechanism remains unknown. Here we aimed to elucidate the function of ECM1 on cell metastasis and glucose metabolism in gastric cancer (GC). The level of ECM1 in sera and tissues of patient with GC were positively correlated with tumor invasion and recurrence. Genetic manipulation of ECM1 expression affected cell metastasis and glucose metabolism in GC cell lines. Enhanced ECM1 expression facilitated gene expression levels associated with epithelial-mesenchymal transition (EMT) and glucose metabolism. Interestingly, our results indicated that ECM1 directly interacted with integrin β4 (ITGB4) and activated ITGB4/focal adhesion kinase (FAK)/glycogen synthase kinase 3β signaling pathway, which further induced the expression of transcription factor SOX2. Aberrant expression of SOX2 altered gene expression of EMT factors and glucose metabolism enzymes. Furthermore, SOX2 enhanced hypoxia-inducible factor α (HIF-1α) promoter activity to regulate glucose metabolism. The micro-positron emission tomography/computed tomography imaging of xenograft model showed that ECM1 substantially increased 18F-fluorodeoxyglucose uptake in xenograft tumors. Using in vivo mouse tail vein injection experiments, ECM1 was also found to increase in lung surface metastasis. These findings provide evidence that ECM1 regulates GC cell metastasis and glucose metabolism by inducing ITGB4/FAK/SOX2/HIF-1α signal pathway and have important implications for the development of therapeutic target to prevent tumor metastasis and recurrence.

Inflammatory stimuli clearly contribute to lung cancer development and progression, but the underlying pathogenic mechanisms are not fully understood. We found that the proinflammatory cytokine IL-1β is dramatically elevated in the serum of patients with non–small cell lung cancer (NSCLC). In vitro studies showed that IL-1β promoted the proliferation and migration of NSCLC cells. Mechanistically, IL-1β acted through the COX2–HIF1α pathway to repress the expression of microRNA-101 (miR-101), a microRNA with an established role in tumor suppression. Lin28B was identified as critical effector target of miR-101 with its repression of Lin28B, a critical aspect of tumor suppression. Overall, IL-1β upregulated Lin28B by downregulating miR-101. Interestingly, cyclooxygenase-2 inhibition by aspirin or celecoxib abrogated IL-1β-mediated repression of miR-101 and IL-1β-mediated activation of Lin28B along with their stimulatory effects on NSCLC cell proliferation and migration. Together, our findings defined an IL-1β–miR-101–Lin28B pathway as a novel regulatory axis of pathogenic inflammatory signaling in NSCLC. Cancer Res; 74(17); 4720–30. ©2014 AACR.

Small noncoding RNAs (ncRNAs) are a novel class of gene regulators that modulate gene expression at transcriptional, post-transcriptional, and epigenetic levels, and they play crucial roles in almost all cellular processes in eukaryotes. Recent studies have indicated that several types of small noncoding RNAs, including microRNAs (miRNAs), endo-small interference RNAs (endo-siRNAs), and Piwi-interacting RNAs (piRNAs), are expressed in the male germline and are required for spermatogenesis in animals. In this review, we summarize the recent knowledge of these small noncoding RNAs in male germ cells and their biological functions and mechanisms of action in animal spermatogenesis.

Thyroid hormone promotes slow-to-fast muscle fiber type conversion by inducing miR-133a1 and thereby repressing the expression of the slow muscle determinant TEAD1., It is known that thyroid hormone (TH) is a major determinant of muscle fiber composition, but the molecular mechanism by which it does so remains unclear. Here, we demonstrated that miR-133a1 is a direct target gene of TH in muscle. Intriguingly, miR-133a, which is enriched in fast-twitch muscle, regulates slow-to-fast muscle fiber type conversion by targeting TEA domain family member 1 (TEAD1), a key regulator of slow muscle gene expression. Inhibition of miR-133a in vivo abrogated TH action on muscle fiber type conversion. Moreover, TEAD1 overexpression antagonized the effect of miR-133a as well as TH on muscle fiber type switch. Additionally, we demonstrate that TH negatively regulates the transcription of myosin heavy chain I indirectly via miR-133a/TEAD1. Collectively, we propose that TH inhibits the slow muscle phenotype through a novel epigenetic mechanism involving repression of TEAD1 expression via targeting by miR-133a1. This identification of a TH-regulated microRNA therefore sheds new light on how TH achieves its diverse biological activities.

Expression, modification, and activity of microRNAs are dynamically regulated in early mouse embryos unveiled by sensitive sequencing., Small RNAs play important roles in early embryonic development. However, their expression dynamics and modifications are poorly understood because of the scarcity of RNA that is obtainable for sequencing analysis. Using an improved deep sequencing method that requires as little as 10 ng of total RNA or 50 oocytes, we profile small RNAs in mouse oocytes and early embryos. We find that microRNA (miRNA) expression starts soon after fertilization, and the mature miRNAs carried into the zygote by sperm during fertilization are relatively rare compared to the oocyte miRNAs. Intriguingly, the zygotic miRNAs display a marked increase in 3′ mono- and oligoadenylation in one- to two-cell embryos, which may protect the miRNAs from the massive degradation taking place during that time. Moreover, bioinformatics analyses show that the function of miRNA is suppressed from the oocyte to the two-cell stage and appears to be reactivated after the two-cell stage to regulate genes important in embryonic development. Our study thus provides a highly sensitive profiling method and valuable data sets for further examination of small RNAs in early embryos.

c-Myc dysregulation is one of the most common abnormalities found in human cancer. MicroRNAs (miRNAs) are functionally intertwined with the c-Myc network as multiple miRNAs are regulated by c-Myc, while others directly suppress c-Myc expression. In this work, we identified miR-33b as a primate-specific negative regulator of c-Myc. The human miR-33b gene is located at 17p11.2, a genomic locus frequently lost in medulloblastomas, of which a subset displays c-Myc overproduction. Through a small-scale screening with drugs approved by the US Food and Drug Administration (FDA), we found that lovastatin upregulated miR-33b expression, reduced cell proliferation and impaired c-Myc expression and function in miR-33b-positive medulloblastoma cells. In addition, a low dose of lovastatin treatment at a level comparable to approved human oral use reduced tumour growth in mice orthotopically xenografted with cells carrying miR-33b, but not with cells lacking miR-33b. This work presents a highly promising therapeutic option, using drug repurposing and a miRNA as a biomarker, against cancers that overexpress c-Myc.

Sirtuin-1 (SIRT1) is a potential therapeutic target to combat insulin resistance and type 2 diabetes. This study aims to identify a microRNA (miRNA) targeting SIRT1 to regulate hepatic insulin sensitivity.Luciferase assay combined with mutation and immunoblotting was used to screen and verify the bioinformatically predicted miRNAs. miRNA and mRNA levels were measured by real-time PCR. Insulin signalling was detected by immunoblotting and glycogen synthesis. Involvement of SIRT1 was studied with adenovirus, inhibitor and SIRT1-deficient hepatocytes. The role of miR-181a in vivo was explored with adenovirus and locked nucleic acid antisense oligonucleotides.miR-181a targets the 3' untranslated region (3'UTR) of Sirt1 mRNA through a miR-181a binding site, and downregulates SIRT1 protein abundance at the translational level. miR-181a is increased in insulin-resistant cultured hepatocytes and liver, and in the serum of diabetic patients. Overexpression of miR-181a decreases SIRT1 protein levels and activity, and causes insulin resistance in hepatic cells. Inhibition of miR-181a by antisense oligonucleotides increases SIRT1 protein levels and activity, and improves insulin sensitivity in hepatocytes. Ectopic expression of SIRT1 abrogates the effect of miR-181a on insulin sensitivity, and inhibition of SIRT1 activity or SIRT1 deficiency markedly attenuated the improvement in insulin sensitivity induced by antisense miR-181a. In addition, overexpression of miR-181a by adenovirus impairs hepatic insulin signalling, and intraperitoneal injection of locked nucleic acid antisense oligonucleotides for miR-181a improves glucose homeostasis in diet-induced obesity mice.miR-181a regulates SIRT1 and improves hepatic insulin sensitivity. Inhibition of miR-181a might be a potential new strategy for treating insulin resistance and type 2 diabetes.

Cancer cells preferentially metabolize glucose through aerobic glycolysis. This phenomenon, known as the Warburg effect, is an anomalous characteristic of glucose metabolism in cancer cells. Chronic inflammation is a key promoting factor of tumourigenesis. It remains, however, largely unexplored whether and how pro-tumourigenic inflammation regulates glucose metabolism in cancer cells. Here, we show that pro-inflammatory cytokines promote glycolysis in breast cancer cells, and that the inflammation-induced miR-155 functions as an important mediator in this process. We further show that miR-155 acts to upregulate hexokinase 2 (hk2), through two distinct mechanisms. First, miR-155 promotes hk2 transcription by activation of signal transducer and activator of transcription 3 (STAT3), a transcriptional activator for hk2. Second, via targeting C/EBPβ (a transcriptional activator for mir-143), miR-155 represses mir-143, a negative regulator of hk2, thus resulting in upregulation of hk2 expression at the post-transcriptional level. The miR-155-mediated hk2 upregulation also appears to operate in other types of cancer cells examined. We suggest that the miR-155/miR-143/HK2 axis may represent a common mechanism linking inflammation to the altered metabolism in cancer cells.

ErbB2 is a vital breast cancer gene and its overexpression has a decisive role in breast tumor initiation and malignant progression. However, the molecular mechanisms that underlie ErbB2 dysregulation in breast cancer cells remain incompletely understood. In this study, we found that ErbB2 expression is inversely correlated with the level of miR-155, a well-documented oncogenic miRNA, in ErbB2-positive breast tumors. We further determined that miR-155 potently suppresses ErbB2 in breast cancer cells. Mechanistically, miR-155 acts to downregulate ErbB2 via two distinct mechanisms. First, miR-155 represses ErbB2 transcription by targeting HDAC2, a transcriptional activator of ErbB2. Second, miR-155 directly targets ErbB2 via a regulatory element in its coding region. Intriguingly, miR-155 is upregulated by trastuzumab and in turn leads to a reduction of ErbB2 expression in trastuzumab-treated ErbB2-positive breast cancer cells. Functional studies showed that miR-155 inhibits ErbB2-induced malignant transformation of human breast epithelial cells. Thus, our findings reveal an intriguing miR-155-ErbB2 context in regulating the malignant transformation of breast epithelial cells, and thereby indicate a novel mode of action for miR-155 in ErbB2-positive breast cancer.

The roles of miRNAs as important post-transcriptional regulators in the circadian clock have been suggested in several studies. But the search for circadian miRNAs has led to disparate results. Here we demonstrated that at least 57 miRNA primary transcripts are rhythmically transcribed in mouse liver. Most of these transcripts are under the regulation of circadian transcription factors such as BMAL1/CLOCK and REV-ERBα/β. However, the mature miRNAs derived from these transcripts are either not oscillating or oscillating at low amplitudes, which could explain the inconsistency of different circadian miRNA studies. In order to show that these circadian primary transcripts can give rise to miRNAs with circadian functions, we over-expressed one of them, miR-378, in mouse by adenovirus injection. We found a significant over-representation of circadian oscillating genes under-expressed by miR-378 over-expression in liver. In particular, we observed that miR-378 modulates the oscillation amplitudes of Cdkn1a in the control of cell cycle and Por in the regulation of oxidation reduction by forming partnership with different circadian transcription factors. Our study suggests that circadian transcription of miRNA at primary transcript level can be a good indicator for circadian miRNA functions.

Genetic studies have elucidated critical roles of Piwi proteins in germline development in animals, but whether Piwi is an actual disease gene in human infertility remains unknown. We report germline mutations in human Piwi (Hiwi) in patients with azoospermia that prevent its ubiquitination and degradation. By modeling such mutations in Piwi (Miwi) knockin mice, we demonstrate that the genetic defects are directly responsible for male infertility. Mechanistically, we show that MIWI binds the histone ubiquitin ligase RNF8 in a Piwi-interacting RNA (piRNA)-independent manner, and MIWI stabilization sequesters RNF8 in the cytoplasm of late spermatids. The resulting aberrant sperm show histone retention, abnormal morphology, and severely compromised activity, which can be functionally rescued via blocking RNF8-MIWI interaction in spermatids with an RNF8-N peptide. Collectively, our findings identify Piwi as a factor in human infertility and reveal its role in regulating the histone-to-protamine exchange during spermiogenesis.

MicroRNA-155 (miR-155) is overexpressed in many human cancers; however, the mechanisms by which miR-155 functions as a putative oncomiR are largely unknown. Here, we report that the tumor suppressor gene suppressor of cytokine signaling 1 (socs1) is an evolutionarily conserved target of miR-155 in breast cancer cells. We found that mir-155 expression is inversely correlated with socs1 expression in breast cancer cell lines as well as in a subset of primary breast tumors. We also identified a 24A→G mutation in the miR-155 binding site of the SOCS1 3′ untranslated region in a breast tumor that reduced miR-155 repression, implicating a mechanism for miRNA targets to avoid repression. Ectopic expression of miR-155 significantly promoted the proliferation of breast cancer cells, the formation of soft agar foci in vitro, and the development of tumors in nude mice. In breast cancer cells, RNA interference silencing of socs1 recapitulates the oncogenic effects of miR-155, whereas restoration of socs1 expression attenuates the protumorigenesis function of miR-155, suggesting that miR-155 exerts its oncogenic role by negatively regulating socs1. Overexpression of miR-155 in breast cancer cells leads to constitutive activation of signal transducer and activator of transcription 3 (STAT3) through the Janus-activated kinase (JAK) pathway, and stimulation of breast cancer cells by the inflammatory cytokines IFN-γ and interleukin-6 (IL-6), lipopolysaccharide (LPS), and polyriboinosinic:polyribocytidylic acid [poly(I:C)] significantly upregulates mir-155 expression, suggesting that miR-155 may serve as a bridge between inflammation and cancer. Taken together, our study reveals that miR-155 is an oncomiR in breast cancer and that miR-155 may be a potential target in breast cancer therapy. Cancer Res; 70(8); 3119–27. ©2010 AACR.

The function of microRNAs (miRNAs) in hematopoietic stem cells (HSCs), committed progenitors, and leukemia stem cells (LSCs) is poorly understood. We show that miR-29a is highly expressed in HSC and down-regulated in hematopoietic progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors results in acquisition of self-renewal capacity by myeloid progenitors, biased myeloid differentiation, and the development of a myeloproliferative disorder that progresses to acute myeloid leukemia (AML). miR-29a promotes progenitor proliferation by expediting G1 to S/G2 cell cycle transitions. miR-29a is overexpressed in human AML and, like human LSC, miR-29a-expressing myeloid progenitors serially transplant AML. Our data indicate that miR-29a regulates early hematopoiesis and suggest that miR-29a initiates AML by converting myeloid progenitors into self-renewing LSC.

We here report that miR-17-92 cluster is a novel target for p53-mediated transcriptional repression under hypoxia. We found the expression levels of miR-17-92 cluster were reduced in hypoxia-treated cells containing wild-type p53, but were unchanged in hypoxia-treated p53-deficient cells. The repression of miR-17-92 cluster under hypoxia is independent of c-Myc. Luciferase reporter assays mapped the region responding to p53-mediated repression to a p53-binding site in the proximal region of the miR-17-92 promoter. Chromatin immunoprecipitation (ChIP), Re-ChIP and gel retardation assays revealed that the binding sites for p53- and the TATA-binding protein (TBP) overlap within the miR-17-92 promoter; these proteins were found to compete for binding. Finally, we show that pri-miR-17-92 expression correlated well with p53 status in colorectal carcinomas. Over-express miR-17-92 cluster markedly inhibits hypoxia-induced apoptosis, whereas blocked miR-17-5p and miR-20a sensitize the cells to hypoxia-induced apoptosis. These data indicated that p53-mediated repression of miR-17-92 expression likely has an important function in hypoxia-induced apoptosis, and thus further our understanding of the tumour suppressive function of p53.

Lung cancer and colorectal cancer account for over one-third of all cancer deaths in the United States. MicroRNA-301a (miR-301a) is an activator of both nuclear factor-κB (NF-κB) and Stat3, and is overexpressed in both deadly malignancies. In this work, we show that genetic ablation of miR-301a reduces Kras-driven lung tumorigenesis in mice. And miR-301a deficiency protects animals from dextran sodium sulfate-induced colon inflammation and colitis-associated colon carcinogenesis. We also demonstrate that miR-301a deletion in bone marrow-derived cells attenuates tumor growth in the colon carcinogenesis model. Our findings ascertain that one microRNA-miR-301a-activates two major inflammatory pathways (NF-κB and Stat3) in vivo, generating a pro-inflammatory microenvironment that facilitates tumorigenesis.

The piRNA machinery is known for its role in mediating epigenetic silencing of transposons. Recent studies suggest that this function also involves piRNA-guided cleavage of transposon-derived transcripts. As many piRNAs also appear to have the capacity to target diverse mRNAs, this raises the intriguing possibility that piRNAs may act extensively as siRNAs to degrade specific mRNAs. To directly test this hypothesis, we compared mouse PIWI (MIWI)-associated piRNAs with experimentally identified cleaved mRNA fragments from mouse testes, and observed cleavage sites that predominantly occur at position 10 from the 5' end of putative targeting piRNAs. We also noted strong biases for U and A residues at nucleotide positions 1 and 10, respectively, in both piRNAs and mRNA fragments, features that resemble the pattern of piRNA amplification by the 'ping-pong' cycle. Through mapping of MIWI-RNA interactions by CLIP-seq and gene expression profiling, we found that many potential piRNA-targeted mRNAs directly interact with MIWI and show elevated expression levels in the testes of Miwi catalytic mutant mice. Reporter-based assays further revealed the importance of base pairing between piRNAs and mRNA targets and the requirement for both the slicer activity and piRNA-loading ability of MIWI in piRNA-mediated target repression. Importantly, we demonstrated that proper turnover of certain key piRNA targets is essential for sperm formation. Together, these findings reveal the siRNA-like function of the piRNA machinery in mouse testes and its central requirement for male germ cell development and maturation.

Spermatogenesis in mammals is characterized by two waves of piRNA expression: one corresponds to classic piRNAs responsible for silencing retrotransponsons and the second wave is predominantly derived from nontransposon intergenic regions in pachytene spermatocytes, but the function of these pachytene piRNAs is largely unknown. Here, we report the involvement of pachytene piRNAs in instructing massive mRNA elimination in mouse elongating spermatids (ES). We demonstrate that a piRNA-induced silencing complex (pi-RISC) containing murine PIWI (MIWI) and deadenylase CAF1 is selectively assembled in ES, which is responsible for inducing mRNA deadenylation and decay via a mechanism that resembles the action of miRNAs in somatic cells. Such a highly orchestrated program appears to take full advantage of the enormous repertoire of diversified targeting capacity of pachytene piRNAs derived from nontransposon intergenic regions. These findings suggest that pachytene piRNAs are responsible for inactivating vast cellular programs in preparation for sperm production from ES.

Region-specific gene expression is an intriguing feature of the mammalian epididymis. This unique property is essential for sperm maturation and storage, and it also implicates stringent and multi-level regulations of gene expression. Over the past decade, the androgen-driven activation of epididymal gene transcription has been extensively studied. However, it still remains largely unexplored whether and how other regulatory mechanisms, such as miRNAs and DNA methylation, are involved in controlling regional gene expression in the epididymis. Using microarray-based approaches, we studied the regional miRNA expression and DNA methylation profiles in 4 distinct epididymal regions (initial segment, caput, corpus and cauda) of rats. We found that the miR-200 family members were more expressed in caput, compared with cauda. By GSEA analysis, the differential expression of miR-200 family between caput and cauda was shown to be negatively correlated with their predicted target genes, among which 4 bona fide targets were verified by luciferase reporter assay. Predicted target genes of miR-200 family have enriched functions in anti-apoptosis, cell transportation and development, implying the regional diversity in epididymal functions. On the other hand, we revealed epididymal DNA methylation of 2002 CpG islands and 2771 gene promoters (-3.88-0.97 kb), among which 1350 (67.43%) CpG islands and 2095 (75.60%) promoters contained region-specific DNA methylation. We observed significant and distinct functional enrichment in genes with specifically methylated promoters in each epididymal regions, but these DNA methylations did not show significant correlation with repressed gene transcription in the mature epididymis. Conclusively, we investigated the regional miRNA expression and DNA methylation in the rat epididymis and revealed a potential role of miR-200 family in gene expression regulation between caput and cauda. This may contribute to the distinct physiological function in sperm maturation / storage of caput / cauda epididymis.

Understanding the regulation of insulin signalling in tissues provides insights into carbohydrate and lipid metabolism in physiology and disease. Here we show that hepatic miR-378/378* expression changes in response to fasting and refeeding in mice. Mice overexpressing hepatic miR-378/378* exhibit pure hepatic insulin resistance. miR-378 inhibits hepatic insulin signalling through targeting p110α, a subunit of PI3K and hence a critical component of insulin signalling. Knockdown of hepatic p110α mimics the effect of miR-378, while restoration of p110α expression abolishes the action of miR-378 on insulin signalling as well as its systemic effects on glucose and lipid homeostasis. miR-378/378* knockout mice display hypoglycemia and increased hepatic triglyceride level with enhanced insulin sensitivity. Inhibition of hepatic p110α in miR-378/378* knockout mice corrects the abnormal glucose tolerance. Finally, we show that overexpression of hepatic miR-378/378* ameliorates hepatic steatosis in ob/ob mice without exacerbating hyperglycemia. Our findings establish fasting-responsive miR-378 as a critical regulator of hepatic insulin signalling.

The cyclic AMP receptor protein (CRP), which activates transcription from the wild-type lacP1 promoter and most of its mutants, represses productive RNA synthesis from a lacP1 promoter variant that contains an extended -10 element, although CRP enhances RNA polymerase binding as well as open complex formation in both promoters. Moreover, abortive RNA synthesis, which is already higher in the extended -10 variant compared with the parent promoter, was further enhanced by CRP. These results, together with the observed decrease in productive RNA synthesis, indicate that CRP, while facilitating the earlier steps of initiation, inhibits transcription from the extended -10 lacP1 by hindering promoter clearance. We propose that CRP decreases energetic barriers to RNA polymerase binding, isomerization, and abortive RNA synthesis but stabilizes the abortive RNA initiating complex, which results in increasing the activation energy of the transition state before the elongation complex. The results demonstrate for the first time that a DNA-binding regulatory protein acts as an activator or a repressor in different steps of the transcription initiation pathway because of the energetic differences of the intermediate complex in the same promoter.

The mechanism of isomerization (basepair openings) during transcription initiation by RNA polymerase at the galP1 promoter of Escherichia coli was investigated by 2-aminopurine (2,AP) fluorescence. The fluorescence of 2,AP is quenched in DNA duplex and enhanced when the basepair is distorted or deformed. The increase of 2,AP fluorescence was used to monitor basepair distortion at several individual positions in the promoter. We observed that basepair distortions during isomerization are a multi-step process. Three distinct hitherto unresolved steps in kinetic terms were observed, where significant fluorescence change occurs: a fast step with a half-life of around 1 s, which is followed by two slower steps occurring with a half-life in the range of minutes at 25 degrees C. Contrary to commonly held expectations, basepairs at different positions opened by 2,AP assays without any obvious pattern, suggesting that basepair opening is an asynchronous multi-step process. cAMP.CRP, which activates transcription at galP1, enhanced the rate-limiting step.

ArgRS (arginyl-tRNA synthetase) belongs to the class I aaRSs (aminoacyl-tRNA synthetases), though the majority of ArgRS species lack the canonical KMSK sequence characteristic of class I aaRSs. A DNA fragment of the ArgRS gene from Bacillus stearothermophilus was amplified using primers designed according to the conserved regions of known ArgRSs. Through analysis of the amplified DNA sequence and known tRNA(Arg)s with a published genomic sequence of B. stearothermophilus, the gene encoding ArgRS ( argS ') was amplified by PCR and the gene encoding tRNA(Arg) (ACG) was synthesized. ArgRS contained 557 amino acid residues including the canonical KMKS sequence. Recombinant ArgRS and tRNA(Arg) (ACG) were expressed in Escherichia coli. ArgRS purified by nickel-affinity chromatography had no ATPase activity. The kinetics of ArgRS and cross-recognition between ArgRSs and tRNA(Arg)s from B. stearothermophilus and E. coli were studied. The activities of B. stearothermophilus ArgRS mutated at Lys(382) and Lys(385) of the KMSK sequence and at Gly(136) upstream of the HIGH loop were determined. From the mutation results, we concluded that there was mutual compensation of Lys(385) and Gly(136) for the amino acid-activation activity of B. stearothermophilus ArgRS.

The cyclic AMP receptor protein (CRP) acts as a transcription activator at many promoters of Escherichia coli. We have examined the kinetics of open complex formation at the lacP1 promoter using tryptophan fluorescence of RNA polymerase and DNA fragments with 2-aminopurine substituted at specific positions. Apart from the closed complex formation and promoter clearance, we were able to detect three steps. The first step after the closed complex formation leads to a rapid increase of 2-aminopurine fluorescence. This was followed by another rapid step in which quenching of tryptophan fluorescence of RNA polymerase was observed. The slowest step detected by 2-aminopurine fluorescence increase is assigned to the final open complex formation. We have found that CRP not only enhances RNA polymerase binding at the promoter, but also enhances the slowest isomerization step by about 2-fold. Furthermore, potassium permanganate probing shows that the conformation of the open complex in the presence of CRP appears qualitatively and quantitatively different from that in the absence of CRP, suggesting that contact with RNA polymerase is maintained throughout the transcription initiation.

Genetic studies have elucidated critical roles of Piwi proteins in germline development in animals, but whether Piwi is an actual disease gene in human infertility remains unknown. We report germline mutations in human Piwi (Hiwi) in patients with azoospermia that prevent its ubiquitination and degradation. By modeling such mutations in Piwi (Miwi) knockin mice, we demonstrate that the genetic defects are directly responsible for male infertility. Mechanistically, we show that MIWI binds the histone ubiquitin ligase RNF8 in a Piwi-interacting RNA (piRNA)-independent manner, and MIWI stabilization sequesters RNF8 in the cytoplasm of late spermatids. The resulting aberrant sperm show histone retention, abnormal morphology, and severely compromised activity, which can be functionally rescued via blocking RNF8-MIWI interaction in spermatids with an RNF8-N peptide. Collectively, our findings identify Piwi as a factor in human infertility and reveal its role in regulating the histone-to-protamine exchange during spermiogenesis.

Small noncoding RNAs (ncRNAs) are a novel class of gene regulators that modulate gene expression at transcriptional, post-transcriptional, and epigenetic levels, and they play crucial roles in almost all cellular processes in eukaryotes. Recent studies have indicated that several types of small noncoding RNAs, including microRNAs (miRNAs), endo-small interference RNAs (endo-siRNAs), and Piwi-interacting RNAs (piRNAs), are expressed in the male germline and are required for spermatogenesis in animals. In this review, we summarize the recent knowledge of these small noncoding RNAs in male germ cells and their biological functions and mechanisms of action in animal spermatogenesis.

MicroRNA-155 broadly orchestrates inflammation-induced changes of microRNA expression in breast cancer

Increased glucose utilization is a hallmark of cancer, and tumor metabolism is emerging as anticancer target for therapeutic intervention. Triple-negative breast cancers TNBC are highly glycolytic and show poor clinical outcomes. We previously identified hexokinase 2, the major glycolytic enzyme, as a target gene of miR-143 in TNBC. Here, we developed a therapeutic formulation using cholesterol-modified miR-143 agomir encapsulated in a neutral lipid-based delivery agent that blocked tumor growth and glucose metabolism in TNBC tumor-bearing mice when administered systemically. The antioncogenic effects were accompanied by a reduction in the direct target hexokinase 2 and [(18)F]-fluorodeoxyglucose ((18)F-FDG) uptake based on positron emission tomography/computed tomography. Treatment with miR-143 formulation has minimal toxic effects and mice tolerated it well. Thus, we demonstrated that miR-143 is a robust inhibitor of the Warburg effect and an effective therapeutic target for TNBC. In addition, (18)F-FDG positron emission tomography/computed tomography can be used to specifically monitor the response of TNBC to miR-143-based therapeutics by targeting tumor glycolysis.

The PIWI/PIWI-interacting RNA (piRNA) machinery has been well documented to maintain genome integrity by silencing transposons in animal germ cells. Recent studies have advanced our understanding of the biogenesis and function of this machinery; however, its metabolism has remained largely unexplored. Here, we show that murine PIWI (MIWI) is degraded through the APC/C-26S proteasome pathway and that piRNAs play an indispensable role in this process by enhancing MIWI interaction with an APC/C substrate-binding subunit. Interestingly, piRNA-triggered MIWI destruction occurs in late spermatids, which in turn leads to piRNA elimination, suggesting a feedforward mechanism for coordinated removal of the MIWI/piRNA machinery at a specific developmental stage. Importantly, the proper removal of MIWI/piRNA is essential for sperm maturation. Together, our results reveal a role for piRNAs in regulating the clearance of the MIWI/piRNA machinery via the ubiquitin-proteosome pathway and demonstrate the critical importance of proper temporal regulation of MIWI/piRNA in male germ cell development.

MicroRNA 21 (miR-21) is overexpressed in virtually all types of carcinomas and various types of hematological malignancies. To determine whether miR-21 promotes tumor development in vivo, we knocked out the miR-21 allele in mice. In response to the 7,12-dimethylbenz[ a ]anthracene (DMBA)/12- O -tetradecanoylphorbol-13-acetate mouse skin carcinogenesis protocol, miR-21 -null mice showed a significant reduction in papilloma formation compared with wild-type mice. We revealed that cellular apoptosis was elevated and cell proliferation was decreased in mice deficient of miR-21 compared to wild-type animals. In addition, we found that a large number of validated or predicted miR-21 target genes were up-regulated in miR-21 -null keratinocytes, which are precursor cells to skin papillomas. Specifically, up-regulation of Spry1 , Pten , and Pdcd4 when miR-21 was ablated coincided with reduced phosphorylation of ERK, AKT, and JNK, three major downstream effectors of Ras activation that plays a predominant role in DMBA-initiated skin carcinogenesis. These results provide in vivo evidence that miR-21 exerts its oncogenic function through negatively regulating its target genes.

MicroRNAs (miRNAs) are 19 to 26-nucleotide RNAs that regulate gene expression. More than 2000 novel human miRNA genes have been identified, and new ones are continuously being discovered. Recent computational methods indicate that up to 92% of human genes may be regulated by miRNA. In this article, we discuss recent progress in miRNA biology and their roles in human disease pathogenesis, particularly in cancer, as well as opportunities and challenges of miRNA-based therapies.

microRNAs (miRNAs) are identified as a class of non-protein regulators and a new source for broad control of gene expression in eukaryotes. The past years have witnessed substantial progress in understanding miRNA functions and mechanisms, although a few controversies remain. Various hypotheses and models have been suggested for the mechanisms of miRNA repression, including translational inhibition at the level of initiation or elongation, rapid degradation of the nascent peptide, mRNA degradation, and mRNA sequestration into P bodies (processing bodies) and SGs (stress granules) for degradation or/and storage. Recently, some noncanonical miRNA regulation, such as miRNA activation and de-repression of miRNA inhibition, have been uncovered. This review discusses some recent advances about how miRNAs regulate their targets and various modes of miRNA function.

Hepatic glucokinase (GCK) is a key enzyme in glucose utilisation. Downregulation of its activity is associated with insulin resistance and type 2 diabetes mellitus. However, it is unknown whether hepatic Gck expression is influenced by age and is involved in ageing-mediated diabetes, and whether the degree of methylation of the hepatic Gck promoter is correlated with the transcription of Gck. To address the question, we evaluated hepatic Gck transcription and promoter methylation in young (14 weeks), adult (40 weeks) and aged (80 weeks) rats. Hepatic glycogen, Gck expression and the kinase activity of GCK were measured in three age groups. The CpG methylation status was determined by both bisulphite direct sequencing and clone sequencing of the PCR amplificates of Gck promoter. The causal relationship between Gck methylation and mRNA expression was confirmed by treating rat primary hepatocytes with 5-aza-2′-deoxycytidine (5-Aza-CdR). We have shown an age-associated decline in hepatic glycogen, Gck expression levels and the kinase activity of hepatic GCK. The eleven CpG sites studied displayed age-related progressive methylation changes in hepatic Gck promoter, which were confirmed by two methods: direct and clone sequencing. After 5-Aza-CdR treatment of rat primary hepatocytes, there was a fourfold increase in Gck expression. Our results demonstrate that an age-related increase in methylation is negatively associated with hepatic Gck expression, suggesting that DNA methylation could be involved in increasing age-dependent susceptibility to hepatic insulin resistance and diabetes. Thus, the epigenetic modification of the hepatic Gck promoter may represent an important marker for diabetogenic potential during the ageing process.

GalR represses the galP1 promoter by a DNA looping-independent mechanism. Equilibrium binding of GalR and RNA polymerase to DNA, and real-time kinetics of base-pair distortion (isomerization) showed that the equilibrium dissociation constant of RNA polymerase-P1 closed complexes is largely unaffected in the presence of saturating GalR, indicating that mutual antagonism (steric hindrance) of the regulator and the RNA polymerase does not occur at this promoter. In fluorescence kinetics with 2-AP labeled P1 DNA, GalR inhibited the slower of the two-step base-pair distortion process. We isolated a negative control GalR mutant, S29R, which while bound to the operator DNA was incapable of repression of P1. Based on these results and previous demonstration that repression requires the C-terminal domain of the alpha subunit (alpha-CTD) of RNA polymerase, we propose that GalR establishes contact with alpha-CTD at the last resolved isomerization intermediate, forming a kinetic trap.

Previous studies showed that the gene argS encoding the arginyl-tRNA synthetase(ArgRS) from Escherichia coli(E.coli), was overexpressed 1 000 folds in the E.coli transformant TG1/pUC-argS, while the gene leuS, encoding the leucyl-tRNA synthetase(LeuRS) from E.coli, was only overproduced 35-fold in the same case. To investigate why the expression of these two aminoacyl-tRNA synthetase genes is so different, a fused gene (termed parg-leuS) was constructed by replacement of the 5' flanking region of leuS to 5' flanking region of argS. In the E.coli transformant TG1/pUC-parg-leuS, the activity of LeuRS was only improved 8.5-fold, which was much lower than that of the transformant harboring the recombinant plasmid pUC18-leuS or pKK-leuS. However, by RNA dot hybridization the amount of mRNA produced in the transcription of parg-leuS was about 5 times than that of the wild type leuS, and was similar to that of pKK-leuS, suggesting that the promoter of argS is very strong. Analysis of the secondary structure around the initiation codon among three mRNAs showed that the secondary structure of the mRNA from parg-leuS was the strongest of the three mRNAs. From the results, it could be deduced that expression of the fused gene parg-leuS might be controlled at the translational level and the strong secondary structure of this mRNA may hinder translation initiation and result in a low translation efficiency.

An Asn(2) deleted mutant of Escherichia coli arginyl-tRNA synthetase deleted Asn(2) and a chimera mutant, in which the N-terminal 23 amino acid residues of yeast arginyl-tRNA synthetase were appended to the N-terminus of Escherichia coli synthetase, were synthesized and studied. The expression of the deletion and chimera mutants in Escherichia coli formed inclusion bodies, presumably due to improper folding of the proteins. Relative to the native enzyme, the deletion mutant showed full amino acid activation activity and a 26% reduction in aminoacylation activity, while the chimera mutant lost 93% and 96% activities in aminoacid activation and aminoacylation, respectively, and did not aminoacylate yeast tRNA(Arg) at all. The mutant deleted Asn(2) and Ile(3) was able to be expressed in Escherichia coli but not stable to be purified. The emission maximum wavelength in the fluorescence spectra of the chimera mutants shifted to longer one and the corresponding intensities decreased, when compared with those of the native enzyme. The data show that the conformation of the mutants are different and the tryptophan residues in the mutants are more exposed than those in the native enzyme. An estimate of the secondary structure of the mutant enzymes from their far ultraviolet CD spectra showed that the chimera mutant contained less alpha-helix, more beta-sheet and slightly higher fraction of random coil, as compared with the native enzyme. The results indicate that an intact N-terminal domain of E.coli arginyl-tRNA synthetase is important to its activity and correct folding.

A long and ever-expanding roster of small (∼20-30 nucleotides) RNAs has emerged during the last decade, and most can be subsumed under the three main headings of microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and short interfering RNAs (siRNAs). Among the three categories, miRNAs is the most quickly expanded group. The most recent number of identified miRNAs is 16,772 (Sanger miRbase, April 2011). However, there are insufficient publications on their primary forms, and no tissue-specific small RNAs precursors have been reported in the epididymis. Here, we report the identification in rats of an epididymis-specific, chimeric, noncoding RNA that is spliced from two different chromosomes (chromosomes 5 and 19), which we named HongrES2. HongrES2 is a 1.6 kb mRNA-like precursor that gives rise to a new microRNA-like small RNA (mil-HongrES2) in rat epididymis. The generation of mil-HongrES2 is stimulated during epididymitis. An epididymis-specific carboxylesterase named CES7 had 100% cDNA sequence homology at the 3'end with HongrES2 and its protein product could be downregulated by HongrES2 via mil-HongrES2. This was confirmed in vivo by initiating mil-HongrES2 over-expression in rats and observing an effect on sperm capacitation.

Materials/Methods: During April 2002 to July 2008, 97 consecutive and nonselected patients with stage T3-4, or N+ gastric and gastroesophageal junction cancer patients were treated postoperatively. Of them 51 (53%) underwent D2 dissection. Seventy-seven percent (77/97) patients received adjuvant concurrent chemoradiotherapy (5-fluorouracil or Capecitabine) and 23% patients were with radiation only. The radiation dose ranged from 43.2-50.4 Gy (median 45Gy). All patients received triple regimen adjuvant chemotherapy of ECF and variant. Patients were divided into two groups according to the cycles of chemotherapy received. Sixty-one percent (59/97) patients received $4cycles were recorded as group 1 and the rest 38 were in the group 2.

Background Young neurons in the developing brain establish a polarized morphology for proper migration. The PIWI family of piRNA processing proteins are considered to be restrictively expressed in germline tissues and several types of cancer cells. They play important roles in spermatogenesis, stem cell maintenance, piRNA biogenesis, and transposon silencing. Interestingly a recent study showed that de novo mutations of PIWI family members are strongly associated with autism. Results Here, we report that PIWI-like 1 (PIWIL1), a PIWI family member known to be essential for the transition of round spermatid into elongated spermatid, plays a role in the polarization and radial migration of newborn neurons in the developing cerebral cortex. Knocking down PIWIL1 in newborn cortical neurons by in utero electroporation of specific siRNAs resulted in retardation of the transition of neurons from the multipolar stage to the bipolar stage followed by a defect in their radial migration to the proper destination. Domain analysis showed that both the RNA binding PAZ domain and the RNA processing PIWI domain in PIWIL1 were indispensable for its function in neuronal migration. Furthermore, we found that PIWIL1 unexpectedly regulates the expression of microtubule-associated proteins in cortical neurons. Conclusions PIWIL1 regulates neuronal polarization and radial migration partly via modulating the expression of microtubule-associated proteins (MAPs). Our finding of PIWIL1’s function in neuronal development implies conserved functions of molecules participating in morphogenesis of brain and germline tissue and provides a mechanism as to how mutations of PIWI may be associated with autism. Electronic supplementary material The online version of this article (doi:10.1186/s13041-015-0131-0) contains supplementary material, which is available to authorized users.

Background The morphogenesis of the cerebral cortex depends on the precise control of gene expression during development. Small non-coding RNAs, including microRNAs and other groups of small RNAs, play profound roles in various physiological and pathological processes via their regulation of gene expression. A systematic analysis of the expression profile of small non-coding RNAs in developing cortical tissues is important for clarifying the gene regulation networks mediating key developmental events during cortical morphogenesis. Results Global profiling of the small RNA transcriptome was carried out in rat cerebral cortex from E10 till P28 using next-generation sequencing technique. We found an extraordinary degree of developmental stage-specific expression of a large group of microRNAs. A group of novel microRNAs with functional hints were identified, and brain-enriched expression and Dicer-dependent production of high-abundant novel microRNAs were validated. Profound editing of known microRNAs at “seed” sequence and flanking sequence was observed, with much higher editing events detected at late postnatal stages than embryonic stages, suggesting the necessity of microRNA editing for the fine tuning of gene expression during the formation of complicated synaptic connections at postnatal stages. Conclusion Our analysis reveals extensive regulation of microRNAs during cortical development. The dataset described here will be a valuable resource for clarifying new regulatory mechanisms for cortical development and diseases and will greatly contribute to our understanding of the divergence, modification, and function of microRNAs.