Qipeng Zhang

Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA

Our previous studies have demonstrated that stable microRNAs (miRNAs) in mammalian serum and plasma are actively secreted from tissues and cells and can serve as a novel class of biomarkers for diseases, and act as signaling molecules in intercellular communication. Here, we report the surprising finding that exogenous plant miRNAs are present in the sera and tissues of various animals and that these exogenous plant miRNAs are primarily acquired orally, through food intake. MIR168a is abundant in rice and is one of the most highly enriched exogenous plant miRNAs in the sera of Chinese subjects. Functional studies in vitro and in vivo demonstrated that MIR168a could bind to the human/mouse low-density lipoprotein receptor adapter protein 1 (LDLRAP1) mRNA, inhibit LDLRAP1 expression in liver, and consequently decrease LDL removal from mouse plasma. These findings demonstrate that exogenous plant miRNAs in food can regulate the expression of target genes in mammals.

Enhanced chilling tolerance in Zoysia matrella by pre-treatment with salicylic acid, calcium chloride, hydrogen peroxide or 6-benzylaminopurine

Following leaf application of salicylic acid (SA), calcium chloride, hydrogen peroxide and 6-benzylaminopurine (BA), Manila grass (Zoysia matrella) plants were exposed to day/night temperature of 7/2 °C for 120 h in a growth chamber. The lower content of malondialdehyde (MDA) and H2O2 and higher activities of ascorbate peroxidase (APX), guaiacol peroxidase (POD) and catalase (CAT) during exposure to low temperature in pre-treated plants in comparison with control plants demonstrated that these compounds improved the chilling tolerance of Manila grass.

Synaptosomes secrete and uptake functionally active microRNAs via exocytosis and endocytosis pathways

In this study, we first characterized synaptosome microRNA (miRNA) profiles using microarray and qRT‐PCR. MicroRNAs were detected in isolated synaptic vesicles, and Ago2 immunoprecipitation studies revealed an association between miRNAs and Ago2. Second, we found that miR‐29a, miR‐99a, and miR‐125a were significantly elevated in synaptosome supernatants after depolarization. MiRNA secretion by the synaptosome was Ca2+‐dependent and was inhibited by the exocytosis inhibitor, okadaic acid. Furthermore, application of nerve growth factor increased miRNA secretion without altering the spontaneous release of miRNAs. Conversely, kainic acid decreased miRNA secretion and enhanced the spontaneous release of miRNAs. These results indicate that synaptosomes could secrete miRNAs. Finally, synthesized miRNAs were taken up by synaptosomes, and the endocytosis inhibitor Dynasore blocked this process. After incubation with miR‐125a, additional miR‐125a was bound to Ago2 in the synaptosome, and expression of the miR‐125a target gene (PSD95 mRNA) was decreased; these findings suggest that the ingested miRNAs were assembled in the RNA‐induced silencing complex, resulting in the degradation of target mRNAs. To our knowledge, this is the first study that demonstrates the secretion of miRNAs by synaptosomes under physiological stimulation and demonstrates that secreted miRNAs might be functionally active after being taken up by the synaptic fraction via the endocytic pathway.

Identification and Characterization of the MicroRNA Profile in Aging Rats with Erectile Dysfunction

INTRODUCTION Aging-related erectile dysfunction (A-ED) is a neurovascular and refractory disorder with complicated pathophysiological mechanisms and a high prevalence. MicroRNAs (miRNAs), which modulate a variety of cell functions, may be involved in the pathophysiological processes of this disorder. AIM To investigate the miRNA profile in the corpus cavernosum (CC) of aging rats with ED, and to analyze the target genes and signaling pathways regulated by the dysregulated miRNAs. METHODS According to the apomorphine test, the experimental animals were divided into three groups: aging rats with ED (group AE), aging rats with normal erectile function (group AN), and young rats as normal controls (group YN). After the erectile functional test, CCs from each group were then collected for histological and molecular measurements. MAIN OUTCOME MEASURES Intracavernous pressure response to electric stimulation of the cavernous nerve was used to evaluate erectile function. Histological changes within CC were evaluated using immunofluorescent staining. GeneChip array was used to analyze the miRNA expression profiling. The miRNA profilings were further validated by real-time polymerase chain reaction. The TargetScan or DAIAN web platform and DAVID were used for bioinformatic analysis. RESULTS Accompanied with significantly decreased erectile function, the content of smooth muscle and endothelium within the CC of rats with A-ED was significantly decreased compared with both AN group and YN group. miR-1, miR-200a, miR-203, and miR-206 were found and validated up-regulating with above twofold change in AE group. According to the bioinformatics analysis, the four up-expressing miRNAs could regulate eNOS/NO/PKG and PGE1/PKA pathways through regulating 13 target genes. CONCLUSIONS Four miRNAs were found up-regulated significantly in the CC of rats with A-ED. The four miRNAs might play important roles in the development of A-ED by regulating the eNOS/NO/PKG and PGE1/PKA pathways despite lots of experiments still need to be validated.

BAP1 suppresses lung cancer progression and is inhibited by miR-31.

BRCA1-associated protein-1 (BAP1) is an important nuclear-localized deubiquitinating enzyme that serves as a tumor suppressor in lung cancer; however, its function and its regulation are largely unknown. In this study, we found that BAP1 protein levels were dramatically diminished in lung cancer tissues while its mRNA levels did not differ significantly, suggesting that a post-transcriptional mechanism was involved in BAP1 regulation. Because microRNAs (miRNAs) are powerful post-transcriptional regulators of gene expression, we used bioinformatic analyses to search for miRNAs that could potentially bind BAP1. We predicted and experimentally validated miR-31 as a direct regulator of BAP1. Moreover, we showed that miR-31 promoted proliferation and suppressed apoptosis in lung cancer cells and accelerated the development of tumor growth in xenograft mice by inhibiting BAP1. Taken together, this study highlights an important role for miR-31 in the suppression of BAP1 in lung cancer cells and may provide insights into the molecular mechanisms of lung carcinogenesis.

MicroRNA-200a is up-regulated in aged rats with erectile dysfunction and could attenuate endothelial function via SIRT1 inhibition

MiR-200a was shown to be upregulated in the corpus cavernosum (CC) of rats with aging-related erectile dysfunction (A-ED) in our previous study. Among its target genes, SIRT1 was also reported as a protective factor in erectile function by our groups previously. Thus, miR-200a might attenuate the erectile function in A-ED via SIRT1 inhibition. In the present study, three animal groups were included: aged rats with ED (group AE, n = 8), aged rats with normal erectile function (group AN, n = 8), and young rats as normal controls (group YN, n = 8). CCs from each group were collected for histological and molecular measurements to validate the dysregulation of miR-200a and SIRT1. After that, the cavernous endothelial cells (CECs) from CC of aged rats with normal erectile function were transfected with miR-200a in vitro. Then the expression of SIRT1 and molecules within the eNOS/NO/PKG pathway were measured to investigate whether the transfection could imitate the attenuated process of erectile function in the aged. As a result, miR-200a was upregulated while the SIRT1, the levels of eNOS and cGMP were all downregulated in the CCs from AE group. After transfection in vitro, the miR-200a was upregulated while the SIRT1 and levels of eNOS and cGMP were obviously downregulated. Finally, based on the results of our previous study, we further verify that up-regulation of miR-200a could participate in the mechanisms of A-ED via SIRT1 inhibition, and mainly attenuate endothelial function via influencing the eNOS/NO/PKGpathway.

Sustained high protein-tyrosine phosphatase 1B activity in the sperm of obese males impairs the sperm acrosome reaction.

Background: Obesity can cause male infertility or subfertility. The underlying mechanism, however, remains unclear. Results: PTP1B level/activity in obese sperm are significantly higher than those in non-obese sperm. Sustained high PTP1B activity causes a prolonged NSF dephosphorylation, which impedes reassembly of the trans-SNARE complexes. Conclusion: PTP1B serves as a link between male obesity and infertility/subfertility. Significance: Identifying PTP1B as a novel therapeutic target in obesity-related male infertility/subfertility. Evidence of a causal link between male obesity and subfertility or infertility has been demonstrated previously. However, the mechanism underlying this link is incompletely understood. Here, we report that sustained high protein-tyrosine phosphatase 1B (PTP1B) activity in sperm of obese donors plays an essential role in coupling male obesity and subfertility or infertility. First, PTP1B level and activity were significantly higher in sperm from ob/ob mice than in wild-type littermates. High PTP1B level and activity in sperm was also observed in obese patients compared with non-obese donors. The enhanced sperm PTP1B level and activity in ob/ob mice and obese patients correlated with a defect of the sperm acrosome reaction (AR). Second, treating sperm from male ob/ob mice or obese men with a specific PTP1B inhibitor largely restored the sperm AR. Finally, blockade of sperm AR by enhanced PTP1B activity in male ob/ob mice or obese men was due to prolonged dephosphorylation of N-ethylmaleimide-sensitive factor by PTP1B, leading to the inability to reassemble the trans-SNARE complexes, which is a critical step in sperm acrosomal exocytosis. In summary, our study demonstrates for the first time that a sustained high PTP1B level or activity in the sperm of obese donors causes a defect of sperm AR and that PTP1B is a novel potential therapeutic target for male infertility treatment.

Selective secretion of microRNA in CNS system

MicroRNAs (miRNAs) are a diverse class of endogenous small non-coding RNAs, which posttranscriptionally regulate gene expression by interacting with their binding sites in target mRNAs. Recently, several studies have demonstrated that miRNAs are also detectable outside cells, and these miRNAs may be called extracellular miRNAs. Extracellular miRNAs are found in various body fluids, including plasma and serum (Chen et al., 2008), saliva (Park et al., 2009), amniotic fl uid (Weber et al., 2010), Bronchial lavage (Molina-Pinelo et al., 2012; Weber et al., 2010), sputum (Xie et al., 2010), tears and urine (Weber et al., 2010), bile (Shigehara et al., 2011), seminal plasma (Wang et al., 2011), breast milk (Zhou et al., 2012), peritoneal fluid (Chen et al., 2012a). Finding the extracellular miRNAs extended the research field of miRNAs, and got a lot of fruitful research fi ndings. Given the diffi culty in accessing brain tissue in vivo or postmortem samples, plasma or serum were employed in most studies, which try to fi nd possible biomarkers for various CNS disorders. However, peripheral blood may not closely reflect the alteration of gene expression in the brain. Cerebrospinal fluid (CSF) is another important body fluid which may reflect more special changes of patient brains. Therefore, the miRNAs in CSF become a research focus of biomarkers in CNS diseases. Extracellular miRNAs were found in CSF and were supposed to be potential excellent biomarkers for various CNS disorders. For example, miRNAs in CSF were reported to be suitable biomarker for Alzheimer’s disease (Cogswell et al., 2008), primary diffuse large B-cell lymphoma of the central nervous system (Baraniskin et al., 2012) and malignant gliomas (Teplyuk et al., 2012) etc. Recently, Gallego et al. (2012) examined miRNAs in CSF and peripheral blood in patients with psychiatric disorders (chizophrenia). This was the fi rst study that compared miRNA expression profi les in CSF and peripheral blood drawn from the same subjects. Interestingly, they found the levels of 35 miRNAs detected in both CSF and blood samples from all subjects were poorly correlated, which means that miRNAs in CSF may refl ect more information of CNS system (Gallego et al., 2012).

The Transcription Factor C-Myc Suppresses MiR-23b and MiR-27b Transcription during Fetal Distress and Increases the Sensitivity of Neurons to Hypoxia-Induced Apoptosis

Previous studies reported that the expression of miR-23b-27b cluster was downregulated in embryonic brain cortices during hypoxia-induced neuronal apoptosis. However, the mechanism underlying this downregulation is not completely understood. Here, we report that the transcription factor c-Myc plays an important role in regulating the expression of miR-23b-27b cluster during hypoxia. First, the c-Myc protein level was significantly elevated in embryonic brain cortices in a mouse model of fetal distress. Second, forced overexpression or knockdown of c-Myc could suppress or increase the expression of miR-23b-27b cluster polynucleotides. Third, we identified 2 conserved c-Myc binding sites (E-boxes) in the enhancer and promoter regions of miR-23b-27b cluster in the mouse genome. Finally, we showed that elevated c-Myc expression led to an increase in the Apaf-1 level by suppressing miR-23b-27b cluster expression and that this enhanced neuronal sensitivity to apoptosis. In summary, our study demonstrates that c-Myc may suppress the expression of the miR-23b-27b cluster, resulting in additional neuronal apoptosis during hypoxia.

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

MicroRNAs (miRNAs ) 是小 RNA 分子(约 22 核苷酸渴望) 参加基因表示的 post-transcriptional 规定(陈和 Rajewsky， 2007 ) 。自从他们的发现，千这些 RNA 被报导了。这些小分子被显示了在大量生物、病理学的过程起一个重要规章的作用(Brennecke 等， 2003；Cuellar 和 McManus， 2005；Lim 等， 2005 ) 。最近， miRNAs 在浆液，血浆，尿和其它被发现了身体液体(陈等， 2008；威伯等， 2010 ) 。有趣地，这些细胞外的 miRNAs 的表示侧面与各种各样的疾病被相关；他们可以在诊断并且监视人的疾病被用作 biomarkers (陆等， 2005 ) 。