Differential expression of microRNAs in TM3 Leydig cells of mice treated with brain‐derived neurotrophic factor

Brain‐derived neurotrophic factor (BDNF) is a neurotrophin that can promote the development and proliferation of neurons. BDNF has been found to be involved in male reproduction. Leydig cells in testicular interstitial tissues can secrete testosterone in a luteinizing hormone‐dependent manner. We showed that BDNF and its receptor TrkB were expressed in mice TM3 Leydig cells in the present study. Furthermore, BDNF can promote proliferation of mouse TM3 Leydig cells in vitro. Results of microRNA (miRNA) deep sequencing showed that BDNF can alter the expression profile of miRNAs in TM3 Leydig cells. Eighty‐three miRNAs were significantly different in the BDNF‐treated and control groups (fold change of >2.0 or <0.5, P < 0.05) wherein 40 were upregulated and 43 were downregulated. The expression levels of miR‐125a‐5p, miR‐22‐5p, miR‐342‐59, miR‐451a, miR‐148a‐5p, miR‐29b‐3p, miR‐199b‐5p, and miR‐145a‐5p were further confirmed by quantitative real‐time polymerase chain reaction. Bioinformatic analysis revealed that miRNAs regulated a large number of genes with different functions. Pathway analysis indicated that miRNAs participate in the pathways involved in signal transduction, cancer, metabolism, endocrine system, immune system, and nerve system. This study indicated that miRNAs might be involved in the BDNF‐regulated cellular functions of Leydig cells.     

MiR‐139‐5p,miR‐940 and miR‐193a‐5p inhibit the growth of hepatocellular carcinoma by targeting SPOCK1

The study was aimed to screen out miRNAs with differential expression in hepatocellular carcinoma (HCC), and to explore the influence of the expressions of these miRNAs and their target gene on HCC cell proliferation, invasion and apoptosis. MiRNAs with differential expression in HCC were screened out by microarray analysis. The common target gene of these miRNAs (miR‐139‐5p, miR‐940 and miR‐193a‐5p) was screened out by analysing the target genes profile (acquired from Targetscan) of the three miRNAs. Expression levels of miRNAs and SPOCK1 were determined by quantitative real time polymerase chain reaction (qRT‐PCR). The target relationships were verified by dual luciferase reporter gene assay and RNA pull‐down assay. Through 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide,thiazolyl blue tetrazolium bromide (MTT) and transwell assays and flow cytometry, HCC cell viability, invasion and apoptosis were determined. In vivo experiment was conducted in nude mice to investigate the influence of three miRNAs on tumour growth. Down‐regulation of miR‐139‐5p, miR‐940 and miR‐193a‐5p was found in HCC. Overexpression of these miRNAs suppressed HCC cell viability and invasion, promoted apoptosis and inhibited tumour growth. SPOCK1, the common target gene of miR‐139‐5p, miR‐940 and miR‐193a‐5p, was overexpressed in HCC. SPOCK1 overexpression promoted proliferation and invasion, and restrained apoptosis of HCC cells. MiR‐139‐5p, miR‐940 and miR‐193a‐5p inhibited HCC development through targeting SPOCK1.     

Altered stress responsiveness and hypothalamic‐pituitary‐adrenal axis function in male rat offspring of socially isolated parents

Social isolation in male rats at weaning results in reduced basal levels of the neuroactive steroid 3α,5α‐tetrahydroprogesterone (3α,5α‐TH PROG) in the brain and plasma as well as increased anxiety‐like behavior. We now show that socially isolated female rats also manifest a reduced basal cerebrocortical concentration of 3α,5α‐TH PROG as well as an anxiety‐like profile in the elevated plus‐maze and Vogel conflict tests compared with group‐housed controls. In contrast, despite the fact that they were raised under normal conditions, adult male offspring of male and female rats subjected to social isolation before mating exhibited an increased basal cerebrocortical level of 3α,5α‐TH PROG but no difference in emotional reactivity compared with the offspring of group‐housed parents. These animals also showed an increased basal activity of the hypothalamic‐pituitary‐adrenal axis as well as reduced abundance of corticotropin‐releasing factor in the hypothalamus and of corticotropin‐releasing factor receptor type 1 in the pituitary. Moreover, negative feedback regulation of hypothalamic‐pituitary‐adrenal axis activity by glucocorticoid was enhanced in association with up‐regulation of glucocorticoid receptor expression in the hippocampus. There was also attenuation of corticosterone release induced by foot‐shock stress in the offspring of socially isolated parents. The increase in the brain concentration of 3α,5α‐TH PROG induced by acute stress was also blunted in these animals. Our results thus show that a stressful experience before mating can influence neuroendocrine signaling in the next generation.     

Inhibition of miR‐103a‐3p suppresses lipopolysaccharide‐induced sepsis and liver injury by regulating FBXW7 expression

Inflammation, apoptosis, and oxidative stress are involved in septic liver dysfunction. Herein, the role of miR‐103a‐3p/FBXW7 axis in lipopolysaccharides (LPS)‐induced septic liver injury was investigated in mice. Hematoxylin‐eosin staining was used to evaluate LPS‐induced liver injury. Quantitative real‐time polymerase chain reaction was performed to determine the expression of microRNA (miR) and messenger RNA, and western blot analysis was conducted to examine the protein levels. Dual‐luciferase reporter assay was used to confirm the binding between miR‐103a‐3p and FBXW7. Both annexin V‐fluoresceine isothiocyanate/propidium iodide staining and caspase‐3 activity were employed to determine cell apoptosis. First, miR‐103a‐3p was upregulated in the septic serum of mice and patients with sepsis, and miR‐103a‐3p was elevated in the septic liver of LPS‐induced mice. Then, interfering miR‐103a‐3p significantly decreased apoptosis by suppressing Bax expression and upregulating Bcl‐2 levels in LPS‐induced AML12 and LO2 cells, and septic liver of mice. Furthermore, inhibition of miR‐103a‐3p repressed LPS‐induced inflammation by downregulating the expression of tumor necrosis factor, interleukin 1β, and interleukin 6 in vitro and in vivo. Meanwhile, interfering miR‐103a‐3p obviously attenuated LPS‐induced overactivation of oxidation via promoting expression of antioxidative enzymes, including catalase, superoxide dismutase, and glutathione in vitro and in vivo. Moreover, FBXW7 was a target of miR‐103a‐3p, and overexpression of FBXW7 significantly ameliorated LPS‐induced septic liver injury in mice. Finally, knockdown of FBXW7 markedly reversed anti‐miR‐103a‐3p‐mediated suppression of septic liver injury in mice. In conclusion, interfering miR‐103a‐3p or overexpression of FBXW7 improved LPS‐induced septic liver injury by suppressing apoptosis, inflammation, and oxidative reaction.     

Hormone replacement therapy enhances IGF‐1 signaling in skeletal muscle by diminishing miR‐182 and miR‐223 expressions: a study on postmenopausal monozygotic twin pairs

MiRNAs are fine‐tuning modifiers of skeletal muscle regulation, but knowledge of their hormonal control is lacking. We used a co‐twin case–control study design, that is, monozygotic postmenopausal twin pairs discordant for estrogen‐based hormone replacement therapy (HRT) to explore estrogen‐dependent skeletal muscle regulation via miRNAs. MiRNA profiles were determined from vastus lateralis muscle of nine healthy 54–62‐years‐old monozygotic female twin pairs discordant for HRT (median 7 years). MCF‐7 cells, human myoblast cultures and mouse muscle experiments were used to confirm estrogen's causal role on the expression of specific miRNAs, their target mRNAs and proteins and finally the activation of related signaling pathway. Of the 230 miRNAs expressed at detectable levels in muscle samples, qPCR confirmed significantly lower miR‐182, miR‐223 and miR‐142‐3p expressions in HRT using than in their nonusing co‐twins. Insulin/IGF‐1 signaling emerged one common pathway targeted by these miRNAs. IGF‐1R and FOXO3A mRNA and protein were more abundantly expressed in muscle samples of HRT users than nonusers. In vitro assays confirmed effective targeting of miR‐182 and miR‐223 on IGF‐1R and FOXO3A mRNA as well as a dose‐dependent miR‐182 and miR‐223 down‐regulations concomitantly with up‐regulation of FOXO3A and IGF‐1R expression. Novel finding is the postmenopausal HRT‐reduced miRs‐182, miR‐223 and miR‐142‐3p expression in female skeletal muscle. The observed miRNA‐mediated enhancement of the target genes' IGF‐1R and FOXO3A expression as well as the activation of insulin/IGF‐1 pathway signaling via phosphorylation of AKT and mTOR is an important mechanism for positive estrogen impact on skeletal muscle of postmenopausal women.     

miR‐34b‐5p inhibition attenuates lung inflammation and apoptosis in an LPS‐induced acute lung injury mouse model by targeting progranulin

Inflammation and apoptosis play important roles in the initiation and progression of acute lung injury (ALI). Our previous study has shown that progranulin (PGRN) exerts lung protective effects during LPS‐induced ALI. Here, we have investigated the potential roles of PGRN‐targeting microRNAs (miRNAs) in regulating inflammation and apoptosis in ALI and have highlighted the important role of PGRN. LPS‐induced lung injury and the protective roles of PGRN in ALI were first confirmed. The function of miR‐34b‐5p in ALI was determined by transfection of a miR‐34b‐5p mimic or inhibitor in intro and in vivo. The PGRN level gradually increased and subsequently significantly decreased, reaching its lowest value by 24 hr; PGRN was still elevated compared to the control. The change was accompanied by a release of inflammatory mediators and accumulation of inflammatory cells in the lungs. Using bioinformatics analysis and RT‐PCR, we demonstrated that, among 12 putative miRNAs, the kinetics of the miR‐34b‐5p levels were closely associated with PGRN expression in the lung homogenates. The gain‐ and loss‐of‐function analysis, dual‐luciferase reporter assays, and rescue experiments confirmed that PGRN was the functional target of miR‐34b‐5p. Intravenous injection of miR‐34b‐5p antagomir in vivo significantly inhibited miR‐34b‐5p up‐regulation, reduced inflammatory cytokine release, decreased alveolar epithelial cell apoptosis, attenuated lung inflammation, and improved survival by targeting PGRN during ALI. miR‐34b‐5p knockdown attenuates lung inflammation and apoptosis in an LPS‐induced ALI mouse model by targeting PGRN. This study shows that miR‐34b‐5p and PGRN may be potential targets for ALI treatments.     

Activity‐regulated cytoskeleton‐associated protein (Arc/Arg3.1) regulates anxiety‐ and novelty‐related behaviors

The activity‐regulated cytoskeleton‐associated protein (Arc, also known as Arg3.1) regulates glutamatergic synapse plasticity and has been linked to neuropsychiatric illness; however, its role in behaviors associated with mood and anxiety disorders remains unclear. We find that stress upregulates Arc expression in the adult mouse nucleus accumbens (NAc)—a brain region implicated in mood and anxiety behaviors. Global Arc knockout mice have altered AMPAR‐subunit surface levels in the adult NAc, and the Arc‐deficient mice show reductions in anxiety‐like behavior, deficits in social novelty preference, and antidepressive‐like behavior. Viral‐mediated expression of Arc in the adult NAc of male, global Arc KO mice restores normal levels of anxiety‐like behavior in the elevated plus maze (EPM). Consistent with this finding, viral‐mediated reduction of Arc in the adult NAc reduces anxiety‐like behavior in male, but not female, mice in the EPM. NAc‐specific reduction of Arc also produced significant deficits in both object and social novelty preference tasks. Together our findings indicate that Arc is essential for regulating normal mood‐ and anxiety‐related behaviors and novelty discrimination, and that Arc's function within the adult NAc contributes to these behavioral effects.     

Genome‐wide identification of urinary cell‐free microRNAs for non‐invasive detection of bladder cancer

Urinary microRNAs (miRNAs) are emerging as clinically useful tool for early and non‐invasive detection of various types of cancer including bladder cancer (BCA). In this study, 205 patients with BCA and 99 healthy controls were prospectively enrolled. Expression profiles of urinary miRNAs were obtained using Affymetrix miRNA microarrays (2578 miRNAs) and candidate miRNAs further validated in independent cohorts using qRT‐PCR. Whole‐genome profiling identified 76 miRNAs with significantly different concentrations in urine of BCA compared to controls (P < 0.01). In the training and independent validation phase of the study, miR‐31‐5p, miR‐93‐5p and miR‐191‐5p were confirmed to have significantly higher levels in urine of patients with BCA in comparison with controls (P < 0.01). We further established 2‐miRNA‐based urinary DxScore (miR‐93‐5p, miR‐31‐5p) enabling sensitive BCA detection with AUC being 0.84 and 0.81 in the training and validation phase, respectively. Moreover, DxScore significantly differed in the various histopathological subgroups of BCA and decreased post‐operatively. In conclusion, we identified and independently validated cell‐free urinary miRNAs as promising biomarkers enabling non‐invasive detection of BCA.     

Cellular and extracellular miRNAs are blood‐compartment‐specific diagnostic targets in sepsis

Septic shock is a common medical condition with a mortality approaching 50% where early diagnosis and treatment are of particular importance for patient survival. Novel biomarkers that serve as prompt indicators of sepsis are urgently needed. High‐throughput technologies assessing circulating microRNAs represent an important tool for biomarker identification, but the blood‐compartment specificity of these miRNAs has not yet been investigated. We characterized miRNA profiles from serum exosomes, total serum and blood cells (leukocytes, erythrocytes, platelets) of sepsis patients by next‐generation sequencing and RT‐qPCR (n = 3 × 22) and established differences in miRNA expression between blood compartments. In silico analysis was used to identify compartment‐specific signalling functions of differentially regulated miRNAs in sepsis‐relevant pathways. In septic shock, a total of 77 and 103 miRNAs were down‐ and up‐regulated, respectively. A majority of these regulated miRNAs (14 in serum, 32 in exosomes and 73 in blood cells) had not been previously associated with sepsis. We found a distinctly compartment‐specific regulation of miRNAs between sepsis patients and healthy volunteers. Blood cellular miR‐199b‐5p was identified as a potential early indicator for sepsis and septic shock. miR‐125b‐5p and miR‐26b‐5p were uniquely regulated in exosomes and serum, respectively, while one miRNA (miR‐27b‐3p) was present in all three compartments. The expression of sepsis‐associated miRNAs is compartment‐specific. Exosome‐derived miRNAs contribute significant information regarding sepsis diagnosis and survival prediction and could serve as newly identified targets for the development of novel sepsis biomarkers.     

Altered sperm tsRNAs in aged male contribute to anxiety‐like behavior in offspring

Parental age at first pregnancy is increasing worldwide. The offspring of aged father has been associated with higher risk of several neuropsychiatric disorders, such as schizophrenia and autism, but the underlying mechanism remains elusive. Here we report that advanced paternal age in mice alters the profile of transfer RNA‐derived small RNAs (tsRNAs). Injection of sperm tsRNAs from aged male mice into zygotes induced anxiety‐like behaviors in F1 males. RNA sequencing of the cerebral cortex and hippocampus of those F1 male mice altered the gene expression of dopaminergic synapse and neurotrophin. tsRNAs from aged male mice injection also altered the neuropsychiatry‐related gene expression in two‐cell and blastocyst stage embryos. More importantly, the sperm tsRNA profile changes significantly during aging in human. The up‐regulated sperm tsRNA target genes were involved in neurogenesis and nervous system development. These results suggest that aging‐related changes of sperm tsRNA may contribute to the intergenerational transmission of behavioral traits.     

Proximate causes and consequences of intergenerational influences of salient sensory experience

Salient sensory environments experienced by a parental generation can exert intergenerational influences on offspring. While these data provide an exciting new perspective on biological inheritance, questions remain about causes and consequences of intergenerational influences of salient sensory experience. We previously showed that exposing male mice to a salient olfactory experience, like olfactory fear conditioning, resulted in offspring demonstrating a sensitivity to the odor used to condition the paternal generation and possessing enhanced neuroanatomical representation for that odor. In this study, we first injected RNA extracted from sperm of male mice that underwent olfactory fear conditioning into naïve single‐cell zygotes and found that adults that developed from these embryos had increased sensitivity and enhanced neuroanatomical representation for the odor (Odor A) with which the paternal male had been conditioned. Next, we found that female, but not male offspring sired by males conditioned with Odor A show enhanced consolidation of a weak single‐trial Odor A + shock fear conditioning protocol. Our data provide evidence that RNA found in the paternal germline after exposure to salient sensory experiences can contribute to intergenerational influences of such experiences, and that such intergenerational influences confer an element of adaptation to the offspring. In so doing, our study of intergenerational influences of parental sensory experience adds to existing literature on intergenerational influences of parental exposures to stress and dietary manipulations and suggests that some causes (sperm RNA) and consequences (behavioral flexibility) of intergenerational influences of parental experiences may be conserved across a variety of parental experiences.     

Progesterone modulation of α5 nAChR subunits influences anxiety‐related behavior during estrus cycle

Smokers often report an anxiolytic effect of cigarettes. In addition, stress‐related disorders such as anxiety, post‐traumatic stress syndrome and depression are often associated with chronic nicotine use. To study the role of the α5 nicotinic acetylcholine receptor subunit in anxiety‐related responses, control and α5 subunit null mice (α5^?/?) were subjected to the open field activity (OFA), light–dark box (LDB) and elevated plus maze (EPM) tests. In the OFA and LDB, α5^?/? behaved like wild‐type controls. In the EPM, female α5^?/? mice displayed an anxiolytic‐like phenotype, while male α5^?/? mice were undistinguishable from littermate controls. We studied the hypothalamus–pituitary–adrenal axis by measuring plasma corticosterone and hypothalamic corticotropin‐releasing factor. Consistent with an anxiolytic‐like phenotype, female α5^?/? mice displayed lower basal corticosterone levels. To test whether gonadal steroids regulate the expression of α5, we treated cultured NTera 2 cells with progesterone and found that α5 protein levels were upregulated. In addition, brain levels of α5 mRNA increased upon progesterone injection into ovariectomized wild‐type females. Finally, we tested anxiety levels in the EPM during the estrous cycle. The estrus phase (when progesterone levels are low) is anxiolytic‐like in wild‐type mice, but no cycle‐dependent fluctuations in anxiety levels were found in α5^?/? females. Thus, α5‐containing neuronal nicotinic acetylcholine receptors may be mediators of anxiogenic responses, and progesterone‐dependent modulation of α5 expression may contribute to fluctuations in anxiety levels during the ovarian cycle.     

Birth of mice after intracytoplasmic injection of single purified sperm nuclei and detection of messenger RNAs and MicroRNAs in the sperm nuclei

We have developed a method that effectively removes all of the perinuclear materials of a mouse sperm head, including the acrosome, plasma membrane, perinuclear theca, and nuclear envelope. By injection of a single purified sperm head into a metaphase II mouse oocyte followed by activation with strontium chloride, 93% of the zygotes developed into two-cell embryos. Although only approximately 17% of the transferred two-cell embryos were born alive, all live pups developed into adults, and they appeared to be normal in reproduction and behavior. We detected RNA species, including mRNAs and miRNAs from the purified sperm heads. Our data demonstrate that pure membrane-free sperm heads are sufficient to produce normal offspring through intracytoplasmic sperm injection and that at least part of the RNA molecules are deeply embedded in the sperm nucleus.     

MicroRNA‐15b promotes neurogenesis and inhibits neural progenitor proliferation by directly repressing TET3 during early neocortical development

MicroRNAs (miRNAs) are important regulators of mouse brain development. However, their precise roles in this context remain to be elucidated. Through screening of expression profiles from a miRNA microarray and experimental analysis, we show here that miR‐15b controls several aspects of cortical neurogenesis. miR‐15b inhibits cortical neural progenitor cell (NPC) proliferation and promotes cell‐cycle exit and neuronal differentiation. Additionally, miR‐15b expression decreases the number of apical progenitors and increases basal progenitors in the VZ/SVZ. We also show that miR‐15b binds to the 3′ UTR of TET3, which plays crucial roles during embryonic development by enhancing DNA demethylation. TET3 promotes cyclin D1 expression, and miR‐15b reduces TET3 expression and 5hmC levels. Notably, TET3 expression rescues miR‐15b‐induced impaired NPC proliferation and increased cell‐cycle exit in vivo. Our results not only reveal a link between miRNAs, TET, and DNA demethylation but also demonstrate critical roles for miR‐15b and TET3 in maintaining the NPC pool during early neocortical development.     

MiR‐485‐3p modulates neural stem cell differentiation and proliferation via regulating TRIP6 expression

Recent references have showed crucial roles of several miRNAs in neural stem cell differentiation and proliferation. However, the expression and role of miR‐485‐3p remains unknown. In our reference, we indicated that miR‐485‐3p expression was down‐regulated during NSCs differentiation to neural and astrocytes cell. In addition, the TRIP6 expression was up‐regulated during NSCs differentiation to neural and astrocytes cell. We carried out the dual‐luciferase reporter and found that overexpression of miR‐485‐3p decreased the luciferase activity of pmirGLO‐TRIP6‐wt but not the pmirGLO‐TRIP6‐mut. Ectopic expression of miR‐485‐3p decreased the expression of TRIP6 in NSC. Ectopic miR‐485‐3p expression suppressed the cell growth of NSCs and inhibited nestin expression of NSCs. Moreover, elevated expression of miR‐485‐3p decreased the ki‐67 and cyclin D1 expression in NSCs. Furthermore, we indicated that miR‐485‐3p reduced proliferation and induced differentiation of NSCs via targeting TRIP6 expression. These data suggested that a crucial role of miR‐485‐3p in self‐proliferation and differentiation of NSCs. Thus, altering miR‐485‐3p and TRIP6 modulation may be one promising therapy for treating with neurodegenerative and neurogenesis diseases.     

MicroRNA‐551b‐3p inhibits tumour growth of human cholangiocarcinoma by targeting Cyclin D1

MicroRNAs (miRNAs) are powerful regulators in the tumorigenesis of cholangiocarcinoma (CCA). Previous studies report that miR‐551b‐3p acts as an oncogenic factor in ovarian cancer, but plays a tumour suppressive role in gastric cancer. However, the expression pattern and potential function of miR‐551b‐3p were still unclear in CCA. Therefore, this study aimed to explore the expression of miR‐551b‐3p and its role as well as molecular mechanism in CCA. Analysis of TCGA dataset suggested that miR‐551b‐3p was under‐expressed in CCA tissues compared to normal bile duct tissues. Furthermore, our data confirmed the decreased levels of miR‐551b‐3p in CCA samples and cell lines. Interestingly, TCGA data suggested that low miR‐551b‐3p level indicated reduced overall survival of CCA patients. Gain‐ and loss‐of‐function experiments found that miR‐551b‐3p inhibited the proliferation, G1‐S phase transition and induced apoptosis of CCA cells. In vivo experiments revealed that ectopic expression of miR‐551b‐3p inhibited tumour growth of CCA in mice. Further investigation demonstrated that miR‐551b‐3p directly bond to the 3′‐UTR of Cyclin D1 (CCND1) mRNA and negatively regulated the abundance of CCND1 in CCA cells. An inverse correlation between miR‐551b‐3p expression and the level of CCND1 mRNA was detected in CCA tissues from TCGA dataset. Notably, CCND1 knockdown showed similar effects to miR‐551b‐3p overexpression in HuCCT‐1 cells. CCND1 restoration rescued miR‐551b‐3p‐induced inhibition of proliferation, G1 phase arrest and apoptosis in HuCCT‐1 cells. In summary, miR‐551b‐3p inhibits the expression of CCND1 to suppress CCA cell proliferation and induce apoptosis, which may provide a theoretical basis for improving CCA treatment.