m6A RNA methylation regulates mitochondrial function.

RNA methylation of N6-methyladenosine (m6A) is emerging as a fundamental regulator of every aspect of RNA biology. RNA methylation directly impacts protein production to achieve quick modulation of dynamic biological processes. However, whether RNA methylation regulates mitochondrial function is not known, especially in neuronal cells which require a high energy supply and quick reactive responses. Here we show that m6A RNA methylation regulates mitochondrial function through promoting nuclear-encoded mitochondrial complex subunit RNA translation. Conditional genetic knockout of m6A RNA methyltransferase Mettl14 (Methyltransferase like 14) by Nestin-Cre together with metabolomic analysis reveals that Mettl14 knockout-induced m6A depletion significantly downregulates metabolites related to energy metabolism. Furthermore, transcriptome-wide RNA methylation profiling of wild type and Mettl14 knockout mouse brains by m6A-Seq shows enrichment of methylation on mitochondria-related RNA. Importantly, loss of m6A leads to a significant reduction in mitochondrial respiratory capacity and membrane potential. These functional defects are paralleled by the reduced expression of mitochondrial electron transport chain complexes, as well as decreased mitochondrial super-complex assembly and activity. Mechanistically, m6A depletion decreases the translational efficiency of methylated RNA encoding mitochondrial complex subunits through reducing their association with polysomes, while not affecting RNA stability. Together, these findings reveal a novel role for RNA methylation in regulating mitochondrial function. Given that mitochondrial dysfunction and RNA methylation have been increasingly implicate in neurodegenerative disorders, our findings not only provide insights into fundamental mechanisms regulating mitochondrial function, but also open up new avenues for understanding the pathogenesis of neurological diseases.

Mitigating aberrant Cdk5 activation alleviates mitochondrial defects and motor neuron disease symptoms in spinal muscular atrophy.

Spinal muscular atrophy (SMA), the top genetic cause of infant mortality, is characterized by motor neuron degeneration. Mechanisms underlying SMA pathogenesis remain largely unknown. Here, we report that the activity of cyclin-dependent kinase 5 (Cdk5) and the conversion of its activating subunit p35 to the more potent activator p25 are significantly up-regulated in mouse models and human induced pluripotent stem cell (iPSC) models of SMA. The increase of Cdk5 activity occurs before the onset of SMA phenotypes, suggesting that it may be an initiator of the disease. Importantly, aberrant Cdk5 activation causes mitochondrial defects and motor neuron degeneration, as the genetic knockout of p35 in an SMA mouse model rescues mitochondrial transport and fragmentation defects, and alleviates SMA phenotypes including motor neuron hyperexcitability, loss of excitatory synapses, neuromuscular junction denervation, and motor neuron degeneration. Inhibition of the Cdk5 signaling pathway reduces the degeneration of motor neurons derived from SMA mice and human SMA iPSCs. Altogether, our studies reveal a critical role for the aberrant activation of Cdk5 in SMA pathogenesis and suggest a potential target for therapeutic intervention.

New perspective on the regulation of acetylcholinesterase via the aryl hydrocarbon receptor.

Acetylcholinesterase (AChE, EC 3.1.1.7) plays important roles in cholinergic neurotransmission and has been widely recognized as a biomarker for monitoring pollution by organophosphate (OP) and carbamate pesticides. Dioxin is an emerging environmental AChE disruptor and is a typical persistent organic pollutant with multiple toxic effects on the nervous system. Growing evidence has shown that there is a significant link between dioxin exposure and neurodegenerative diseases and neurodevelopmental disorders, most of which involve AChE and cholinergic dysfunctions. Therefore, an in-depth understanding of the effects of dioxin on AChE and the related mechanisms of action might help to shed light on the molecular bases of dioxin impacts on the nervous system. In the past decade, the effects of dioxins on AChE have been revealed in cultured cells of different origins and in rodent animal models. Unlike OP and carbamate pesticides, dioxin-induced AChE disturbance is not due to direct inhibition of enzymatic activity; instead, dioxin causes alterations of AChE expression in certain models. As a widely accepted mechanism for most dioxin effects, the aryl hydrocarbon receptor (AhR)-dependent pathway has become a research focus in studies on the mechanism of action of dioxin-induced AChE dysregulation. In this mini-review, the effects of dioxin on AChE and the diverse roles of the AhR pathway in AChE regulation are summarized. Additionally, the involvement of AhR in AChE regulation during different neurodevelopmental processes is discussed. These AhR-related findings might also provide new insight into AChE regulation triggered by diverse xenobiotics capable of interacting with AhR.

A genome-wide genetic linkage map and reference quality genome sequence for a new race in the wheat pathogen Pyrenophora tritici-repentis.

Pyrenophora tritici-repentis is an ascomycete fungus that causes tan spot of wheat. The disease has a worldwide distribution and can cause significant yield and quality losses in wheat production. The fungal pathogen is homothallic in nature, which means it can undergo sexual reproduction by selfing to produce pseudothecia on wheat stubble for seasonal survival. Since homothallism precludes the development of bi-parental fungal populations, no genetic linkage map has been developed for P. tritici-repentis for mapping and map-based cloning of fungal virulence genes. In this work, we created two heterothallic strains by deleting one of the mating type genes in each of two parental isolates 86-124 (race 2) and AR CrossB10 (a new race) and developed a bi-parental fungal population between them. The draft genome sequences of the two parental isolates were aligned to the Pt-1C-BFP reference sequence to mine single nucleotide polymorphisms (SNPs). A total of 225 SNP markers were developed for genotyping the entire population. Additionally, 75 simple sequence repeat, and two gene markers were also developed and used in the genotyping. The resulting linkage map consisted of 13 linkage groups spanning 5,075.83 cM in genetic distance. Because the parental isolate AR CrossB10 is a new race and produces Ptr ToxC, it was sequenced using long-read sequencing platforms and de novo assembled into contigs. The majority of the contigs were further anchored into chromosomes with the aid of the linkage maps. The whole genome comparison of AR CrossB10 to the reference genome of M4 revealed a few chromosomal rearrangements. The genetic linkage map and the new AR CrossB10 genome sequence are valuable tools for gene cloning in P. tritici-repentis.

The RNA-binding protein FMRP facilitates the nuclear export of N6-methyladenosine-containing mRNAs.

N6-Methyladenosine (m6A) is the most abundant post-transcriptional mRNA modification in eukaryotes and exerts many of its effects on gene expression through reader proteins that bind specifically to m6A-containing transcripts. Fragile X mental retardation protein (FMRP), an RNA-binding protein, has previously been shown to affect the translation of target mRNAs and trafficking of mRNA granules. Loss of function of FMRP causes fragile X syndrome, the most common form of inherited intellectual disability in humans. Using HEK293T cells, siRNA-mediated gene knockdown, cytoplasmic and nuclear fractions, RNA-Seq, and LC-MS/MS analyses, we demonstrate here that FMRP binds directly to a collection of m6A sites on mRNAs. FMRP depletion increased mRNA m6A levels in the nucleus. Moreover, the abundance of FMRP targets in the cytoplasm relative to the nucleus was decreased in Fmr1-KO mice, an effect also observed in highly methylated genes. We conclude that FMRP may affect the nuclear export of m6A-modified RNA targets.

Knockdown of lncRNA-UCA1 inhibits cell viability and migration of human glioma cells by miR-193a-mediated downregulation of CDK6.

BACKGROUND: Long noncoding RNA urothelial carcinoma-associated 1 (lncRNA-UCA1) is generally recognized as an oncogenic molecule in several human malignant tumors. However, the available evidence does not necessarily imply an unequivocal causal function of UCA1 in glioblastoma. The current study was aimed to probe the biological function of lncRNA-UCA1 in human glioblastoma cell lines. Besides, we further investigated the potential mechanisms. METHODS: Cell viability, apoptosis, as well as migration and invasion were measured using a commercial cell counting kit-8, flow cytometry, and 24-Transwell assay, respectively. LncRNA-UCA1, microRNA-193a (miR-193a), and CDK6 at messenger RNA levels were evaluated by quantitative real-time polymerase chain reaction method. Protein level was examined by Western blot analysis. RNA immunoprecipitation was utilized to validate lncRNA-UCA1 associated with miR-193a. Luciferase activity assay was used to identify the miR-193a-targeted CDK6 3'-untranslated region. RESULTS: lncRNA-UCA1 knockdown weakened cell viability, augmented apoptosis progression, as well as suppressed migration and invasion behaviors in glioblastoma cells, whereas lncRNA-UCA1 silence exhibited the opposite functions. lncRNA-UCA1 functioned as an endogenous sponge of miR-193a. miR-193a silence reversed the biological function of lncRNA-UCA1 knockdown on U-118 MG cells. miR-193a negatively regulated the expression of CDK6, and it affected the U-118 MG cells through regulating CDK6 expression. CDK6 overexpression abrogated the blockage of PI3K/AKT, mitogen-activated protein kinase (MAPK), and Notch signaling pathways. Furthermore, lncRNA-UCA1 and miR-193a could affect these signaling cascades through regulating CDK6 expression. The regulatory mechanisms of lncRNA-UCA1 were further consolidated in clinical specimens. CONCLUSION: lncRNA-UCA1 silence reduced cell viability, promoted apoptosis progression, while impeding the migration and invasion of glioblastoma cells by miR-193a-mediated silence of CDK6, with blockage of PI3K/AKT, MAPK, and Notch pathways.

Sirt3 protects dopaminergic neurons from mitochondrial oxidative stress.

Age-dependent elevation in mitochondrial oxidative stress is widely posited to be a major factor underlying the loss of substantia nigra pars compacta (SNc) dopaminergic neurons in Parkinson's disease (PD). However, mechanistic links between aging and oxidative stress are not well understood. Sirtuin-3 (Sirt3) is a mitochondrial deacetylase that could mediate this connection. Indeed, genetic deletion of Sirt3 increased oxidative stress and decreased the membrane potential of mitochondria in SNc dopaminergic neurons. This change was attributable to increased acetylation and decreased activity of manganese superoxide dismutase (MnSOD). Site directed mutagenesis of lysine 68 to glutamine (K68Q), mimicking acetylation, decreased MnSOD activity in SNc dopaminergic neurons, whereas mutagenesis of lysine 68 to arginine (K68R), mimicking deacetylation, increased activity. Introduction of K68R MnSOD rescued mitochondrial redox status and membrane potential of SNc dopaminergic neurons from Sirt3 knockouts. Moreover, deletion of DJ-1, which helps orchestrate nuclear oxidant defenses and Sirt3 in mice led to a clear age-related loss of SNc dopaminergic neurons. Lastly, K68 acetylation of MnSOD was significantly increased in the SNc of PD patients. Taken together, our studies suggest that an age-related decline in Sirt3 protective function is a major factor underlying increasing mitochondrial oxidative stress and loss of SNc dopaminergic neurons in PD.

Characterization of the Aryl Hydrocarbon Receptor (AhR) Pathway in Anabas testudineus and Mechanistic Exploration of the Reduced Sensitivity of AhR2a.

Field investigations have revealed the ability of the climbing perch Anabas testudineus to survive in highly contaminated water bodies. The aryl hydrocarbon receptor (AhR) pathway is vital in mediating the toxicity of aromatic hydrocarbon contaminants, and genotypic variation in the AhR can confer resistance to these contaminants. Thus, we characterized the AhR pathway in A. testudineus in order to understand the mechanism(s) underlying the resistance of this species to contaminants and to broaden current knowledge on teleost AhR. In A. testudineus, four AhRs, two AhR nuclear translocators (ARNTs), and one AhR repressor (AhRR) were found. Transient transfection assays revealed that AhR1a, AhR1b, and AhR2b were functional, whereas AhR2a was poorly activated by the potent agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Two ARNTs (partner of AhR) and one AhRR (repressor of AhR) all were functional with each of the active AhR. As a major form, the insensitivity of AhR2a might serve as a potential mechanism for A. testudineus' reduced sensitivity to severe contamination. We explored the key residues that may account for AhR2a's insensitivity in silico and then functionally validated them in vitro. Two sites (VCS322-324, M370) in its ligand-binding domain (LBD) were proved critical for its sensitivity to TCDD. This systematic exploration of the AhR pathway showed that most members have maintained their traditional functions as expected, whereas a nonfunctionalization event has occurred for AhR2a.

Motor neuron mitochondrial dysfunction in spinal muscular atrophy.

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, predominantly affects high metabolic tissues including motor neurons, skeletal muscles and the heart. Although the genetic cause of SMA has been identified, mechanisms underlying tissue-specific vulnerability are not well understood. To study these mechanisms, we carried out a deep sequencing analysis of the transcriptome of spinal motor neurons in an SMA mouse model, in which we unexpectedly found changes in many genes associated with mitochondrial bioenergetics. Importantly, functional measurement of mitochondrial activities showed decreased basal and maximal mitochondrial respiration in motor neurons from SMA mice. Using a reduction-oxidation sensitive GFP and fluorescence sensors specifically targeted to mitochondria, we found increased oxidative stress level and impaired mitochondrial membrane potential in motor neurons affected by SMA. In addition, mitochondrial mobility was impaired in SMA disease conditions, with decreased retrograde transport but no effect on anterograde transport. We also found significantly increased fragmentation of the mitochondrial network in primary motor neurons from SMA mice, with no change in mitochondria density. Electron microscopy study of SMA mouse spinal cord revealed mitochondria fragmentation, edema and concentric lamellar inclusions in motor neurons affected by the disease. Intriguingly, these functional and structural deficiencies in the SMA mouse model occur during the presymptomatic stage of disease, suggesting a role in initiating SMA. Altogether, our findings reveal a critical role for mitochondrial defects in SMA pathogenesis and suggest a novel target for improving tissue health in the disease.

Numb directs the subcellular localization of EAAT3 through binding the YxNxxF motif.

Excitatory amino acid transporter type 3 (EAAT3, also known as SLC1A1) is a high-affinity, Na(+)-dependent glutamate carrier that localizes primarily within the cell and at the apical plasma membrane. Although previous studies have reported proteins and sequence regions involved in EAAT3 trafficking, the detailed molecular mechanism by which EAAT3 is distributed to the correct location still remains elusive. Here, we identify that the YVNGGF sequence in the C-terminus of EAAT3 is responsible for its intracellular localization and apical sorting in rat hepatoma cells CRL1601 and Madin-Darby canine kidney (MDCK) cells, respectively. We further demonstrate that Numb, a clathrin adaptor protein, directly binds the YVNGGF motif and regulates the localization of EAAT3. Mutation of Y503, N505 and F508 within the YVNGGF motif to alanine residues or silencing Numb by use of small interfering RNA (siRNA) results in the aberrant localization of EAAT3. Moreover, both Numb and the YVNGGF motif mediate EAAT3 endocytosis in CRL1601 cells. In summary, our study suggests that Numb is a pivotal adaptor protein that mediates the subcellular localization of EAAT3 through binding the YxNxxF (where x stands for any amino acid) motif.

The utility of stem cells in pediatric urinary bladder regeneration.

Pediatric patients with a neurogenic urinary bladder, caused by developmental abnormalities including spina bifida, exhibit chronic urological problems. Surgical management in the form of enterocystoplasty is used to enlarge the bladder, but is associated with significant clinical complications. Thus, alternative methods to enterocystoplasty have been explored through the incorporation of stem cells with tissue engineering strategies. Within the context of this review, we will examine the use of bone marrow stem cells and induced pluripotent stem cells (iPSCs), as they relate to bladder regeneration at the anatomic and molecular levels. The use of bone marrow stem cells has demonstrated significant advances in bladder tissue regeneration as multiple aspects of bladder tissue have been recapitulated including the urothelium, bladder smooth muscle, vasculature, and peripheral nerves. iPSCs, on the other hand, have been well characterized and used in multiple tissue-regenerative settings, yet iPSC research is still in its infancy with regards to bladder tissue regeneration with recent studies describing the differentiation of iPSCs to the bladder urothelium. Finally, we examine the role of the Sonic Hedgehog signaling cascade that mediates the proliferative response during regeneration between bladder smooth muscle and urothelium. Taken together, this review provides a current, comprehensive perspective on bladder regeneration.

RBM-5 modulates U2AF large subunit-dependent alternative splicing in C. elegans.

A key step in pre-mRNA splicing is the recognition of 3' splicing sites by the U2AF large and small subunits, a process regulated by numerous trans-acting splicing factors. How these trans-acting factors interact with U2AF in vivo is unclear. From a screen for suppressors of the temperature-sensitive (ts) lethality of the C. elegans U2AF large subunit gene uaf-1(n4588) mutants, we identified mutations in the RNA binding motif gene rbm-5, a homolog of the tumor suppressor gene RBM5. rbm-5 mutations can suppress uaf-1(n4588) ts-lethality by loss of function and neuronal expression of rbm-5 was sufficient to rescue the suppression. Transcriptome analyses indicate that uaf-1(n4588) affected the expression of numerous genes and rbm-5 mutations can partially reverse the abnormal gene expression to levels similar to that of wild type. Though rbm-5 mutations did not obviously affect alternative splicing per se, they can suppress or enhance, in a gene-specific manner, the altered splicing of genes in uaf-1(n4588) mutants. Specifically, the recognition of a weak 3' splice site was more susceptible to the effect of rbm-5. Our findings provide novel in vivo evidence that RBM-5 can modulate UAF-1-dependent RNA splicing and suggest that RBM5 might interact with U2AF large subunit to affect tumor formation.

[Analysis of prognosis of acute myeloid leukemia patients based on genetic mutations].

OBJECTIVE: To correlate the clinical features of patients with acute myeloid leukemia (AML) with mutations of FLT3-ITD, NPM1, CEBPA, c-KIT, DNMT3A and ND4 genes as well as chromosomal aberrations. METHODS: Somatic mutations of aforementioned genes in 412 newly diagnosed AML patients were detected with PCR and direct sequencing. All patients were also subjected to R-banding chromosomal analysis. The results were correlated with the clinical features and prognosis of the patients. RESULTS: The mutation rates of FLT3-ITD, NPM1, CEBPA, c-KIT, DNMT3A and ND4 were 9.0% (26/289), 19.1% (50/262), 18.9% (34/180), 3.4% (7/208), 6.6% (9/137) and 6.9% (4/58), respectively. Patients with poor prognosis based on genetic mutations had lower blood platelet count than those with intermediate and good prognosis (P=0.001 and P=0.001, respectively). None of the three groups attained median overall survival (OS) (P> 0.05). The complete remission (CR) was similar among the three groups (P> 0.05). For patients with different prognosis based on cytogenetic findings, white blood cell count in those with intermediate prognosis was higher than those with good and poor prognosis (P< 0.001 and P=0.004, respectively), while the blood platelet count of the intermediate group was higher than that of the group with good prognosis (P=0.018). No significant difference was found among the three groups in terms of hemoglobin level (P> 0.05). The group with poor prognosis has attained shorter OS compared with those with good and intermediate prognosis (P< 0.001 and P=0.003, respectively). However, the CR rate of the group with good prognosis was higher than that of the intermediate group (P=0.001). For the group with intermediate prognosis, presence of genetic mutations did not correlate with the clinic characteristics such as white blood cell count, blood platelet count, hemoglobin level, OS and CR rate (P> 0.05 for all comparisons). CONCLUSION: Genetic mutations combined with cytogenetic analysis can facilitate the prognosis and personalized treatment for patients with AML.

Non-aggregating tau phosphorylation by cyclin-dependent kinase 5 contributes to motor neuron degeneration in spinal muscular atrophy.

Mechanisms underlying motor neuron degeneration in spinal muscular atrophy (SMA), the leading inherited cause of infant mortality, remain largely unknown. Many studies have established the importance of hyperphosphorylation of the microtubule-associated protein tau in various neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, tau phosphorylation in SMA pathogenesis has yet to be investigated. Here we show that tau phosphorylation on serine 202 (S202) and threonine 205 (T205) is increased significantly in SMA motor neurons using two SMA mouse models and human SMA patient spinal cord samples. Interestingly, phosphorylated tau does not form aggregates in motor neurons or neuromuscular junctions (NMJs), even at late stages of SMA disease, distinguishing it from other tauopathies. Hyperphosphorylation of tau on S202 and T205 is mediated by cyclin-dependent kinase 5 (Cdk5) in SMA disease condition, because tau phosphorylation at these sites is significantly reduced in Cdk5 knock-out mice; genetic knock-out of Cdk5 activating subunit p35 in an SMA mouse model also leads to reduced tau phosphorylation on S202 and T205 in the SMA;p35(-/-) compound mutant mice. In addition, expression of the phosphorylation-deficient tauS202A,T205A mutant alleviates motor neuron defects in a zebrafish SMA model in vivo and mouse motor neuron degeneration in culture, whereas expression of phosphorylation-mimetic tauS202E,T205E promotes motor neuron defects. More importantly, genetic knock-out of tau in SMA mice rescues synapse stripping on motor neurons, NMJ denervation, and motor neuron degeneration in vivo. Altogether, our findings suggest a novel mechanism for SMA pathogenesis in which hyperphosphorylation of non-aggregating tau by Cdk5 contributes to motor neuron degeneration.

Molecular mechanisms and animal models of spinal muscular atrophy.

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is characterized by the degeneration of spinal motor neurons and muscle atrophy. Although the genetic cause of SMA has been mapped to the Survival Motor Neuron1 (SMN1) gene, mechanisms underlying selective motor neuron degeneration in SMA remain largely unknown. Here we review the latest developments and our current understanding of the molecular mechanisms underlying SMA pathogenesis, focusing on the animal model systems that have been developed, as well as new diagnostic and treatment strategies that have been identified using these model systems. This article is part of a special issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis.

Excessive Wnt/beta-catenin signaling promotes midbrain floor plate neurogenesis, but results in vacillating dopamine progenitors.

The floor plate (FP), a ventral midline structure of the developing neural tube, has differential neurogenic capabilities along the anterior-posterior axis. The midbrain FP, unlike the hindbrain and spinal cord floor plate, is highly neurogenic and produces midbrain dopaminergic (mDA) neurons. Canonical Wnt/beta-catenin signaling, at least in part, is thought to account for the difference in neurogenic capability. Removal of beta-catenin results in mDA progenitor specification defects as well as a profound reduction of neurogenesis. To examine the effects of excessive Wnt/beta-catenin signaling on mDA specification and neurogenesis, we have analyzed a model wherein beta-catenin is conditionally stabilized in the Shh+domain. Here, we show that the Foxa2+/Lmx1a+ domain is extended rostrally in mutant embryos, suggesting that canonical Wnt/beta-catenin signaling can drive FP expansion along the rostrocaudal axis. Although excess canonical Wnt/beta-catenin signaling generally promotes neurogenesis at midbrain levels, less tyrosine hydroxylase (Th)+, mDA neurons are generated, particularly impacting the Substantia Nigra pars compacta. This is likely because of improper progenitor specification. Excess canonical Wnt/beta-catenin signaling causes downregulation of net Lmx1b, Shh and Foxa2 levels in mDA progenitors. Moreover, these progenitors assume a mixed identity to that of Lmx1a+/Lmx1b+/Nkx6-1+/Neurog1+ progenitors. We also show by lineage tracing analysis that normally, Neurog1+ progenitors predominantly give rise to Pou4f1+ neurons, but not Th+ neurons. Accordingly, in the mutant embryos, Neurog1+ progenitors at the midline generate ectopic Pou4f1+ neurons at the expense of Th+ mDA neurons. Our study suggests that an optimal dose of Wnt/beta-catenin signaling is critical for proper establishment of the mDA progenitor character. Our findings will impact embryonic stem cell protocols that utilize Wnt pathway reagents to derive mDA neuron models and therapeutics for Parkinson's disease.

HAP1 gene expression is associated with radiosensitivity in breast cancer cells.

OBJECTIVES: The purpose of this study was to investigate the relationship between huntingtin-associated protein1 (HAP1) gene and radiation therapy of breast cancer cells. METHODS: HAP1 gene was transfected into breast cancer MCF-7 cells, which was confirmed by quantitative reverse transcription-polymerase chain reaction analysis (qRT-PCR) and Western blot in vitro. The changes of cell radiosensitivity were assessed by colony formation assay. Apoptosis were examined by flow cytometry. The expressions of two radiation-induced genes were evaluated by Western blot. Tumor growth was investigated in nude mice xenograft models in vivo. RESULTS: Our data showed that HAP1 gene expression was significantly increased in HAP1-transfected MCF-7 cells in comparison with the parental cells or negative control cells. The survival rate in MCF-7/HAP1 cells was significantly decreased after irradiation (0, 2, 4, 6, 8Gy), compared to cells in MCF-7 and MCF-7/Pb groups in vitro. HAP1 gene increased apoptosis in MCF-7 cells after irradiation. Additionally, the tumor volume and weight in MCF-7/HAP1+RT group were observably lower than in MCF-7/HAP1 group and MCF-7/Pb+RT group. CONCLUSION: The present study indicated that HAP1 gene expression was related to the radiosensitivity of breast cancer cells and may play an important role in the regulation of cellular radiosensitivity.

Effect of Matrine on HPAC cell migration by down-regulating the expression of MT1-MMP via Wnt signaling.

AIM: This study sought to explore the exact mechanism of Matrine inhibited migration and invasion of human pancreatic cancer cells. METHODS: HPAC or Capan-1 cells were cultured in completed RPMI-1640 medium, contained with 50 µg/ml Matrine or 0.05 µg/ml docetaxel, respectively. Cell viability was evaluated by spectrophotometric analysis using MTT assay. Wound healing assay and transwell approach were used to detect the effects of Matrine on HPAC cell migration and invasion. Western Blot and RT-PCR were performed to detect the expressions of MT1-MMP, Wnt and ß-Catenin. CHIP assay was used to detect whether the MT1-MMP transcription activity correlated with Wnt signaling pathway. RESULTS: MTT results indicated that cell proliferration was inhibited by Matrine at a range of concentrations, especially at high dose. We further found that Matrine treatment significantly induced cell migration and invasion decreased. Interestingly, the expression of MT1-MMP decreased evidently upon Matrine treatment, paralleled with the expressions of Wnt and ß-Catenin detected by Western Blot and RT-PCR assay. Further analysis of MT1-MMP transcription activity revealed that Matrine reduced the expression of MT1-MMP mediated by Wnt signaling pathway. CONCLUSION: Matrine play a vital role in inhibiting HPAC cellular migration and invasion through down-regulating the expression of MT1-MMP via Wnt signaling pathway.

Effect of Furin inhibitor on lung adenocarcinoma cell growth and metastasis.

BACKGROUND: To investigate the mechanisms of lung adenocarcinoma cell metastasis and provide a theoretical basis for the in-depth study of lung adenocarcinoma. METHODS: A549 cells are incubated with different concentrations of Furin inhibitor for indicated times. The proliferation and migration were confirmed with MTT, colony formation, wound Healing and Transwell assayes. Hochest 33342 / PI double staining was used to detect apoptosis. Cell migration and apoptosis associated proteins were analysed by enzyme-linked immunosorbent assay (ELISA) and western blot. RESULTS: We have found that Furin inhibitor play a significant role in inhibition A549 cell growth. And we also found cell migration was inhibited significantly upon Furin inhibitor treatment. CONCLUSION: The proliferration and migration of A549 cell were inhibited by Furin inbitor through down-regulation the expression of migration and apoptosis related proteins.

The survival motor neuron gene smn-1 interacts with the U2AF large subunit gene uaf-1 to regulate Caenorhabditis elegans lifespan and motor functions.

Spinal muscular atrophy (SMA), the most frequent human congenital motor neuron degenerative disease, is caused by loss-of-function mutations in the highly conserved survival motor neuron gene SMN1. Mutations in SMN could affect several molecular processes, among which aberrant pre-mRNA splicing caused by defective snRNP biogenesis is hypothesized as a major cause of SMA. To date little is known about the interactions of SMN with other splicing factor genes and how SMN affects splicing in vivo. The nematode Caenorhabditis elegans carries a single ortholog of SMN, smn-1, and has been used as a model for studying the molecular functions of SMN. We analyzed RNA splicing of reporter genes in an smn-1 deletion mutant and found that smn-1 is required for efficient splicing at weak 3' splice sites. Genetic studies indicate that the defective lifespan and motor functions of the smn-1 deletion mutants could be significantly improved by mutations of the splicing factor U2AF large subunit gene uaf-1. In smn-1 mutants we detected a reduced expression of U1 and U5 snRNAs and an increased expression of U2, U4 and U6 snRNAs. Our study verifies an essential role of smn-1 for RNA splicing in vivo, identifies the uaf-1 gene as a potential genetic modifier of smn-1 mutants, and suggests that SMN-1 has multifaceted effects on the expression of spliceosomal snRNAs.