The role and mechanism of hnRNP A2/B1 in MCF-7 Cells

核不均一核糖核蛋白A2/B1（hnRNPA2/B1）在很多肿瘤中高表达，但其在 乳腺癌发生发展中的作用还不明确。为了探究其在乳腺癌中的具体作用与机制， 我们通过CRISPR-Cas9基因编辑技术构建敲除hnRNPA2/B1的MCF-7细胞系， 检测其对生物学功能的影响，并研究hnRNPA2/B1对部分基因的可变剪接的作用。 还通过亲和纯化技术结合液质联用质谱（HPLC-MS），构建与分析hnRNPA2/B1 相互作用蛋白网络，利用生物信息学方法对hnRNPB1相互作用蛋白进行KEGG 信号通路及功能分析，进一步来研究hnRNPA2/B1在乳腺癌细胞中的作用与机制。 MTT法检测发...An emerging body of data shows the overexpression of hnRNPA2/B1 in many cancers, but its specific molecular mechanism in tumors is still poorly understand. In order to explore the mechanism of hnRNPA2/B1 and find valid therapeutic target in breast cancer. We used CRISPR-Cas9 to construct the stable MCF-7 cells in which hnRNPA2/B1 were konck-out, and explored the change of proliferation, migrat...学位：理学硕士院系专业：医学院_微生物学学号：2452013115343

The role and mechanism of hnRNP A2/B1 in breast cancer

核不均一核糖核蛋白A2/B1（hnRNPA2/B1）是hnRNP蛋白家族成员，在肿瘤细胞增殖与凋亡过程中具有重要作用。本实验为了探究hnRNPA2/B1蛋白在乳腺癌组织中的表达及其在乳腺癌细胞中的定位。研究hnRNPA2/B1在乳腺癌细胞中所涉及的主要功能、信号通路以及可变剪接和蛋白融合过程，并探究hnRNPA2/B1对细胞增殖、凋亡、迁移等生物功能的影响。从而阐明hnRNPA2/B1在乳腺癌中的作用机制，为肿瘤的机理研究、临床诊断和治疗提供一个新靶点。本实验用免疫组化方法检测hnRNPA2/B1在大鼠乳腺组织中的变化，并结合免疫荧光检测hnRNPA2/B1在姜黄素诱导人乳腺癌细胞凋亡过程中的...Heterogeneous nuclear ribonucleoproteins(hnRNP A2/B1),a member of the hnRNP family, plays a key role in cell life process, such as differentiation, apoptosis. To investigate the difference of expression of hnRNP A2/B1 between the normal and cancer tissue and the change of location of hnRNP A2/B1 during the apoptosis procession. To research main biological function, pathways, splicing and gene fus...学位：理学硕士院系专业：医学院_微生物学学号：2452012115319

hnRNPA2/B1 regulates apoptosis-related gene PLK3 expression and BIM alternative splicing in breast cancer cells

目的：核糖核不均一蛋白（hnRNPA2/B1）作为一个可变剪切子，在乳腺癌组织中过度表达，参与细胞多种生理过程。本文研究乳腺癌细胞中hnRNPA2/B1调控凋亡相关基因PLK3表达和BIM的可变剪切。 方法：通过CRISPSR-Cas9技术敲除乳腺癌细胞MCF-7和MDA-MB-231中hnRNPA2/B1基因后，验证凋亡相关基因的表达；结合转录组测序分析以及RTPCR验证凋亡相关基因是否发生可变剪切，并通过RNA免疫沉淀技术检测hnRNPA2/B1蛋白是否与凋亡相关基因的pre-mRNA结合。体内实验中用Tunel法检测MCF-7细胞hnRNPA2/B1敲除前后小鼠移植瘤凋亡变化。通过CC...Objectives: As a splicing regulator, Previous studies found hnRNP A2/B1 overexpression in breast cancers associated with various physiological processes of cells. Our studies are to illustrate hnRNPA2/B1 regulates apoptosis-related gene PLK3 expression and BIM alternative splicing in breast cancer cells. Methods: We knocked out hnRNP A2/B1 by CRISPSR-Cas9 technique in MCF-7 and MDA-MB-231 cell, c...学位：理学硕士院系专业：医学院_微生物学学号：2452014115346

Regulation mechanism of HnRNP A2/B1 inhibiting breast carcinoma cell epithelial-mesenchymal transition and metastasis

乳腺癌转移是乳腺癌引发死亡的主要原因。上皮间充质转化（EMT）和乳腺癌的转移密切相关。HnRNPA2/B1作为重要的功能调控蛋白，在人类多种癌症进程中起到关键作用，但对乳腺癌转移研究及其分子机制尚不明确。 本文从细胞、动物以及临床水平研究了HnRNPA2/B1的表达水平与乳腺癌转移的相关性：利用CRISPR/Cas9基因编辑系统，构建稳定敲除HnRNPA2/B1基因的MDA-MB-231和MCF-7细胞系，从细胞水平上检测HnRNPA2/B1对乳腺癌细胞EMT以及侵袭和转移的影响；通过构建裸鼠实验性肺转移模型和自发性肺转移模型，在体内检测分析HnRNPA2/B1的表达变化对乳腺癌细胞转移能力...Breast cancer metastasis is the leading cause of death in breast cancer. Epithelial mesenchymal transition (EMT) is closely related to the metastasis of breast cancer.As an important functional regulatory protein,HnRNP A2/B1 plays a key role in the process of many human cancers, but its study on breast cancer metastasis and molecular mechanism is not clear. In this paper, the relationship betwee...学位：理学硕士院系专业：医学院_微生物学学号：2452014115346

核内不均一核糖核蛋白A2/B1的研究进展

核内不均一核糖核蛋白A2/B1(hnRNPA2/B1)是核不均一核糖核蛋白家族的成员,其在细胞生命活动中起着非常重要的作用,主要参与pre-mRNA的可变剪接和转录调控、端粒维持及细胞增殖、迁移过程,在很多肿瘤中呈高表达的状态。本文就hnRNPA2/B1在肿瘤中的作用与机制及其所涉及的其他功能作一综述。国家自然科学基金(81272245

Modulation of PKM alternative splicing by PTBP1 promotes gemcitabine resistance in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and incurable disease. Poor prognosis is due to multiple reasons, including acquisition of resistance to gemcitabine, the first-line chemotherapeutic approach. Thus, there is a strong need for novel therapies, targeting more directly the molecular aberrations of this disease. We found that chronic exposure of PDAC cells to gemcitabine selected a subpopulation of cells that are drug-resistant (DR-PDAC cells). Importantly, alternative splicing (AS) of the pyruvate kinase gene (PKM) was differentially modulated in DR-PDAC cells, resulting in promotion of the cancer-related PKM2 isoform, whose high expression also correlated with shorter recurrence-free survival in PDAC patients. Switching PKM splicing by antisense oligonucleotides to favor the alternative PKM1 variant rescued sensitivity of DR-PDAC cells to gemcitabine and cisplatin, suggesting that PKM2 expression is required to withstand drug-induced genotoxic stress. Mechanistically, upregulation of the polypyrimidine-tract binding protein (PTBP1), a key modulator of PKM splicing, correlated with PKM2 expression in DR-PDAC cell lines. PTBP1 was recruited more efficiently to PKM pre-mRNA in DR- than in parental PDAC cells. Accordingly, knockdown of PTBP1 in DR-PDAC cells reduced its recruitment to the PKM pre-mRNA, promoted splicing of the PKM1 variant and abolished drug resistance. Thus, chronic exposure to gemcitabine leads to upregulation of PTBP1 and modulation of PKM AS in PDAC cells, conferring resistance to the drug. These findings point to PKM2 and PTBP1 as new potential therapeutic targets to improve response of PDAC to chemotherapy.Oncogene advance online publication, 3 August 2015; doi:10.1038/onc.2015.270

Heterozygous frameshift variants in HNRNPA2B1 cause early-onset oculopharyngeal muscular dystrophy

Missense variants in RNA-binding proteins (RBPs) underlie a spectrum of disease phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, and inclusion body myopathy. Here, we present ten independent families with a severe, progressive muscular dystrophy, reminiscent of oculopharyngeal muscular dystrophy (OPMD) but of much earlier onset, caused by heterozygous frameshift variants in the RBP hnRNPA2/B1. All disease-causing frameshift mutations abolish the native stop codon and extend the reading frame, creating novel transcripts that escape nonsense-mediated decay and are translated to produce hnRNPA2/B1 protein with the same neomorphic C-terminal sequence. In contrast to previously reported disease-causing missense variants in HNRNPA2B1, these frameshift variants do not increase the propensity of hnRNPA2 protein to fibrillize. Rather, the frameshift variants have reduced affinity for the nuclear import receptor karyopherin β2, resulting in cytoplasmic accumulation of hnRNPA2 protein in cells and in animal models that recapitulate the human pathology. Thus, we expand the phenotypes associated with HNRNPA2B1 to include an early-onset form of OPMD caused by frameshift variants that alter its nucleocytoplasmic transport dynamics

Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene

Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as “splicing mutations,” but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002–1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5′- and 3′-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002–1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75

The Role of Forebrain Cholinergic Signalling In Regulating Hippocampal Function And Neuropathology

Cholinergic dysfunction has been associated with cognitive abnormalities in a variety of neurodegenerative and neuropsychiatric disorders, including Alzheimer’s Disease (AD). Cumulative use of drugs with anticholinergic activity is associated with increased risk for dementia and AD. Also, cholinergic function has been implicated in predicting the development of key neuropathological hallmarks seen in AD. However, the relationship between cholinergic dysfunction and conservation of cognitive ability as well as neuronal cell maintenance is not fully understood. Here, we tested how information processing and distinct molecular mechanisms associated with AD are regulated by cholinergic tone in genetically-modified mice in which cholinergic transmission was altered by targeting the vesicular acetylcholine transporter (VAChT), a protein required for acetylcholine storage and release. We assessed the long-term consequences of loss of central cholinergic signalling for hippocampal vulnerability to age-induced stress. We show that deletion of forebrain-specific ACh release leads to age-related increases in neuronal vulnerability, protein aggregation, tau Thr-231 phosphorylation and misfolding, and neuroinflammation. Moreover, inhibition of forebrain cholinergic neurotransmission led to a disturbance in adult hippocampal neurogenesis, highlighted by decreased proliferation and cell survival in neural precursor cells. Additionally, we measured long-term potentiation of Schaffer collateral-CA1 synapses in vivo and assessed information processing by using a mouse touchscreen version of Paired Associates Learning task (PAL). Acquisition in the mouse PAL task was impaired in forebrain-specific VAChT-deficient mice, suggesting a critical role for cholinergic tone. Accordingly, synaptic plasticity in the hippocampus in vivo was disturbed, but not completely abolished, by decreased hippocampal cholinergic signalling. In contrast, spatial memory was relatively preserved. Moreover, we assessed the functional consequence of impaired neurogenesis by testing pattern separation using a Location Discrimination task. Mice with compromised cholinergic signalling were impaired when stimuli were presented with small separation, but not when stimuli were presented with high separation, suggesting that deficient cholinergic tone has major consequences on pattern separation. The pathological changes in the hippocampus we observed in VAChT-deficient mice have important consequences as they presented age-related deterioration in spatial navigation. Our findings provide a refined understanding of the importance of acetylcholine in modulating molecular mechanisms and key cognitive behaviours involved in AD

Prion-like domain mutations in hnRNPs cause multisystem proteinopathy and ALS

Summary Algorithms designed to identify canonical yeast prions predict that ~250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbor a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here, we define pathogenic mutations in PrLDs of hnRNPA2/B1 and hnRNPA1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and a case of familial ALS. Wild-type hnRNPA2 and hnRNPA1 display an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a ‘steric zipper’ motif in the PrLD, which accelerates formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Importantly, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant ‘steric zipper’ motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs must be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone