A candidate protective factor in amyotrophic lateral sclerosis:heterogenous nuclear ribonucleoprotein G

Heterogenous nuclear ribonucleoprotein G is down-regulated in the spinal cord of the Tg(SOD1*G93A)1Gur(TG)amyotrophic lateral sclerosis mouse model.However,most studies have only examined heterogenous nuclear ribonucleoprotein G expression in the amyotrophic lateral sclerosis model and heterogenous nuclear ribonucleoprotein G effects in amyotrophic lateral sclerosis pathogenesis such as in apoptosis are unknown.In this study,we studied the potential mechanism of heterogenous nuclear ribonucleoprotein G in neuronal death in the spinal cord of TG and wild-type mice and examined the mechanism by which heterogenous nuclear ribonucleoprotein G induces apoptosis.Heterogenous nuclear ribonucleoprotein G in spinal cord was analyzed using immunohistochemistry and western blotting,and cell proliferation and proteins(TAR DNA binding protein 43,superoxide dismutase 1,and Bax)were detected by the Cell Counting Kit-8 and western blot analysis in heterogenous nuclear ribonucleoprotein G siRNA-transfected PC12 cells.We analyzed heterogenous nuclear ribonucleoprotein G distribution in spinal cord in the amyotrophic lateral sclerosis model at various time points and the expressions of apoptosis and proliferation-related proteins.Heterogenous nuclear ribonucleoprotein G was mainly localized in neurons.Amyotrophic lateral sclerosis mice were examined at three stages:preonset(60-70 days),onset(90-100 days)and progression(120-130 days).The number of heterogenous nuclear ribonucleoprotein G-positive cells was significantly higher in the anterior horn of the lumbar spinal cord segment of TG mice at the preonset stage than that of control group but lower than that of the control group at the onset stage.The number of heterogenous nuclear ribonucleoprotein G-positive cells in both central canal and surrounding gray matter of the whole spinal cord of TG mice at the onset stage was significantly lower than that in the control group,whereas that of the lumbar spinal cord segment of TG mice was significantly higher than that in the control group at preonset stage and significantly lower than that in the control group at the progression stage.The numbers of heterogenous nuclear ribonucleoprotein G-positive cells in the posterior horn of cervical and thoracic segments of TG mice at preonset and progression stages were significantly lower than those in the control group.The expression of heterogenous nuclear ribonucleoprotein G in the cervical spinal cord segment of TG mice was significantly higher than that in the control group at the preonset stage but significantly lower at the progression stage.The expression of heterogenous nuclear ribonucleoprotein G in the thoracic spinal cord segment of TG mice was significantly increased at the preonset stage,significantly decreased at the onset stage,and significantly increased at the progression stage compared with the control group.heterogenous nuclear ribonucleoprotein G expression in the lumbar spinal cord segment of TG mice was significantly lower than that of the control group at the progression stage.After heterogenous nuclear ribonucleoprotein G gene silencing,PC12 cell survival was lower than that of control cells.Both TAR DNA binding protein 43 and Bax expressions were significantly increased in heterogenous nuclear ribonucleoprotein G-silenced cells compared with control cells.Our study suggests that abnormal distribution and expression of heterogenous nuclear ribonucleoprotein G might play a protective effect in amyotrophic lateral sclerosis development via preventing neuronal death by reducing abnormal TAR DNA binding protein 43 generation in the spinal cord.

Protein and gene expression characteristics of heterogeneous nuclear ribonucleoprotein H1 in esophageal squamous cell carcinoma

AIM To investigate the expression characteristics of heterogeneous nuclear ribonucleoprotein H1(HNRNPH1) m RNA and protein in cell lines and tissues of esophageal squamous cell carcinoma(ESCC). METHODS Western blotting was used to assess the expression of HNRNPH1 protein in seven ESCC cell lines and 30 paired fresh tissue specimens. The subcellular localization of HNRNPH1 was determined by immunofluorescence in ESCC cells. The RNA sequencing data from 87 patients with ESCC were obtained from the cancer genome atlas(TCGA), and the expression and clinical characteristics analysis of different transcript variants of HNRNPH1 were evaluated in this dataset. In addition, immunohistochemistry was carried out to detect the expression of HNRNPH1 protein in 125 patients.RESULTS The expression of HNRNPH1 protein varied across different ESCC cell lines. It was exclusively restricted to the nucleus of the ESCC cells. There are two transcript variants of the HNRNPH1 gene. Variant 1 was constitutively expressed, and its expression did not change during tumorigenesis. In contrast, levels of variant 2 were low in non-tumorous tissues and were dramatically increased in ESCC(P = 0.0026). The high levels of variant 2 were associated with poorer differentiated tumors(P = 0.0287). Furthermore, in paired fresh tissue specimens, HNRNPH1 protein was overexpressed in 73.3%(22/30) of neoplastic tissues. HNRNPH1 was significantly upregulated in ESCC, with strong staining in 43.2%(54/125) of tumor tissues and 22.4%(28/125) of matched non-cancerous tissues(P = 0.0005). Positive HNRNPH1 expression was significantly associated with poor tumor differentiation degree(P = 0.0337).CONCLUSION The different alternative transcript variants of HNRNPH1 exhibited different expression changes during tumorigenesis. Its m RNA and protein were overexpressed in ESCC and associated with poorer differentiation of tumor cells. These findings highlight the potential of HNRNPH1 in the therapy and diagnosis of ESCC.

The heterodimeric structure of heterogeneous nuclear ribonucleoprotein C1/C2 dictates 1,25-dihydroxyvitamin D-directed transcriptional events in osteoblasts

Heterogeneous nuclear ribonucleoprotein （hnRNP） C plays a key role in RNA processing but also exerts a dominant negative effect on responses to 1,25-dihydroxyvitamin D （1,25（OH）2D） by functioning as a vitamin D response element-binding protein （VDRE-BP）. hnRNPC acts a tetramer of hnRNPC1 （huC1） and hnRNPC2 （huC2）, and organization of these subunits is critical to in vivo nucleic acid-binding. Overexpression of either huC1 or huC2 in human osteoblasts is sufficient to confer VDRE-BP suppression of 1,25（OH）2D-mediated transcription. However, huC1 or huC2 alone did not suppress 1,25（OH）2D-induced transcription in mouse osteoblastic cells. By contrast, overexpression of huC1 and huC2 in combination or transfection with a bone-specific polycistronic vector using a ＂self-cleaving＂ 2A peptide to co-express huC1/C2 suppressed 1,25D-mediated induction of osteoblast target gene expression. Structural diversity of hnRNPC between human/NWPs and mouse/rat/rabbit/dog was investigated by analysis of sequence variations within the hnRNP CLZ domain. The predicted loss of distal helical function in hnRNPC from lower species provides an explanation for the altered interaction between huC1/C2 and their mouse counterparts. These data provide new evidence of a role for hnRNPC1/C2 in 1,25（OH）2D-driven gene expression, and further suggest that species-specific tetramerization is a crucial determinant of its actions as a regulator of VDR-directed transactivation.

Dengue virus non-structural 1 protein interacts with heterogeneous nuclear ribonucleoprotein H in human monocytic cells

Objective: To study protein-protein interaction between heterogeneous nuclear ribonucleoprotein H(hn RNP H) and Dengue virus(DENV) proteins. Methods: DENV proteins were screened against the host hn RNP H protein, in order to identify the host-viral protein-protein interactions in DENV infected THP-1 cells by co-immunoprecipitation. The co-localization of the interacting proteins was further confirmed by immunofluorescence microscopy. Results: The host protein hn RNP H was found to interact with DENV nonstructural 1 protein and help the virus to multiply in the cell. Conclusions: The non-structural 1 glycoprotein is a key modulator of host immune response and is also involved in viral replication. Therefore, disruption of this key interaction between hn RNP H and DENV nonstructural 1 could be an important therapeutic strategy for management of DENV infection.

Gene Expression Changes Associated with the Loss of Heterogeneous Nuclear Ribonucleoprotein M Function

Advanced glycation endproducts (AGEs) are formed by the nonenzymatic reaction of sugars with proteins. Glycation may adversely affect proteins, such as by inducing a loss of function. It has been shown that glyceraldehyde-derived AGEs (Glycer-AGEs) accumulate in the liver of patients with nonalcoholic steatohepatitis (NASH). Previously, we showed the formation of intracellular Glycer-AGEs upon exposure of hepatocytes to fructose in vitro, and identified an RNA-binding protein, heterogeneous nuclear ribonucleoprotein M (HNRNPM), as a target for glycation. However, the impact of glycated HNRNPM in NASH remains poorly understood. In this study, we examined gene expression changes caused by HNRNPM knockdown, and investigated the up- and down-regulated genes as noninvasive biomarker candidates for NASH. Microarray analysis after HNRNPM knockdown showed that the levels of 138 transcripts were increased, while those of 100 transcripts were decreased as compared with those in the control. Gene Ontology-based functional analysis showed that 14 upregulated and 9 downregulated genes were associated with the extracellular space, which may enable their detection using blood tests. Among these, six of the up- and down-regulated genes were associated with the extracellular exosome. These results suggest that the loss of HNRNPM function by glycation is reflected extracellularly. Therefore, the identified genes may serve as noninvasive biomarkers for Glycer-AGEs-related NASH.

Significance of the antigenic epitopes in heterogenous nuclear ribonucleoproteins-Ⅰfor the diagnosis and prognosis of systematic sclerosis

To assess the presence of autoantibodies against epitopes of heterogenous nuclear ribonucleoprotein-Ⅰ(hnRNP-Ⅰ) in systematic sclerosis (SSc) and to analyze their clinical significance, polypeptides of hnRNP-Ⅰwere designed by biological technical software and analyzed with both the Wonderful Biology Information System and DNA Star-Protean Analysis Software at the same time. In these ways, two polypeptides of hnRNP-Ⅰwere obtained based on their amino acid sequences, folding features, hydrophilic, curl style, dough kneading sensation and the possibility on the surface of proteins. They are named as hnRNP-Ⅰ-1 ( NVKYNNDKSRDYTRPDLPSGDSQPSLDQT, 264-292 aa) and hnRNP-Ⅰ-2 (QLP4REGQEDQGLTKDYGNSOL, 441-461 aa), simply designated asⅠ-1 andⅠ-2. The autoantibodies against hnRNPs were detected by means of ELISA using the synthetic epitopes polypeptides as antigen. It was found that the positive rate of detection for anti-Ⅰ-1 and anti-Ⅰ-2 autoantibodies were rather higher in SSc patients than that in other CTDs and the sensitivities and specificities of the testing with ELJSA for anti-Ⅰ-1 and anti-Ⅰ-2 antibodies in SSc patients were 47. 62%/93. 43% and 38. 1%/ 91.08% , without any significant difference between these two groups of testings. Also, there was no significant difference in the clinical features and laboratory findings, such as age, involvements in digestive and respiratory tracts and erythrocyte sedimentation rate etc., between the anti-Ⅰ-1-positive and -negative groups in SSc patients. However, the hnRNP-Ⅰ-autoantibody-positive group of patients had obviously shorter duration of disease course compared with that of the autoantibody-negative group. Anti-Ⅰ-1 and anti-Ⅰ-2 autoantibodies also had no association with antinuclear antibody, anti-Scl70 and anti-centromere antibody (ACA) in SSc patients. So, it is apparent that the autoantibodies related with SSc may act through different pathways in the pathogenesis of SSc, and the hnRNP-Ⅰautoantibody is a new type antibody occuring during the early stage of disease and appearing to have diagnostic and prognostic significances .

Engineered extracellular vesicles efficiently deliver CRISPR-Cas9 ribonucleoprotein(RNP)to inhibit herpes simplex virus1 infection in vitro and in vivo

Extracellular vesicles(EVs)have recently emerged as a promising delivery platform for CRISPR/Cas9 ribonucleoproteins(RNPs),owing to their ability to minimize off-target effects and immune responses.However,enhancements are required to boost the efficiency and safety of Cas9 RNP enrichment within EVs.In response,we employed the Fc/Spa interaction system,in which the human Fc domain was fused to the intracellular domain of PTGFRN-Δ687 and anchored to the EV membrane.Simultaneously,the B domain of the Spa protein was fused to the C domain of cargos such as Cre or spCas9.Due to the robust interaction between Fc and Spa,this method enriched nearly twice the amount of cargo within the EVs.EVs loaded with spCas9 RNP targeting the HSV1 genome exhibited significant inhibition of viral replication in vitro and in vivo.Moreover,following neuron-targeting peptide RVG modification,the in vivo dosage in neural tissues substantially increased,contributing to the clearance of the HSV1 virus in neural tissues and exhibiting a lower off-target efficiency.These findings establish a robust platform for efficient EV-based SpCas9 delivery,offering potential therapeutic advantages for HSV1 infections and other neurological disorders.

Novel miR-490-3p/hnRNPA1-b/PKM2 axis mediates the Warburg effect and proliferation of colon cancer cells via the PI3K/AKT pathway

BACKGROUND Heterogeneous ribonucleoprotein A1(hnRNPA1)has been reported to enhance the Warburg effect and promote colon cancer(CC)cell proliferation,but the role and mechanism of the miR-490-3p/hnRNPA1-b/PKM2 axis in CC have not yet been elucidated.AIM To investigate the role and mechanism of a novel miR-490-3p/hnRNPA1-b/PKM2 axis in enhancing the Warburg effect and promoting CC cell proliferation through the PI3K/AKT pathway.METHODS Paraffin-embedded pathological sections from 220 CC patients were collected and subjected to immunohistochemical analysis to determine the expression of hnRNPA1-b.The relationship between the expression values and the clinicopathological features of the patients was investigated.Differences in mRNA expression were analyzed using quantitative real-time polymerase chain reaction,while differences in protein expression were analyzed using western blot.Cell proliferation was evaluated using the cell counting kit-8 and 5-ethynyl-2’-deoxyuridine assays,and cell cycle and apoptosis were detected using flow cytometric assays.The targeted binding of miR-490-3p to hnRNPA1-b was validated using a dual luciferase reporter assay.The Warburg effect was evaluated by glucose uptake and lactic acid production assays.RESULTS The expression of hnRNPA1-b was significantly increased in CC tissues and cells compared to normal controls(P<0.05).Immunohistochemical results demonstrated significant variations in the expression of the hnRNPA1-b antigen in different stages of CC,including stage I,II-III,and IV.Furthermore,the clinicopathologic characterization revealed a significant correlation between hnRNPA1-b expression and clinical stage as well as T classification.HnRNPA1-b was found to enhance the Warburg effect through the PI3K/AKT pathway,thereby promoting proliferation of HCT116 and SW620 cells.However,the proliferation of HCT116 and SW620 cells was inhibited when miR-490-3p targeted and bound to hnRNPA1-b,effectively blocking the Warburg effect.CONCLUSION These findings suggest that the novel miR-490-3p/hnRNPA1-b/PKM2 axis could provide a new strategy for the diagnosis and treatment of CC.

AGPS在神经胶质瘤细胞中的相互作用蛋白及作用模式

目的探讨胶质瘤细胞(U251细胞)中癌基因烷基甘油酮磷酸合酶(AGPS)的相互作用蛋白及作用模式。方法常规培养U251细胞并随机分为对照组、shR-AGPS-1组、shR-AGPS-2组,分别加入阴性对照慢病毒和不同沉默水平的AGPS shRNA慢病毒,用Western blotting法检测AGPS蛋白表达以验证沉默效率。通过免疫共沉淀技术和质谱技术鉴定AGPS相互作用的蛋白,用Western blotting法进行验证;用计算机模拟技术进行相互作用蛋白的同源模建并推测二者相互作用模式。结果与对照组比较,shR-AGPS-1组、shR-AGPS-2组AGPS表达低(P均<0.05),且shR-AGPS-1组AGPS表达低于shR-AGPS-2组(P<0.05)。将筛选获得的数据进行整理,初步判断不均一核糖核蛋白K(HNRNPK)为AGPS的靶蛋白。在AGPS抗体免疫共沉淀的细胞裂解物中可同时检测到AGPS蛋白、HNRNPK蛋白,表明AGPS与HNRNPK可形成复合体,二者均在细胞核中表达。计算机模拟结果显示AGPS和HNRNPK通过氨基酸残基以氢键和共轭、疏水键和静电力形式相互作用。结论胶质瘤细胞中HNRNPK是AGPS的相互作用蛋白,氢键和共轭、疏水键和静电力相互作用可能是其主要的作用模式。

基于HNRNPA2B1及其调控miRNAs构建肺腺癌TP53突变人群的预后模型

目的基于癌症基因组图谱(TCGA)数据库多组学数据构建肺腺癌TP53突变人群的预后模型,探讨核内不均一核糖核蛋白A2/B1(HNRNPA2B1)与肺腺癌TP53突变之间的相关性。方法通过生物信息学方法搜集TCGA和基因表达数据库(GEO)中的突变数据,分析TP53突变对肺腺癌病人HNRNPA2B1表达及预后的影响;将病人随机分为训练集和验证集(7∶3),筛选潜在的受HNRNPA2B1调控的miRNAs构建模型、绘制ROC曲线,并通过列线图可视化。结果HNRNPA2B1在TP53突变肺腺癌中显著高表达(P<0.001),且高表达病人预后不良(P=0.031)。筛选出9个受HNRNPA2B1调控且与预后相关的miRNAs构建预后模型,结果表明列线图对预后模型具有较好的区分度和准确度(χ^(2)=9.443,P=0.306)。结论HNRNPA2B1与肺腺癌TP53突变存在正相关,基于HNRNPA2B1调控的miRNAs可建立预测TP53突变肺腺癌病人预后的良好模型。

Enhanced Bombyx genome editing via Cas9 ribonucleoprotein injection

Dear Editor, The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has become a popular and powerful method for manipulating genomes with considerably little effort and high efficiency. It allows for the generation of targeted indels, genomic structure variations, and insertion of foreign fragments. However, increasing concerns have been raised about possible off-target effects.

Efficient genome editing in Claviceps purpurea using a CRISPR/Cas9 ribonucleoprotein method

Claviceps purpurea produces many pharmacologically important ergot alkaloids(EAS),which are widely used to treat migraine and hypertension and to aid childbirth.Although an EAS biosynthetic cluster of C.purpurea has been discovered more than 20 years ago,the complete biosynthetic pathway of EAS has not been fully characterized until now.The main obstacle to elucidating this pathway and strain modification is the lack of efficient genome-editing tools for C.purpurea.The conventional gene manipulation method for C.purpurea relies on homologous recombination(HR),although the efficiency of HR in C.purpurea is very low(~1-5%).Consequently,the disruption of target genes is laborious and time-consuming.Although CRISPR/Cas9 genome-editing methods based on in vivo Cas9 expression and gRNA transcription have been reported recently,their gene-disruption efficiency is still very low.Here,we developed an efficient genome-editing system in C.purpurea based on in vitro assembled CRISPR/Cas9 gRNA ribonucleoprotein complexes.As proof of principle,three target genes were efficiently knocked out using this CRISPR/Cas9 ribonucleoprotein complex-mediated HR system,with editing efficiencies ranging from 50%to 100%.Inactivation of the three genes,which are closely related to uridine biosynthesis(ura5),hypha morphology(rac),and EAS production(easA),resulted in a uridine auxotrophic mutant,a mutant with a drastically different phenotype in axenic culture,and a mutant that did not produce EAS,respectively.Our ribonucleoprotein-based genome-editing system has a great advantage over conventional and in vivo CRISPR/Cas9 methods for genome editing in C.purpurea,which will greatly facilitate elucidation of the EAS biosynthetic pathway and other future basic and applied research on C.purpurea.

HNRNPF促进前列腺癌的增殖、迁移和侵袭

目的研究核不均一核糖核蛋白F(heterogeneous nuclear ribonucleoproteins F,HNRNPF)在前列腺癌中的表达、临床相关性及其对前列腺癌细胞增殖、迁移和侵袭能力的影响。方法使用TCGA和GEO数据库分析HNRNPF在前列腺癌中的表达特征、免疫浸润特征及其与前列腺癌患者临床病理特征的相关性。使用RNA干扰在前列腺癌细胞PC-3和DU145中沉默HNRNPF基因,使用CCK-8、EdU和集落形成实验检测细胞增殖能力的改变,使用Transwell和划痕愈合实验检测细胞迁移、侵袭能力的改变。结果相比正常前列腺组织,HNRNPF在前列腺癌组织中的表达量显著升高。HNRNPF的表达量与前列腺癌患者的T分期、Gleason评分、前列腺特异性抗原以及多种免疫细胞的浸润水平显著相关。HNRNPF高表达的患者总生存期和疾病特异性生存期较短。沉默HNRNPF基因显著抑制了PC-3和DU145细胞的增殖、迁移、侵袭能力。结论HNRNPF是一个在前列腺癌中高表达的基因,具有显著临床相关性,且能够促进前列腺癌细胞增殖、迁移和侵袭。

重楼皂苷Ⅵ通过调控SNRPD1抑制肝癌细胞增殖的机制研究

目的探讨重楼皂苷Ⅵ通过调控小核核糖核蛋白D1(SNRPD1)抑制肝癌细胞增殖的作用机制。方法①21只雄性裸鼠,采用皮下注射人肝癌Huh7细胞建立裸鼠肝癌移植瘤模型。裸鼠随机分为模型组(药物溶剂)、5 mg/kg重楼皂苷Ⅵ组、10 mg/kg重楼皂苷Ⅵ组,每组7只。连续腹腔注射给予相应干预10 d,观察各组裸鼠瘤体体积变化。②将人肝癌Huh7和JHH7细胞分为对照组和重楼皂苷Ⅵ组,采用重楼皂苷Ⅵ(0~15μmol/L)给予相应干预后,用细胞计数试剂盒(CCK-8)法和克隆形成实验检测细胞增殖情况,流式细胞术检测细胞周期情况,Western blot法和实时荧光定量逆转录聚合酶链式反应(RT-qPCR)法检测SNRPD1、细胞周期蛋白D1(Cyclin D1)、周期蛋白依赖性激酶4(CDK4)蛋白和基因的表达情况。采用分子对接技术预测重楼皂苷Ⅵ和SNRPD1蛋白的潜在靶向结合情况。结果①与模型组比较,10 mg/kg重楼皂苷Ⅵ能抑制Huh7细胞裸鼠移植瘤生长(P<0.05)。②与对照组比较,重楼皂苷Ⅵ能抑制人肝癌Huh7和JHH7细胞的增殖(P<0.05)、细胞克隆形成(P<0.05)及诱导细胞周期的阻滞(P<0.05)。分子对接结果显示,重楼皂苷Ⅵ潜在靶向SNRPD1蛋白;Western blot和RT⁃qPCR结果显示,与对照组比较,重楼皂苷Ⅵ可以下调SNRPD1的蛋白表达水平(P<0.05),但不能下调SNRPD1 mRNA表达水平(P>0.05)。与对照组比较,重楼皂苷Ⅵ能下调SNRPD1的下游细胞周期蛋白Cyclin D1和CDK4的表达水平(P<0.05)。结论重楼皂苷Ⅵ可能通过调控SNRPD1发挥抗肝癌作用。

Cloning and characterization of a novel deletion mutant of heterogeneous nuclear ribonucleoprotein M4 from human dendritic cells

To identify differentially expressed genes from antigen-stimulated human dendritic cells (DC), subtractive cloning was adopted and more than ten novel genes differentially expressed were cloned. One is a deletion mutant of heterogeneous nuclear ribonucleoprotein (hnRNP) M4 in which the residues from 159 to 197 of hnRNP M4 have been absent. The deletion mutant was shown to- be co-expressed with hnRNP M4 in cell lines. The mutant was expressed in antigen-stimulated DC but not in normal DC. Northern blot analysis revealed the presence of a major hnRNP M4 deletion mutant mRNA transcript of 2.4 kilobase with the highest levels in peripheral lymphocytes, lung, liver and spleen. It was also expressed in bone marrow-derived stromal cells (BMSC), BMSC treated with several cytokines but not in BMSC treated with TNF-a. The results revealed a new member of hnRNP family and suggested that hnRNP would participate in antigen process and presentation.

Overexpression of c-Myc-dependent heterogeneous nuclear ribonucleoprotein A1 promotes proliferation and inhibits apoptosis in NOTCH1-mutated chronic lymphocytic leukemia cells

Background: NOTCH1 mutation is an essential molecular biologic aberration in chronic lymphocytic leukemia (CLL). CLL patients withNOTCH1 mutation have shown an unfavorable survival and a poor response to chemoimmunotherapy. This study aims to present the mechanisms of adverse prognosis caused byNOTCH1 mutation from the perspective of the splicing factor heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1).Methods: The microarray data in Gene Expression Omnibus datasets were analyzed by bioinformatics and the function of hnRNPA1 was checked by testing the proliferation and apoptosis of CLL-like cell lines. Afterward, quantitative reverse transcription-polymerase chain reaction and Western blotting were applied to explore the relationship among NOTCH1, c-Myc, and hnRNPA1.Results: RNA splicing was found to play a vital part inNOTCH1-mutated CLL cells;hence, hnRNPA1 was selected as the focus of this study. Higher expression of hnRNPA1 validated in primaryNOTCH1-mutated CLL samples could promote proliferation and inhibit apoptosis in CLL. The expression of hnRNPA1 increased when NOTCH1 signaling was activated by transfection with NOTCH1 intracellular domain (NICD)-overexpressed adenovirus vector and declined after NOTCH1 signaling was inhibited by NOTCH1-shRNA. Higher expression of c-Myc was observed in NICD-overexpressed cells and hnRNPA1 expression was downregulated after applying c-Myc inhibitor 10058-F4. Moreover, in NICD-overexpressed cells, hnRNPA1 expression decreased through c-Myc inhibition.Conclusion: Overexpression of c-Myc-dependent hnRNPA1 could promote proliferation and inhibit apoptosis inNOTCH1- mutated CLL cells, which might partly account for the poor prognosis of patients withNOTCH1 mutation.

长链非编码RNA MALAT1对氧糖剥夺/复氧诱导的人脑微血管内皮细胞血管生成的影响

目的:探讨长链非编码RNA (lncRNA)肺腺癌转移相关转录本1 (MALAT1)对氧糖剥夺/复氧(OGD/R)诱导的人脑微血管内皮细胞(HBMECs)血管生成的影响,并阐明其潜在的分子机制。方法:采用生物信息学方法预测MALAT1、不均一核糖核蛋白K (hnRNPK)和血管内皮生长因子A (VEGFA)的结合位点。HBMECs进行OGD/R处理构建脑缺血细胞模型,分为对照组、OGD/R模型组、OGD/R+siNC组、OGD/R+沉默MALAT1 (OGD/R+siMALAT1)组和OGD/R+siMALAT1+过表达VEGFA (OGD/R+siMALAT1+VEGFA)组。采用小干扰RNA (siRNA)沉默MALAT1的表达,采用pcDNA载体构建VEGFA过表达载体,将构建的siMALAT1和pcDNA VEGFA载体分别或同时转染至HBMECs中。利用管形成实验检测各组细胞血管形成能力,Western blotting法检测各组细胞中VEGFA蛋白表达水平。6周龄健康雄性C57BL/6 J小鼠20只,随机分为假手术组、大脑中动脉闭塞(MCAO)模型组、MCAO+NC空载体(MCAO+NC)组和MCAO+过表达MALAT1 (MCAO+MATAL1)组,每组5只,除假手术组外,其余各组小鼠利用线栓法构建MCAO小鼠模型,MCAO+NC组和MCAO+MATAL1组小鼠右脑室内分别注射NC空载体和MALAT1过表达载体。采用2,3,5-氯化三苯基四氮唑(TTC)染色检测各组小鼠脑梗死面积百分率,Western blotting法检测各组小鼠脑组织中VEGFA蛋白表达水平。结果:生物信息学预测,MALAT1与hnRNPK及hnRNPK与VEGFA均存在结合位点。管形成实验和Western blotting法检测,与对照组比较,OGD/R模型组细胞成管数明显增加(P<0.01),细胞中VEGFA蛋白表达水平明显升高(P<0.01);与OGD/R模型组比较,OGD/R+siMALAT1组细胞成管数明显减少(P<0.01),细胞中VEGFA蛋白表达水平明显降低(P<0.01);与OGD/R+siMALAT1组比较,OGD/R+siMALAT1+VEGFA组细胞成管数明显增加(P<0.01),细胞中VEGFA蛋白表达水平明显升高(P<0.01)。在MCAO模型小鼠实验中,TTC染色和Western blotting法检测,与假手术组比较,MCAO模型组小鼠脑梗死面积百分率和脑组织中VEGFA蛋白表达水平明显升高(P<0.01);与MCAO模型组比较,MCAO+MALAT1组小鼠脑梗死面积百分率明显降低(P<0.01),脑组织中VEGFA蛋白表达水平明显升高(P<0.01)。结论:LncRNA MALAT1可增强靶基因VEGFA的稳定性并上调其表达,进而促进缺血性脑卒中小鼠的脑血管生成,为缺血性脑卒中的治疗提供了新的思路。

老年结直肠癌患者组织AIM2、hnRNP AB与血清CEA及病理参数的相关性

目的探讨老年结直肠癌患者组织黑色素瘤缺乏因子2(AIM2)、核不均一核糖核蛋白AB(hnRNP AB)和血清癌胚抗原(CEA)及病理参数的相关性。方法采集2017年6月至2020年6月收治的78例老年结直肠癌患者的癌组织及癌旁正常组织标本。采用免疫组化SP法检测癌组织及癌旁正常组织标本中的AIM2、hnRNP AB阳性表达情况,采用电化学发光法检测患者术前血清CEA水平。收集患者的临床病理参数,分析结直肠癌组织组织中AIM2、hnRNP AB表达及术前血清CEA与临床病理参数的关系。采用Kendall相关分析结直肠癌组织中AIM2、hnRNP AB表达与术前血清CEA的相关性。结果免疫组化显示,AIM2主要分布于肿瘤组织细胞质内,hnRNP AB主要分布于细胞核内;78例肿瘤组织中AIM2阳性29例(37.18%),明显低于癌旁组织的63例(80.77%),hnRNP AB阳性55例(70.51%),明显高于癌旁组织的17例(21.79%),术前CEA水平阳性34例(43.59%),明显高于癌旁组织的5例(6.41%),差异均有统计学意义(P<0.05)。结直肠癌组织中AIM2、hnRNP AB表达及术前血清CEA水平均与性别、年龄、肿瘤直径、肿瘤部位、分化程度、大体分型、远处转移等无关(P>0.05),而与浸润程度、TNM分期、淋巴结转移等有关(P<0.05)。Kendall相关分析显示,结直肠癌组织中AIM2表达与术前血清CEA水平呈负相关(r=-0.211,P<0.01),而结直肠癌组织中hnRNP AB表达与术前血清CEA水平呈正相关(r=0.291,P<0.01)。结论老年结直肠癌患者组织AIM2、hnRNP AB表达与血清CEA水平密切相关,浸润程度越深,TNM分期越晚,发生淋巴结转移的结直肠癌组织中AIM2表达降低、hnRNP AB表达升高,AIM2、hnRNP AB可能成为结直肠癌靶向治疗的新靶点。

小核核糖核蛋白多肽A对肝细胞癌细胞恶性生物学行为的影响及其机制

目的:探究小核核糖核蛋白多肽A(SNRPA)在肝细胞癌(HCC)组织和细胞中的表达及其调控HCC细胞HepG2和Hep3B恶性生物学行为的作用及其机制。方法:数据库分析SNRPA在泛癌组织中的表达及其与病理分期、HCC患者预后的相关性。常规培养HepG2和Hep3B细胞,将si-NC,si-SNRPA#1、si-SNRPA#2转染HepG2和Hep3B细胞,实验分为si-NC组、si-SNRPA#1组和si-SNRPA#2组;将SNRPA-vector和SNRPA-oe载体转染LO2细胞,分为SNRPA-vector组和SNRPA-oe组。qPCR法检测正常肝细胞和肝癌细胞以及转染各组HepG2和Hep3B细胞中SNRPA mRNA的表达,MTT法、Transwell法和WB法分别检测转染后各组HepG2和Hep3B细胞的增殖、迁移和侵袭能力以及EMT相关蛋白表达的变化。结果:数据库分析显示,SNRPA mRNA在多数肿瘤组织中均呈高表达(均P<0.001)且与病理分期有关联(P<0.05或P<0.01)。SNRPA在HCC组织和细胞中均呈高表达(P<0.05或P<0.01),且与HCC患者的预后有关联(P<0.01)。敲减SNRPA表达明显抑制HepG2和Hep3B细胞增殖(P<0.05或P<0.01)而过表达SNRPA则能促进LO2细胞增殖(P<0.01),敲减SNRPA表达明显抑制HepG2和Hep3B细胞的迁移和侵袭能力(均P<0.01),明显促进E-cadherin的表达上调(P<0.01),而抑制N-cadherin、vimentin的表达(P<0.01)。结论:SNRPA在HCC组织及细胞中呈明显高表达,其可能通过调控上皮间质转化(EMT)进程进而促进HepG2和Hep3B细胞的增殖、迁移和侵袭。

SpCas9蛋白的高效可溶表达及应用

规律间隔成簇短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)/CRISPR相关蛋白9(CRISPR-associated protein 9,Cas9)主要元件Cas9蛋白一般采用大肠杆菌表达,但在表达纯化过程中易出现形成包涵体形式的不溶解性蛋白,内毒素含量高、蛋白过大导致蛋白折叠不正确、产量低等问题。为实现化脓性链球菌(Streptococcus pyogenes)Cas9蛋白(SpCas9)在大肠杆菌中的高效可溶表达,进一步促进其应用及编辑技术的推广,本研究应用GB1促溶标签提高Cas9蛋白表达量及溶解度,同时通过使用多重启动子策略进一步提高了Cas9蛋白表达量。两种策略的组合使Cas9蛋白表达量提升了2.52倍。体外酶切分析显示融合GB1标签的Cas9蛋白功能活性不受影响。进一步组装RNP复合物转化黑曲霉宿主成功破坏了pyrG基因。