Bioinformatic identification of key candidate genes and pathways in axon regeneration after spinal cord injury in zebrafish

Zebrafish and human genomes are highly homologous;however,despite this genomic similarity,adult zebrafish can achieve neuronal proliferation,regeneration and functional restoration within 6–8 weeks after spinal cord injury,whereas humans cannot.To analyze differentially expressed zebrafish genes between axon-regenerated neurons and axon-non-regenerated neurons after spinal cord injury,and to explore the key genes and pathways of axonal regeneration after spinal cord injury,microarray GSE56842 was analyzed using the online tool,GEO2R,in the Gene Expression Omnibus database.Gene ontology and protein-protein interaction networks were used to analyze the identified differentially expressed genes.Finally,we screened for genes and pathways that may play a role in spinal cord injury repair in zebrafish and mammals.A total of 636 differentially expressed genes were obtained,including 255 up-regulated and 381 down-regulated differentially expressed genes in axon-regenerated neurons.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment results were also obtained.A protein-protein interaction network contained 480 node genes and 1976 node connections.We also obtained the 10 hub genes with the highest correlation and the two modules with the highest score.The results showed that spectrin may promote axonal regeneration after spinal cord injury in zebrafish.Transforming growth factor beta signaling may inhibit repair after spinal cord injury in zebrafish.Focal adhesion or tight junctions may play an important role in the migration and proliferation of some cells,such as Schwann cells or neural progenitor cells,after spinal cord injury in zebrafish.Bioinformatic analysis identified key candidate genes and pathways in axonal regeneration after spinal cord injury in zebrafish,providing targets for treatment of spinal cord injury in mammals.

NDRG4通过Wnt/β-catenin信号转导通路抑制卵巢癌细胞的迁移和侵袭能力

目的探讨N-myc downstream regulated gene 4(NDRG4)对卵巢癌SKOV3细胞迁移和侵袭能力的影响及其分子机制。方法应用脂质体转染法将NDRG4过表达载体pcDNA3.1-NDRG4质粒(pcDNA3.1-NDRG4质粒组)和空白pcDNA3.1质粒(空白质粒组)转染至SKOV3细胞中。采用实时荧光定量PCR和Western blot法验证NDRG4过表达效果,应用transwell小室法检测细胞迁移和侵袭能力的变化,Western blot法检测卵巢癌细胞中上皮间质转化相关蛋白(E-cadherin、N-cadherin和Vimentin)以及Wnt/β-catenin信号通路相关蛋白(β-catenin和c-myc)的表达水平。结果转染48 h后,与空白质粒组比较,pcDNA3.1-NDRG4质粒组细胞迁移和侵袭能力均下降(P=0.006,P=0.023)。转染72 h后,与空白质粒组比较,pcDNA3.1-NDRG4质粒组E-cadherin表达水平上升(P=0.023),N-cadherin和Vimentin的表达水平均下降(P=0.013,P=0.021)。转染72 h后,与空白质粒组比较,pcDNA3.1-NDRG4质粒组β-catenin和c-myc的表达水平均下降(P=0.035,P=0.006)。结论NDRG4蛋白抑制卵巢癌细胞迁移和侵袭能力,并抑制上皮间质转化过程。该作用可能是通过抑制Wnt/β-catenin细胞信号通路实现。

Efficacy of Yisui granule(益髓颗粒)on myelodysplastic syndromes in SKM-1 mouse xenograft model through suppressing Wnt/β-catenin signaling pathway

OBJECTIVE:To unmask the underlying mechanisms of Yisui granule(益髓颗粒,YSG)for the treatment of Myelodysplastic syndromes(MDS).METHODS:Our study used an SKM-1 mouse xenograft model of MDS to explore the anti-tumor potential of YSG and its safety,assess its effect on overall survival(OS),and evaluate whether its mechanism is associated with the demethylation of the secreted frizzled related protein 5(s FRP5)gene and suppressing Wnt/β-catenin pathway.Bisulfite amplicon sequencing was applied to detect the level of methylation of the s FRP5 gene;western blotting,immunofluorescence staining,and real-time Polymerase Chain Reaction were performed to detect DNA methyltransferase 1(DNMT1),s FRP5,and other Wnt/β-catenin pathway-related m RNA and protein expression.RESULTS:The results showed that high-dosage YSG exerted an anti-tumor effect similar to that of decitabine,improved OS,and reduced long-term adverse effects in the long term.Mechanically,YSG reduced the expression of DNMT1 methyltransferase,decreased the methylation,and increased the expression of the Wnt/β-catenin pathway antagonist-s FRP5.Furthermore,components of the Wnt/β-catenin pathway,including Wnt3a,β-catenin,c-Myc,and cyclin D1,were down-regulated in response to YSG,suggesting that YSG could treat MDS by demethylating the s FRP5 gene and suppressing the Wnt/β-catenin pathway.CONCLUSIONS:Our findings demonstrated that YSG could be used alone or in combination with decitabine to improve outcomes in the MDS animal model,providing an alternative solution for treating MDS.

重亚硫酸氢盐测序法检测Wnt信号通路抑制基因在急性早幼粒细胞白血病细胞中甲基化变化

目的:应用重亚硫酸氢盐测序法(bisulfite sequencing PCR,BSP)检测急性早幼粒细胞白血病细胞株(NB4)Wnt信号通路抑制基因启动子区CPG岛的甲基化状态,筛选NB4细胞中高甲基化的Wnt信号通路抑制基因,并探讨BSP法作为定量研究基因甲基化方法的优缺点。方法:以NB4细胞为研究对象,20例健康人单个核细胞为对照;常规提取DNA并用亚硫酸氢盐处理,然后用PCR扩增目标序列,应用重亚硫酸氢盐测序法(BSP)分析在NB4细胞中Wnt信号通路抑制基因基因的甲基化状态。并通过与甲基化特异性PCR(methylation specific PCR,M SP)、焦磷酸测序技术(pyrosequencing)比较、评价BSP法检测NB4细胞Wnt信号通路抑制基因甲基化状态的优缺点。结果:BSP产物克隆测序检测NB4细胞Wnt信号通路抑制基因甲基化发生率分别为:WIF-1基因95.26%,DKK3基因86%,SFRP1基因81.67%,SFRP2基因95.71%,SFRP4基因85%,SFRP5基因95%;20例健康人单个核细胞为对照组中Wnt信号通路抑制基因甲基化发生率分别为:WIF-1基因1.5%,DKK3基因4.2%,SFRP1基因0%,SFRP2基因0.9%,SFRP4基因2.5%,SFRP5基因1.75%。与正常人M NC相比,NB4细胞的Wnt信号通路相关抑制基因启动子甲基化率明显增高。结论:急性早幼粒细胞株NB4细胞中存在Wnt信号通路多个抑制基因高甲基化状态。此类基因异常甲基化有可能成为急性早幼粒细胞白血病的早期诊断指标和治疗靶点。

DKK3、Wnt/β-连环蛋白信号通路与宫颈癌关系的研究进展

宫颈癌为女性恶性肿瘤之一,对于其发病机制及所涉及的通路分子的研究越来越深入。其中Wnt/β-连环蛋白(β-catenin)信号通路的研究受到高度关注。Wnt/β-catenin信号通路涉及广泛的生理和病理过程,包括细胞生长、分化、个体发育、迁移、遗传稳定性、凋亡、干细胞自我更新和维持成人组织稳态。稳定的β-catenin在细胞质中积累并转移到细胞核,激活Wnt/β-catenin信号通路,是多种癌症的诱发因素之一。β-catenin的积累在Wnt/β-catenin信号通路占据核心位置。β-catenin通过Wnt/β-catenin信号通路驱动靶基因,参与宫颈癌多步骤的发生及发展。而Dickkopf相关蛋白3(DKK3)可能通过降解β-catenin、阻断β-catenin的核转运,抑制Wnt/β-catenin信号通路,在抑制宫颈癌的发生、发展中发挥潜在作用。因此,深入研究DKK3、Wnt/β-catenin信号通路与宫颈癌的关系,显得尤为重要。

MicroRNA changes of bone marrow-derived mesenchymal stem cells differentiated into neuronal-like cells by Schwann cell-conditioned medium

Bone marrow-derived mesenchymal stem cells differentiate into neurons under the induction of Schwann cells. However, key microRNAs and related pathways for differentiation remain unclear. This study screened and identified differentially expressed microRNAs in bone marrow- derived mesenchymal stem cells induced by Schwann cell-conditioned medium, and explored targets and related pathways involved in their differentiation into neuronal-like cells. Primary bone marrow-derived mesenchymal stem cells were isolated from femoral and tibial bones, while primary Schwann cells were isolated from bilateral saphenous nerves. Bone marrow-derived mesenchymal stem cells were cultured in unconditioned (control group) and Schwann cell-conditioned medium (bone marrow-derived mesenchymal stem cell + Schwann cell group). Neuronal differentiation of bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium was observed by time-lapse imaging. Upon induction, the morphology of bone marrow-derived mesencaymal stem cells changed into a neural shape with neurites. Results of quantitative reverse transcription-polymerase chain reaction revealed that nestin mRNA expression was upregulated from 1 to 3 days and downregulated from 3 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. Compared with the control group, microtubule-associated protein 2 mRNA expression gradually increased from 1 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. After 7 days of induction, microRNA analysis iden:ified 83 significantly differentially expressed microRNAs between the two groups. Gene Ontology analysis indicated enrichment of microRNA target genes for neuronal projection development, regulation of axonogenesis, and positive regulation of cell proliferation. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that Hippo, Wnt, transforming growth factor-beta, and Hedgehog signaling pathv/ays were potentially associated with neural differentiation of bone marrow-derived mesenchymal stem cells. This study, which carried out successful microRNA analysis of neuronal-like cells differentiated from bone marrow-derived mesenchymal stem cells by Schwann cell induction, revealed key microRNAs and pathways involved in neural differentiation of bone marrow-derived mesenchymal stem cells. All protocols were approved by the Animal Ethics Committee of Institute of Radiation Medicine, Chinese Academy of Medical Sciences on March 12, 2017 (approval number: DWLI-20170311).

Epigenetic regulation of Wnt signaling pathway gene SRY-related HMG-box 17 in papillary thyroid carcinoma

Background SRY-related HMG-box 17 （SOX17） encodes a member of the SOX （SRY-related HMG-box） family of transcription factors involved in the regulation of embryonic development and in the determination of the cell fate. Recently, it was considered as a tumor suppressor gene to inhibit canonical Wnt/β-catenin signaling pathway in several malignancies. However, the function of SOX17 in thyroid cancer was unknown. Therefore, we investigated the epigenetic changes and the function of SOX17 in thyroid cancer. Methods The methylation status of the promoter region of SOX17 was detected using methylation-specific PCR in 63 papillary thyroid carcinoma （PTC） tissue, 10 normal thyroid tissue, and two thyroid cancer cell lines. Semi-quantitative RT-PCR was used to assess mRNA expression of SOX17 before and after 5-aza-2＇-deoxycytidine treatment in thyroid cancer cell lines. Expression of SOX17 and β-catenin were detected by immunohistochemistry in PTC and adjacent tissue. Luciferase reporter assay, colony formation, transfection, and Western blotting were employed to analyze the effect of SOX17 on thyroid cancer cell proliferation and the function of SOX17 in the Wnt signal pathway. Results Loss of SOX17 expression was correlated to the promoter region hypermethylation in thyroid cancer cell lines. Re-expression of SOX17 was found in TPC-1 cell line after 5-aza-2＇-deoxycytidine treatment. In primary thyroid cancer, 60.3% （38/63） were methylated and 39.7% （25/63） unmethylated. But no methylation was found in noncancerous thyroid tissues. Methylation of SOX17 was associated reversely with β-catenin expression in the cytoplasm or nucleus significantly in the PTC （P 〈0.05）. Colony formation was inhibited by re-expression of SOX17 in TPC-1 cells. SOX17 suppressed the Wnt signaling pathway and the HMG domain was essential for this effect. Conclusions SOX17 was frequently methylated in human PTC. Loss of SOX17 expression was induced by promoter region hypermethylation. SOX17 inhibited thyroid cancer proliferation. Methylation of SOX17 activated the Wnt signaling pathway in human thyroid cancer.

LATS2基因通过Wnt信号通路对H9C2心肌细胞保护的作用机制研究

目的探讨大肿瘤抑制因子2(LATS2)基因对缺氧复氧(H/R)诱导的H9C2心肌细胞凋亡及Wnt信号通路的影响。方法 LATS2 siRNA或LATS2过表达质粒转染H9C2心肌细胞,并分别转染阴性对照siRNA及空载体(vector),转染48 h后,通过Western blotting检测转染后的各组细胞中LATS2的蛋白表达;建立H/R模型,细胞分为正常对照组(Control组)、单纯缺氧复氧组(H/R组)、H/R+LATS2-siRNA组和H/R+pcDNA3.1-LATS2组,流式细胞术检测各组细胞凋亡率及活性氧(ROS)含量;Western blotting检测凋亡蛋白含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved caspase-3)及Wnt信号通路β-连环蛋白(β-catenin)和c-myc的蛋白表达。结果 siRNA组和PcDNA3.1组的LATS2蛋白表达与Control组差异无统计学意义(P>0.05),而LATS2-siRNA组LATS2的蛋白表达显著低于Control组(P<0.05),pcDNA3.1-LATS2组LATS2的蛋白表达显著高于Control组(P<0.05);与Control组比较,H/R组细胞凋亡率、ROS含量及cleaved caspase3、β-catenin和c-myc的蛋白表达均显著升高(P<0.05),与H/R组比较,H/R+LATS2-siRNA组细胞凋亡率、ROS含量及cleaved caspase3、β-catenin和c-myc的蛋白表达均显著升高,H/R+pcDNA3.1-LATS2组细胞凋亡率、ROS含量及cleaved caspase3、β-catenin和c-myc的蛋白表达均显著降低(P<0.05)。结论抑制LATS2基因表达可诱导心肌细胞凋亡及上调Wnt信号通路,而抑制LATS2基因表达反之。

Wnt抑制分子Dkk3、SFRP1和SFRP4在子宫内膜癌中的表达分析

目的研究Dickkopf相关蛋白3(Dickkopf-related protein 3,Dkk3)、分泌型卷曲相关蛋白1(secreted frizzled-related protein 1,SFRP1)和SFRP4在子宫内膜癌(endometrial carcinoma,EC)发生、发展中的作用及病理相关性。方法收集EC标本58例,子宫内膜正常组织30例。用RT-PCR、Western blot法检测组织中Dkk3、SFRP1和SFRP4的表达变化。结果Ⅱ、Ⅲ、Ⅳ期子宫内膜癌组织和正常组织比较,Dkk3、SFRP1和SFPRP4的mRNA及蛋白表达水平降低,并随着组织分期程度的升高而降低(均P<0.05)。结论 Dkk3、SFRP1和SFRP4与EC的分期呈负相关,可考虑作为EC的分期标志。

人小细胞肺癌NCI-H446细胞成瘤组织中HSG及WNT/Ca^(2+)信号通路相关基因表达

目的探究细胞增殖抑制基因(HSG)、WNT5A、WNT11对人小细胞肺癌NCI-H446细胞成瘤的影响并探讨其作用机制。方法采用裸鼠成瘤技术在裸鼠皮下培养NCI-H446细胞肿瘤组织,采用实时荧光定量逆转录聚合酶链式反应检测成瘤组织与癌旁组织中HSG、WNT5A、WNT11基因的相对表达量。结果 20只接种NCIH446细胞悬液的裸鼠中,1只裸鼠于操作过程中死亡,15只裸鼠成功成瘤,成瘤率为78.9%。肿瘤组织中WNT5A、WNT11基因的相对表达量明显高于癌旁组织(P﹤0.01);肿瘤组织中HSG基因的相对表达量明显低于癌旁组织(P﹤0.01)。结论 HSG基因可能具有抑制小细胞肺癌成瘤的作用,WNT/Ca2+信号通路在NCI-H446细胞成瘤组织中大量开放并可能参与其成瘤过程。

Linc00891通过竞争性结合并抑制miR-27a-3p在骨肉瘤中发挥抑癌作用

目的探讨Linc00891在骨肉瘤(OS)中发挥的作用并初步分析其机制。方法实时荧光定量逆转录聚合酶链反应(qPCR)检测Linc00891在不同OS细胞系U-2 OS、Saos-2、MG-63、SW 1353及人成骨细胞系hFOB 1.19中的表达,选择Saos-2和MG-63 OS细胞系转染过表达Linc00891载体。CCK-8法、平板克隆法和Transwell小室检测过表达Linc00891对人OS细胞系Saos-2和MG-63增殖、克隆形成能力和迁移能力的影响。Western blot法检测上皮间质转化相关蛋白钙黏着蛋白E(E-cad)、钙黏着蛋白N(N-cad)的表达。RNA pulldown实验检测Linc00891和miR-27a-3p在OS细胞中的结合关系。TCF/LEF报告试剂盒检测过表达Linc00891和上调miR-27a-3p对不同OS细胞系中Wnt/β-catenin信号通路的影响。结果不同OS细胞系U-2 OS、Saos-2、MG-63、SW 1353中Linc00891的表达水平显著低于正常成骨细胞系hFOB 1.19(P<0.05)。过表达Linc00891可抑制人OS细胞系Saos-2和MG-63的增殖、克隆形成能力、迁移能力及上皮间质转化(P<0.05)。Linc00891和miR-27a-3p在OS细胞中存在结合关系。过表达Linc00891能够逆转miR-27a-3p对Wnt/β-catenin信号通路的激活(P<0.05)。结论Linc00891通过竞争性结合并抑制miR-27a-3p,在OS中发挥抑癌作用。

miR-20a-5p靶向调控BRMS1L对人结直肠癌细胞株SW480增殖、凋亡能力的影响及其机制探讨

目的观察微小RNA-20a-5p(miR-20a-5p)靶向调控乳腺癌转移抑制基因1(BRMS1L)对人结直肠癌细胞株SW480增殖、凋亡能力的影响,并探讨其可能机制。方法取SW480细胞,分别将miR-20a-5p抑制物(anti-miR-20a-5p)、miR-20a-5p抑制物阴性对照(anti-miR-NC)、BRMS1L过表达载体(pcDNA-BRMS1L)、BRMS1L过表达载体阴性对照(pcDNA-NC)、miR-20a-5p模拟物(miR-20a-5p mimics)、miR-20a-5p模拟物阴性对照(miR-NC)转染至细胞中,CCK-8法测算细胞存活率,流式细胞术测算细胞凋亡率,Western blotting法检测细胞周期素D1(CyclinD1)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved-Caspase-3)、β-catenin蛋白。取SW480细胞,分别将miR-20a-5p mimics、miR-NC与WT-BRMS1L或MUT-BRMS1L共转染至细胞中,采用双荧光素酶报告基因检测系统检测细胞荧光素酶活性;另取SW480细胞,分别将miR-20a-5p mimics、miR-NC、anti-miR-20a-5p、anti-miR-NC分别转染至细胞中,采用Western blotting法检测细胞BRMS1L蛋白。结果抑制miR-20a-5p表达或过表达pcDNA-BRMS1L,SW480细胞存活率降低,细胞凋亡率升高,CyclinD1蛋白表达降低,Cleaved-Caspase-3蛋白表达升高(P均<0.05);抑制BRMS1L表达部分逆转anti-miR-20a-5p对SW480细胞增殖和凋亡的影响(P均<0.05)。抑制miR-20a-5p表达后SW480细胞中β-catenin蛋白的表达较低,抑制BRMS1L表达部分逆转anti-miR-20a-5p对β-catenin蛋白表达的影响(P均<0.05)。miR-20a-5p靶向BRMS1L,并负性调控其表达(P均<0.05)。结论miR-20a-5p靶向调控BRMS1L抑制SW480细胞增殖,促进细胞凋亡,其机制可能与抑制β-catenin蛋白表达有关。

Research Progress on the Mechanism of Correlation Between Vitamin D and Thyroid Cancer

Vitamin D is a kind of fat-soluble vitamin,which is mainly involved in the metabolism of calcium and bone in the human body.As a metabolic substance,it also has a certain impact on the cellular microenvironment,and vitamin D also inhibits the proliferation of tumor cells.25(OH)D is considered the best index to evaluate the vitamin D level in the human body because of its relatively stable characteristics in the circulation.Thyroid cancer is a common malignant tumor that develops from malignant thyroid nodules.A large number of studies have found that the lower the serum 25(OH)D level,the higher the risk of thyroid nodules.A large number of studies have found that the lower the serum 25(OH)D level,the higher the risk of thyroid nodules.

肿瘤中Dapper蛋白家族表观遗传调控的研究进展

Dapper是一类Wnt信号通路调控蛋白,与肿瘤发生发展密切相关,通常在正常组织中表达,在肿瘤组织中表达降低或缺失。Dapper蛋白表达降低或表达缺失与基因表观遗传调控相关;Dapper家族基因的表观遗传学改变与肿瘤的病理学特征、疾病进展及预后相关。本综述总结了新近的研究发现,聚焦Dapper家族在肿瘤发生发展中的生物学功能,以及Dapper家族表达的表观遗传学调控的新机制。

DKK3与宫颈癌的相关性研究进展

宫颈癌是常见的妇科恶性肿瘤,其发病率和死亡率均较高。DKK3(dickkopf-3)是经典Wnt信号传导通路的拮抗因子,也是近年来发现的抑癌基因。DKK3通过调节β-catenin信号传导通路、逆转上皮-间质转化、抑制肿瘤血管生成等多种途径发挥抗肿瘤功能,是肿瘤治疗中的潜在靶点。基因的表观遗传修饰特别是启动子甲基化在多种恶性肿瘤的发生发展中发挥着非常重要的作用。DKK3基因启动子区的高甲基化是DKK3蛋白失活的重要原因,这使Wnt信号通路异常激活,从而导致肿瘤的发生。宫颈癌组织中DKK3表达常下调,且DKK3基因启动子甲基化导致的DKK3表达沉默是宫颈癌中的频发事件。DKK3的甲基化水平及表达水平与宫颈癌患者预后显著相关,有较好的预测作用。因此,深入研究DKK3与宫颈癌间的关系可能为宫颈癌的机制研究、靶向治疗及判断预后提供新的思路。