RUNX3在结直肠癌中的表达与作用及其与TGF-β/SMAD4信号通路的关系

背景与目的 转化生长因子β（TGF-β）/SMAD4信号传导通路在结直肠癌发生与发展中起了重要作用。runt相关转录因子3（RUNX3）在结直肠癌组织中的表达水平显著降低,且可能发挥抑癌作用。但RUNX3的作用与TGF-β/SMAD4通路的关系尚未见报道。因此,本研究旨在探讨RUNX3与SMAD4在结肠癌中的表达及作用,以及两者的关系。方法 收集98例结直肠癌组织和癌旁正常组织标本,分别用Western blot检测RUNX3和SMAD4蛋白的水平,qRT-PCR检测SMAD4 mRNA的水平,并分析两者表达的相关性。将人结直肠癌细胞SW480分别转染RUNX3过表达载体（RUNX3组）、SMAD4过表达载体（SMAD4组）、RUNX3+SMAD4过表达载体（RUNX3+SMAD4组）,以转染阴性对照质粒的SW480细胞为对照组,分别用Western blot与qRT-PCR检测各组细胞RUNX3与SMAD4表达的变化;用CCK-8实验与Transwell实验分析各组细胞的增殖与侵袭能力的差异。结果 结直肠癌组织中的RUNX3蛋白表达量明显低于癌旁正常组织,而SMAD4的mRNA与蛋白表达水平均明显高于癌旁正常组织（均P<0.05）;结直肠癌组织中RUNX3蛋白表达与SMAD4 mRNA和蛋白表达均呈负相关（r=0.511,P=0.004;r=0.487,P=0.009）。与对照组比较,RUNX3组的RUNX3蛋白水平均明显升高,但SMAD4 mRNA和蛋白水平明显降低（均P<0.05）;SMAD4组的RUNX3蛋白水平无明显变化（P>0.05）,但SMAD4 mRNA和蛋白的表达水平明显升高（均P<0.05）;RUNX3+SMAD4组的RUNX3蛋白水平均明显升高（P<0.05）,SMAD4 mRNA和蛋白水平变化不明显（均P>0.05）。RUNX3组细胞增殖与侵袭能力均明显低于对照组（均P<0.05）,SMAD4组的细胞增殖和侵袭能力均明显高于对照组（均P<0.05）,RUNX3+SMAD4组的细胞增殖和侵袭能力介于RUNX3组与SMAD4组之间,与对照组比较,差异无统计学意义（均P>0.05）。结论 RUNX3在结肠癌组织中表达降低,上调RUNX3的表达对结肠癌细胞的恶性生物学行为有抑制作用,其机制可能与抑制TGF-β/SMAD4通路的活性有关。     

lncRNA MIR31HG和miR-101在分化型甲状腺癌组织中的表达及临床意义

背景与目的 研究表明长链非编码RNA（lncRNA）与微小RNA（miRNA）的表达异常以及两者的相互作用在恶性肿瘤的发生与发展中起了重要作用。本研究探讨分化型甲状腺癌患者癌组织lncRNA MIR31HG与miR-101的表达特点,以及两者表达与患者临床病理特征及预后的关系。方法 用qRT-PCR检测92例分化型甲状腺癌患者的癌组织及癌旁组织手术标本中lncRNA MIR31HG和miR-101表达水平,收集患者的临床资料与随访资料,分析两者表达与患者临床病理因素的关系,用Kaplan-Meier法分析患者生存率,采用Cox比例风险回归模型分析患者预后不良的影响因素。结果 分化型甲状腺癌组织中lncRNA MIR31HG相对表达量明显高于癌旁组织,而miR-101相对表达量明显低于癌旁组织（均P<0.01）。lncRNA MIR31HG和miR-101相对表达量均与患者的临床分期、分化程度、淋巴结转移明显有关（均P<0.05）。lncRNA MIR31HG高表达患者的累积生存率低于lncRNA MIR31HG低表达患者（84.7% vs. 94.6%,χ²=7.032,P=0.016）,miR-101低表达患者累积生存率低于miR-101高表达患者（78.3% vs. 95.6%,χ²=8.482,P=0.004）。临床分期Ⅲ~Ⅳ、分化程度高、癌组织lncRNA MIR31HG相对表达量>1.5和miR-101相对表达量<0.5是分化型甲状腺癌预后不良的危险因素（均P<0.05）。结论 分化型甲状腺癌组织中lncRNA MIR31HG表达上调与miR-101表达下调,是分化型甲状腺癌预后不良危险因素,两者可能成为分化型甲状腺癌预后不良标志物。     

核转运蛋白α2通过ERK信号通路调控乳腺癌细胞生物学行为的研究

背景与目的 核转运蛋白α2（KPNA2）异常表达能增强乳腺癌细胞迁移和侵袭能力,导致肺转移风险,并与乳腺癌患者不良预后相关。本研究进一步分析KPNA2在乳腺癌细胞中的表达情况,并探讨其对乳腺癌细胞生物学行为影响的相关机制。方法 用免疫组化检测62例乳腺癌患者癌组织与癌旁组织标本中KPNA2的表达。将两种人乳腺癌细胞株（MDA-MB-453、MCF-7）分为阴性对照组（转染空白质粒）、KPNA2敲低组（转染KPNA2 siRNA）、ERK抑制剂组（ERK抑制剂U0126处理）、联合组（转染KPNA2 siRNA联合U0126处理）。各组细胞处理48 h后,分别用qRT-PCR与Western blot检测KPNA2 mRNA与蛋白表达,并分别用MTT法、流式细胞术、Transwell实验、Western blot检测细胞增殖、凋亡、侵袭能力,以及ERK1/2通路与凋亡相关蛋白表达的变化。结果 免疫组化结果显示,KPNA2蛋白在乳腺癌组织中表达水平高于癌旁组织（2.48±0.39 vs. 1.28±0.22,P<0.05）。qRT-PCR与Western blot结果显示,两种乳腺癌细胞株的阴性对照组和ERK抑制剂组的KPNA2 mRNA及蛋白表达水平均无明显差异（均P>0.05）,而KPNA2敲低组和联合组KPNA2 mRNA和蛋白表达下调（均P<0.05）。功能实验结果显示,与各自的阴性对照组比较,ERK抑制剂组、KPNA2敲低组和联合组的细胞增殖率降低、凋亡率升高、细胞侵袭能力减弱,其中联合组各项变化最为明显（均P<0.05）。Western blot结果显示,与各自的阴性对照组比较,ERK抑制剂组、KPNA2敲低组和联合组磷酸化ERK1/2、裂解的胱天蛋白酶3蛋白表达均下调,且联合组两者的下调程度最为明显（均P<0.05）。结论 乳腺癌组织中KPNA2表达水平升高,其增强乳腺癌细胞迁移和侵袭能力的作用可能与活化ERK信号通路有关,KPNA2有望作为乳腺癌药物开发的新靶点。     

富亮氨酸α2糖蛋白1在乳腺癌中的表达及其功能的生物信息学分析

背景与目的 富亮氨酸α2糖蛋白1（LRG1）是富亮氨酸重复序列（LRR）家族蛋白成员,近些年研究显示LRG1在恶性肿瘤的发生、上皮间质转化、侵袭转移、异常血管生成、预后预测中发挥重要作用。然而LRG1在乳腺癌中的表达、预后、功能和潜在机制方面尚待阐明。本研究旨在通过生物信息学方法系统分析LRG1在乳腺癌中的表达及意义。方法 使用TCGA、Breast Cancer Gene-Expression Miner、UALCAN、Kaplan-Meier Plotter、GeneMANIA、DAVID等多个数据库对LRG1在乳腺癌中的表达与临床病理特征关系、预后价值、相互蛋白作用网络及功能富集进行综合分析。结果 TCGA数据库分析显示,LRG1 mRNA在乳腺浸润癌中的表达量明显高于正常组织（23.461 vs. 8.357,P<0.001）。在不同分子亚型中,luminal型乳腺癌中LRG1 mRNA表达量为37.462（9.930~74.197）,高于HER-2阳性型乳腺癌和三阴性乳腺癌（TNBC）（均P<0.01）;I、II、III期乳腺癌中LRG1 mRNA的表达水平均高于正常乳腺组织（均P<0.05）。Breast Cancer Gene-Expression Miner数据库分析显示,雌激素受体（ER）和（或）孕激素受体（PR）阳性乳腺癌中LRG1 mRNA的表达水平高于ER和（或）PR阴性乳腺癌,HER-2阴性乳腺癌中LRG1 mRNA表达高于HER-2阳性型乳腺癌（均P<0.05）;淋巴结阳性乳腺癌LRG1 mRNA表达量高于淋巴结阴性乳腺癌（P<0.000 1）。用GEPIA在线平台对TCGA数据库中乳腺癌数据进行生存分析发现,LRG1高表达患者总生存率（OS）及无复发生存率（RFS）均低于低表达患者,但差异无统计学意义（HR=0.81,P=0.200;HR=0.70,P=0.064）;用Kaplan-Meier plotter对TCGA中乳腺癌数据进行生存分析发现,LRG1的表达与luminal A型、luminal B型、HER-2阳性型乳腺癌的OS无明显关系（均P>0.05）,但在basal-like亚型中,LRG1低表达患者OS明显优于LRG1高表达患者（HR=3.12,95% CI=1.54~6.29,P<0.001）。使用GeneMANIA数据库进行分析,共筛选出20个与LRG1相互作用蛋白质,GO富集分析显示,LRG1及与其共表达相关的20个蛋白富集于细胞胞外区,外泌体,血液微粒,受体复合物等结构中,参与细胞的血管生成调控、上皮间充质的转化、缺氧反应等相关生物学过程。结论 LRG1在乳腺癌组织中表达上调,并可预测部分不良乳腺癌亚型的预后,LRG1可能为乳腺癌治疗提供新的靶点。     

ε和δ亚型微管蛋白复合物2在肝细胞癌中的表达及其与患者预后的关系

背景与目的 肝癌（肝细胞癌占75%~85%）每年的新发病例数及死亡例数在我国所有癌症类型中高居第五和第二位,肝癌生长、转移、免疫逃逸及治疗抵抗的分子机制复杂,是目前研究的热点及难点。研究发现ε和δ亚型微管蛋白复合物2（TEDC2）在多种恶性肿瘤中表达上调,且与预后相关,但目前肝癌中的临床意义尚未见报道,因此,本研究探讨TEDC2在肝癌中的表达及其与患者预后的关系。方法 从TCGA和ICGC数据库中下载肝癌患者的转录组数据及临床病理资料,通过GEPIA2网站对TEDC2行泛癌分析,用Kaplan-Meier法分析TEDC2表达与患者预后的关系,用单因素和多因素Cox回归模型分析患者的预后因素,用基因富集分析探索TEDC2表达相关的生物学功能。结果 泛癌分析发现TEDC2是肾上腺皮质癌（ACC）、肾透明细胞癌（KIRC）、肾乳头状细胞癌（KIRP）、急性髓细胞样白血病（LAML）、肝癌（LIHC）和肺腺癌（LUAD）这6种癌症的风险预后因子。TCGA和ICGC数据分析均表明肝癌组织中的TEDC2表达水平高于正常组织（均P<0.05）。TEDC2的表达水平与TNM分期、T分期、组织学分级有关（均P<0.05）,与性别、年龄无关（均P>0.05）。生存分析显示,TEDC2高表达的患者总生存期和无病生存期短于TEDC2低表达的患者（均P<0.05）。单因素和多因素Cox回归分析发现,TNM分期、T分期、TEDC2表达是肝癌预后的风险因素,而仅TEDC2表达在TCGA和ICGC患者中均是肝癌的独立预后因子（均P<0.05）。基因富集分析发现在TEDC2高表达样本中富集细胞增殖相关的通路。结论 TEDC2在肝癌组织中高表达,并且是肝癌的独立预后因子,其高表达与患者的不良预后密切相关,而且,机制可能与细胞增殖活性增加有关。     

ATP结合盒转运蛋白A5在胰腺癌中的预后意义的生物信息学分析与验证

背景与目的 ATP结合盒（ABC）转运蛋白家族的成员ABCA5在多种肿瘤中发挥着重要的作用。然而，ABCA5在胰腺癌中的研究尚不清楚。因此，本研究通过生物信息学分析及临床样本验证，探讨ABCA5在胰腺癌中的表达及与患者预后的关系，同时对ABCA5在胰腺癌中的可能作用机制进行分析。方法 使用TCGA和GEO数据库，分析ABCA5在胰腺癌组织及正常组织中的表达情况，并用Kaplan-Meier方法绘制生存曲线，Cox比例风险模型进行单因素与多因素分析。采用免疫组织化学法检测65例胰腺癌组织和癌旁组织中ABCA5的表达，并分析其与预后及胰腺癌临床病理特征的关系。使用TIMER、STRING和Gene MANIA数据库对ABCA5与免疫细胞浸润、蛋白互相作用网络（PPI）和基因-基因互作网络进行分析。利用基因富集分析（GSEA）和相关性分析对ABCA5在胰腺癌中可能参与的信号通路及其可能的作用机制进行探索。通过癌症药物敏感性基因组学（GDSC）分析ABCA5与治疗药物敏感性的关系。结果 在TCGA和GEO数据集中，ABCA5在胰腺癌组织中的表达明显低于正常组织（均P<0.05）。在TCGA和GSE62452数据集中，ABCA5低表达的患者生存时间明显缩短（均P<0.05）；ABCA5的表达是胰腺癌患者预后的独立影响因素（HR=0.458，P=0.001；HR=0.439，P=0.017）。65例临床病例分析显示，ABCA5在癌组织与癌旁组织相比处于低表达水平，且ABCA5低表达患者的预后更差（均P<0.05），单因素与多因素Cox回归分析表明ABCA5的表达是胰腺癌患者预后的独立影响因素（HR=0.327，P=0.032）。TIMER数据库结果显示，ABCA5表达与免疫浸润密切相关。PPI蛋白互作网络显示，有14个与ABCA5相关的互作蛋白；基因-基因互作关系网络图得到20个与ABCA5相关的互作基因。基因富集分析与相关性分析结果显示，ABCA5在胰腺癌中可能与细胞周期和铁死亡有关。ABCA5高表达患者对5种治疗药物的IC₅₀明显低于ABCA5低表达患者（均P<0.05）。结论 ABCA5在胰腺癌组织中低表达并与患者不良预后相关，其表达水平是胰腺癌患者预后的独立影响因素，ABCA5在胰腺癌中的作用机制可能与细胞周期、免疫调节和铁死亡有关。     

结直肠癌细胞中NF-κB活化与TRAF6、CCR5及PTEN/PI3K通路的关系及作用

背景与目的 NF-κB的活化在多种恶性肿瘤的发生、发展中起了重要的作用。研究发现，趋化因子受体5（CCR5）、肿瘤坏死因子受体相关因子6（TRAF6）以及PTEN/PI3K通路相关蛋白均在恶性肿瘤中异常表达。因此，本研究探讨以上分子在结直肠癌细胞中的作用及相互关系。方法 分别用Western blot、CCK-8实验、Transwell法检测结直肠癌HT29和SW480细胞经Maraviroc（CCR5抑制剂）、MG132（TRAF6抑制剂）和NF-BAY（NF-κB抑制剂）处理后，各蛋白表达的变化，以及增殖、迁移、侵袭能力的变化。结果 在两种结直肠癌细胞中，抑制CCR5蛋白后，PI3K的表达降低，PTEN表达升高（均P<0.05），TRAF6和NF-κB表达无明显变化（均P>0.05）；抑制TRAF6蛋白后，PI3K和CCR5表达降低，PTEN表达升高（均P<0.05），NF-κB的表达无明显变化（均P>0.05）；抑制NF-κB表达后，CCR5、TRAF6和PI3K表达降低，PTEN表达升高（均P<0.05）。三种抑制剂均可明显降低两种结直肠癌细胞的增殖、迁移和侵袭能力（均P<0.05）。结论 结直肠癌细胞中存在NF-κB异常活化，后者可能通过上调TRAF6与CCR5的表达，抑制抑癌分子PTEN的活性，从而导致促癌分子PI3K及其通路的活性升高。     

结直肠癌干细胞中miR-520c-3p的表达及其对丙酮酸代谢的调控机制研究

背景与目的 研究显示,丙酮酸代谢的改变在结直肠癌的发生发展中起重要作用,而结直肠癌干细胞中microRNA（miRNA）表达异常可能与丙酮酸代谢密切相关。笔者前期通过TCGA数据库分析发现,miR-520c-3p在结直肠癌中表达升高,且与预后相关。然而,miR-520c-3p是否参与了丙酮酸代谢尚不清楚。因此,本研究探讨结直肠癌干细胞中miR-520c-3p的表达与丙酮酸代谢的关系。方法 选择人结肠癌细胞株,并从中分离纯化结直肠癌干细胞。检测过表达或敲低miR-520c-3p后,结直肠癌干细胞及结直肠癌细胞增殖能力、丙酮酸氧化水平、乳酸产量的变化。用_D-［U-¹³C］葡萄糖孵育细胞,质量同位素分析追踪葡萄糖衍生碳的命运。通过miRNA序列分析和遗传学手段分析和鉴定miR-520c-3p的功能底物。结果 过表达miR-520c-3p后,结直肠癌干细胞的增殖能力明显增强、丙酮酸氧化水平明显下降、乳酸产量明显升高（均P<0.05）;用_D-（U-¹³C）葡萄糖培养后,未标记的柠檬酸盐（m+0）明显增加,而高阶柠檬酸盐标记（m+1、m+4和m+5）明显减少（均P<0.05）。在敲低miR-520c-3p后,结直肠癌干细胞的上述情况呈反向变化（均P<0.05）。过表达或敲低miR-520c-3p对结直肠癌细胞的上述指标均无明显影响（均P>0.05）。miR-520c-3p可以靶向线粒体丙酮酸载体1（MPC1）mRNA 3''UTR（P<0.05）。过表达miR-520c-3p后,结直肠癌干细胞中MPC1的mRNA与蛋白水平均明显下降,敲低miR-520c-3p后则相反（均P<0.05）。TCGA数据库分析结果显示,低表达MPC1的结直肠癌患者预后较差（P<0.05）。敲低MPC1后,结直肠癌干细胞的丙酮酸氧化水平明显降低、乳酸产量明显增高、增殖能力均明显增强（均P<0.05）;用_D-［U-¹³C］葡萄糖培养后,未标记的柠檬酸盐（m+0）在敲低MPC1的结直肠癌干细胞中明显增加,而高阶柠檬酸盐标记（m+1、m+4和m+5）明显减少（均P<0.05）。同时敲低miR-520c-3p和MPC1后,结直肠癌干细胞丙酮酸氧化水平、乳酸产量、增殖能力均无明显变化（均P>0.05）。结论 结直肠癌中miR-520c-3p的高表达与较差的预后相关,其机制可能是miR-520c-3p靶向MPC1调控结直肠癌干细胞中的丙酮酸代谢水平,促进了结直肠癌干细胞的增殖。     

膜联蛋白A5在胰腺癌中的预后意义及机制的生物信息学分析

背景与目的 胰腺癌是一种诊断较晚、预后差的侵袭性疾病，对于胰腺癌的分子机制研究对改善胰腺癌患者的预后具有重要意义。膜联蛋白A5（ANXA5）与人类肿瘤的发生与发展关系密切，但ANXA5与胰腺癌患者预后的关系及具体机制尚不十分清楚。本研究通过生物信息学分析结合实验验证探讨ANXA5的表达与胰腺癌预后的关系及其作用机制。方法 从TCGA和GEO数据库（GSE15471、GSE16515、GSE21501）下载胰腺癌转录组及临床数据，利用R包分析GEO数据库中ANXA5基因在胰腺癌组织与癌旁正常组织的表达情况，并利用GEPIA在线网站分析TCGA数据库中胰腺癌患者组织与GTEx数据库中正常组织ANXA5基因表达情况。采用Kaplan-Meier的方法分析ANXA5的表达水平对胰腺癌患者总生存时间的影响，然后单因素及多因素Cox分析判断胰腺癌相关危险因素，并利用GSEA分析ANXA5在胰腺癌中可能的信号通路并分析其相关性。最后采用免疫组织化学法检测49例胰腺癌组织和癌旁组织中ANXA5的表达，并分析其与预后及胰腺癌临床病理特征的关系。结果 数据库分析显示，在GSE15471与GSE16515数据集，以及TCGA数据库中ANXA5在胰腺癌组织中的均表达明显升高（均P<0.05）；在GSE21501数据集与TCGA数据库中ANXA5高表达的患者总体生存期明显短于低表达患者（均P<0.05）；TCGA中ANXA5表达是胰腺癌患者预后的独立危险因素（HR=1.819，95% CI=1.058~3.126，P=0.03）；ANXA5基因与胰腺癌TGF-β通路及肿瘤上皮-间质转化（EMT）高度相关。胰腺癌组织样本分析结果显示，ANXA5的表达明显高于癌旁组织（P<0.001），且此基因高表达预示着较差的预后（P=0.008 2）；ANXA5表达是胰腺癌患者预后的独立危险因素（HR=3.06，95% CI=1.046~8.952，P=0.041）；ANXA5的表达与肿瘤临床分期（P=0.000 94）及淋巴结转移（P<0.001）明显相关。结论 ANXA5在胰腺癌中表达升高，其高表达是胰腺癌患者预后的危险因素，其机制可能是通过TGF-β通路及EMT进程促进胰腺癌发展有关。     

长链非编码RNA LINC00313与miR-342-3p/膜联蛋白A2轴在肝细胞癌细胞中的作用与机制

背景与目的 肝细胞癌（HCC）是造成癌症相关性死亡的常见原因之一，研究表明长链非编码RNA（lncRNA）调控微小RNA（miRNA）的表达，进而通过抑制靶mRNA翻译或促进mRNA降解来参与肿瘤发生及进展过程。LINC00313作为一种具有致癌活性的lncRNA参与肿瘤发生及进展过程；膜联蛋白A2（ANXA2）在包括HCC的多种恶性肿瘤中表达上调，促进恶性表型的发生，并可能受上游miR-342-3p的调控。因此，本研究探讨LINC00313、miR-342-3p、ANXA2在HCC细胞中的表达及其相互关系。方法 用qRT-PCR与Western blot检测人肝实质细胞及HCC细胞系（Li-7、HuH-7、Hep3B2.1-7）中LINC00313、miR-342-3p及ANXA2表达。将体外培养的Li-7细胞分为空白对照组（无处理）、LINC00313 siRNA组（转染LINC00313 siRNA）、miR-342-3p模拟物组（转染miR-342-3p模拟物）、共转染阴性对照组（转染阴性siRNA序列与阴性miRNA序列）、共转染组（转染LINC00313 siRNA及miR-342-3p抑制物），用qRT-PCR与Western blot检测各组细胞LINC00313、miR-342-3p及ANXA2表达；MTT实验及平板集落形成实验检测各组细胞增殖；进行TUNEL染色检测各组细胞凋亡；Transwell侵袭及Western blot分别检测各组细胞侵袭数目及上皮-间充质转化（EMT）相关蛋白波形蛋白（vimentin）、E-钙黏蛋白（E-cadherin）表达；免疫荧光染色检测各组细胞Bcl-2关联X蛋白（Bax）/B淋巴细胞瘤-2（Bcl-2）；双荧光素酶报告实验分析Li-7细胞中LINC00313对miR-342-3p、miR-342-3p对ANXA2的靶向调控。建立皮下裸鼠异种移植瘤模型，验证LINC00313沉默对Li-7细胞体内生长的影响。结果 与人肝实质细胞比较，Li-7、HuH-7、Hep3B2.1-7细胞的LINC00313、ANXA2 mRNA及蛋白表达均明显升高，而miR-342-3p表达均明显降低（均P<0.05）。与对照组比较，LINC00313 siRNA组、miR-342-3p模拟物组细胞ANXA2 mRNA及蛋白表达、增殖率、集落生成率、侵袭细胞数目、vimentin蛋白表达均明显降低（P<0.05），miR-342-3p表达、凋亡率、E-cadherin蛋白表达、Bax/Bcl-2比值均明显升高（均P<0.05）；与LINC00313 siRNA组比较，共转染组细胞ANXA2 mRNA及蛋白表达、增殖率、集落生成率、侵袭细胞数目、vimentin蛋白表达均明显升高，而miR-342-3p表达、凋亡率、E-cadherin蛋白表达、Bax/Bcl-2比值均明显降低（均P<0.05）。Li-7细胞中，LINC00313可靶向下调miR-342-3p表达，miR-342-3p可靶向下调其ANXA2表达（均P<0.05）。体内实验结果显示，与无处理的Li-7细胞移植瘤比较，LINC00313敲低的Li-7细胞移植瘤的体积与质量均明显降低，肿瘤组织中LINC00313、ANXA2 mRNA及蛋白表达均明显降低，而miR-342-3p表达明显升高（均P<0.05）。结论 LINC00313在HCC细胞中的表达上调，LINC00313可能通过抑制miR-342-3p而增加后者靶基因ANXA2的表达，进而促进HCC细胞的恶性表型。     

miR-486-5p的靶基因预测及其在胰腺腺癌中作用的生物信息学分析

背景与目的:胰腺癌是癌症相关性死亡的主要原因之一,是消化系统恶性程度最高的肿瘤,其主要的病理类型为胰腺腺癌(PAAD),预后较差。miR-486-5p在不同癌症中起重要的作用,但尚缺乏miR-486-5p在PAAD中的研究报告。本研究通过生物信息学方法探寻miR-486-5p的靶基因并分析靶基因在PAAD中的表达及意义。方法:使用PROGmiRV2数据库分析miR-486-5p与PAAD预后的相关性。综合运用多个数据平台来预测miR-486-5p的靶基因,并使用DAVID在线数据库对筛选出的靶基因进行基因本体论(GO)富集分析和京都基因与基因组百科(KEGG)信号通路分析,再以STRING数据库构建靶基因蛋白质-蛋白质相互作用(PPI)网络,并利用Cytoscape软件进行可视化编辑,筛选PPI网络中的核心基因,最后验证筛选出的核心基因,找出与PAAD预后相关的核心基因。结果:miR-486-5p低表达PAAD患者的总生存时间较miR-486-5p高表达患者明显缩短(P0.05)。得到至少被3个数据库预测到的靶基因数目共187个。GO分析显示,靶基因主要参与蛋白质稳定、蛋白质磷酸化、RNA聚合酶II启动子的转录正调节及凋亡过程的负调节等生物学过程;KEGG分析显示靶基因主要参与FOXO信号通路、p53信号通路、Ras信号通路及PI3K-Akt信号通路等。miR-486-5p潜在靶基因的蛋白网络分析发现,SIRT1、PTEN、SMAD2、CSNK2A1、SE RPINE1是PPI网络中关键靶基因;进一步通过GEPIA验证发现CSNK2A1、SERPINE1在PAAD组织中均明显上调(均P0.05),这些基因的高表达与PAAD患者的总体存活和无病生存相关(均P0.05),CSNK2A1和SERPINE1的高表达的PAAD患者有更差的预后。结论:miR-486-5p通过对靶向基因的调控,作用于PAAD患者体内多种信号通路的网络,参与PAAD的发生和发展,影响PAAD患者的预后。     

Expression of VEGF-D,SMAD4, and SMAD7 and Their Relationship with Lymphangiogenesis and Prognosis in Colon Cancer

Aim

The vascular endothelial growth factor (VEGF) and TGF-β1 pathways play important roles in cancer. However, few studies have evaluated the expression and roles of VEGF-D, SMAD4, and SMAD7 in colon cancer, and the conclusions remain controversial. To clarify the roles of VEGF-D, SMAD4, and SMAD7 in colon cancer, we examined their expression and evaluated correlations with lymphangiogenesis, prognosis, and chemotherapeutic outcome.

Methods

The expression of VEGF-D, SMAD4, and SMAD7 was immunohistochemically examined in 251 primary colon cancer samples obtained from the Harbin Medical University.

Results

The expression of VEGF-D, SMAD4, and SMAD7 was identified in 71.7, 41.0, and 69.7 % of samples, respectively. Positive expression of VEGF-D and SMAD7 and lost expression of SMAD4 were significantly correlated with lymph node metastasis and high lymphatic vessel density. VEGF-D and SMAD7 were found to be independent indicators of prognosis and chemotherapy outcome, and positive expression of either VEGF-D or SMAD7 was associated with significantly shorter overall survival and disease-free survival (OS and DFS) than negative expression in all 251 patients (P?<?0.001 for OS and DFS) and patients following chemotherapy (P?<?0.001 for OS and DFS).

Conclusion

VEGF-D, SMAD4, and SMAD7 were involved in lymphangiogenesis and lymph node metastasis. VEGF-D and SMAD7 can serve as predictors of prognosis and chemotherapeutic outcome in colon cancer.

     

Higher Expression of Receptor Tyrosine Kinase Axl,and Differential Expression of its Ligand,Gas6, Predict Poor Survival in Lung Adenocarcinoma Patients

Background

Downstream activation through receptor tyrosine kinases (RTKs) plays important roles in carcinogenesis. In this study, we assessed the clinical involvement of Axl, an RTK, and its ligand, Gas6, in surgically treated lung adenocarcinoma.

Methods

Axl and Gas6 mRNA and protein expression levels were quantified using quantitative real-time polymerase chain reaction and immunohistochemistry, respectively, in completely resected lung adenocarcinoma tissues (n = 88) and were evaluated for correlation with clinicopathologic features and patient survival.

Results

Higher expressions of Axl mRNA/protein and Gas6 protein were significantly related to worse clinicopathological features and prognosis (5-year overall survival rates: Axl mRNA low: 72.3 %, high: 49.7 %, P = 0.047; Axl protein low: 77.5 %, high: 38.6 %, P < 0.001; and Gas6 protein low: 70.5 %, high: 48 %, P = 0.042). On the contrary, higher Gas6 mRNA expression was related to better clinicopathological features and prognosis (5-year overall survival rates: Gas6 mRNA low: 59.2 %, high: 81.8 %, P = 0.054). Multivariate analysis suggests that high Axl mRNA expression may be an independent factor for poor patient prognosis (P = 0.04).

Conclusions

In lung adenocarcinoma, Axl and Gas6 expression levels were associated with tumor advancement and patient survival, thus rendering them as reliable biomarkers and potential targets for treatment of lung adenocarcinoma.

     

Overexpression of Sox3 is Associated with Diminished Prognosis in Esophageal Squamous Cell Carcinoma

Background

Esophageal squamous cell carcinoma (ESCC) is a lethal malignancy lacking valid prognostic biomarkers. As a member of the High Mobility Group domain-containing DNA-binding proteins, Sox3 has been reported to induce oncogenic transformation of chicken embryo fibroblasts. However, the expression and prognostic value of Sox3 in ESCC remain unclear.

Methods

A total of 30 pairs of ESCC with a corresponding non-neoplastic esophageal epithelium (NE) specimen were investigated for Sox3 expression using RT-PCR and western blot analysis. Tissue microarrays containing 118 ESCC and 30 NE samples were detected for Sox3 expression using immunohistochemical staining. The relationship of Sox3 staining with various clinicopathological characteristics and survival of patients was statistically analyzed.

Results

Sox3 expression in ESCC was 3.1- and 2.7-fold higher than in NE at mRNA (P < 0.001) and protein level (P < 0.001), respectively. Positive staining of Sox3 was observed in 77.1 % of the ESCC and 16.7 % of the NE samples (P < 0.001). High expression of Sox3 was significantly correlated with the regional lymph nodes metastasis (RLNM) (P = 0.022) and advanced TNM stage (P = 0.011). Moreover, high expression of Sox3 was significantly associated with poor overall survival (P < 0.001) and recurrence-free survival (P < 0.001) in ESCC patients. Both Sox3 expression (P < 0.001) and RLNM (P = 0.002) were independent prognostic factors for patients with ESCC.

Conclusions

Sox3 might play a positive role in tumor development and could serve as an independent predictor of poor prognosis for ESCC.

     

Somatic mutations in PIK3CA and activation of AKT in intraductal tubulopapillary neoplasms of the pancreas

Intraductal tubulopapillary neoplasm (ITPN) is a recently recognized rare variant of intraductal neoplasms of the pancreas. Molecular aberrations underlying the neoplasm remain unknown. We investigated somatic mutations in PIK3CA, PTEN, AKT1, KRAS, and BRAF. We also investigated aberrant expressions of phosphorylated AKT, phosphatase and tensin homolog (PTEN), tumor protein 53 (TP53), SMAD4, and CTNNB1 in 11 cases of ITPNs and compared these data with those of 50 cases of intraductal papillary mucinous neoplasm (IPMN), another distinct variant of pancreatic intraductal neoplasms. Mutations in PIK3CA were found in 3 of 11 ITPNs but not in IPMNs (P = 0.005; Fisher exact test). In contrast, mutations in KRAS were found in none of the ITPNs but were found in 26 of the 50 IPMNs (P = 0.001; Fisher exact test). PIK3CA mutations were associated with strong expression of phosphorylated AKT (P < 0.001; the Mann-Whitney U test). Moreover, the expression of phosphorylated AKT was apparent in most ITPNs but only in a few IPMNs (P < 0.001; the Mann-Whitney U test). Aberrant expressions of TP53, SMAD4, and CTNNB1 were not statistically different between these neoplasms. Mutations in PIK3CA and the expression of phosphorylated AKT were not associated with age, sex, tissue invasion, and patients' prognosis in ITPNs. These results indicate that activation of the phosphatidylinositol 3-kinase pathway may play a crucial role in ITPNs but not in IPMNs. In contrast, the mutation in KRAS seems to play a major role in IPMNs but not in ITPNs. The activated phosphatidylinositol 3-kinase pathway may be a potential target for molecular diagnosis and therapy of ITPNs.     

Mechanisms of Hypothermic Machine Perfusion to Decrease Donation After Cardiac Death Graft Inflammation: Through the Pathway of Upregulating Expression of KLF2 and Inhibiting TGF‐β Signaling

Hypothermic machine perfusion (HMP) has been known as an efficient way to improve kidney graft function, but the underlying mechanisms remain unclear. Here, we adopt a rabbit reperfusion mode to investigate the upstream mechanisms of end‐ischemic HMP of kidneys from donors after cardiac death (DCD), with static cold storage (CS) as a control. Eighteen New Zealand healthy male rabbits (12 weeks old, with a weight of 3.0 ± 0.2 kg) were randomly divided into three groups: HMP group, CS group, and Normal group (n = 6). The left kidney of rabbits underwent warm ischemia for 25 min through clamping the left renal pedicle and then reperfusion for 1 h. Then the left kidneys were preserved by CS or HMP (4°C for 4 h) ex vivo respectively, after they were autotransplanted and rabbits were submitted to a right nephrectomy. Twenty‐four hours after reperfusion, all left renal specimens were collected. Finally, the expression of Krüppel‐like factor 2 (KLF2), transforming growth factor‐β (TGF‐β) and SMAD4 protein in renal cortical tissue were detected by immunoblotting, and the TGF‐β and SMAD4 expressions were further confirmed by immunohistochemistry analysis. We found that expression of KLF2 in HMP group was significantly higher than CS group (P = 0.011), while expression of TGF‐β and SMAD4 in HMP group were significantly lower than CS group (P = 0.002, P = 0.01, respectively); Compared with normal group, the expression of TGF‐β and SMAD4 in HMP and CS group significantly increased (P<0.05). Compared with CS group, TGF‐β and SMAD4 protein were equally down‐regulated in glomerular and the tubular epithelial cells in HMP group confirmed by immunohistochemistry. In conclusion, HMP may decrease DCD kidneys inflammation through the pathway of upregulating expression of KLF2 and inhibiting TGF‐β signaling after transplantation.     

PIK3R3在胆囊癌组织中的表达及临床意义

目的 检测胆囊癌患者癌组织及癌旁组织中磷脂酰肌醇3激酶调节亚基3（PIK3R3）的表达,分析其临床意义。方法 回顾性分析上海交通大学医学院附属新华医院2014年1月至2016年12月共50例胆囊癌患者的临床资料;采用门诊及电话的方式对患者进行随访,随访时间截止至2018 年12 月。利用免疫组织化学染色法检测50 例胆囊癌患者癌组织及其癌旁组织中PIK3R3蛋白的表达情况,并用统计学方法分析其与患者临床特征及预后的关系。结果 免疫组化分析表明,PIK3R3 蛋白表达主要位于胆囊癌细胞的细胞质中,胆囊癌组织中PIK3R3蛋白表达阳性率较癌旁组织中明显升高（74.0% vs 28.0%,χ2=7.923,P＜0.05）。而且PIK3R3 蛋白表达阳性率在患者的肿瘤TNM分期和分化程度上差异有统计学意义（χ2=7.033,6.821,P＜0.05）,而在性别、年龄、是否患有胆囊结石、淋巴结转移方面,则无明显统计学差异（P＞0.05）。PIK3R3蛋白高表达者相较于低表达者预后更差（中位生存时间10 个月 vs 19 个月,P＜0.05）;经Cox多因素回归分析发现,PIK3R3表达阳性可作为胆囊癌预后危险因素之一（HR 5.766,95%CI 1.843～18.033,P＜0.05）。结论 PIK3R3在胆囊癌组织中表达升高,且主要表达在细胞质中。PIK3R3表达与胆囊癌发展指标如TNM分期、肿瘤分化程度有关。在患者预后方面,PIK3R3 蛋白高表达的患者具有较差的预后。PIK3R3蛋白的表达可以作为胆囊癌患者预后的指标之一。     

Upregulation of Protein Tyrosine Phosphatase Type IVA Member 3 (PTP4A3/PRL-3) is Associated with Tumor Differentiation and a Poor Prognosis in Human Hepatocellular Carcinoma

Background

Protein tyrosine phosphatase type IVA member 3 (PTP4A3/PRL-3), a metastasis-associated phosphatase, plays multiple roles in cancer metastasis. We investigated PTP4A3/PRL-3 expression and its correlation with the clinicopathological features and prognosis in hepatocellular carcinoma (HCC).

Methods

Gene expression profiles of PTP4A3/PRL-3 were obtained in poorly differentiated HCC tissues. The results were validated independently by TaqMan gene expression assays and immunohistochemical analysis.

Results

According to the microarray profiles, PTP4A3/PRL-3 was upregulated in patients with poorly differentiated disease compared to patients with well-differentiated disease with hepatic backgrounds associated with hepatitis B or C. Validation analysis showed that the PTP4A3/PRL-3 mRNA and protein levels were significantly associated with poor differentiation (P < 0.0001), high serum α-fetoprotein (P < 0.01), high serum protein induced by vitamin K absence/antagonist-II (PIVKA-II), and hepatic vascular invasion (P < 0.05). The expression of PTP4A3/PRL-3 protein was also correlated with advanced cancer stages (P < 0.01); this resulted in a significantly poorer prognosis in both overall (P = 0.0024) and recurrence-free survival (P = 0.0227). According Cox regression univariate analysis, the positive expression of PTP4A3/PRL-3 was a poor risk prognostic factor (OS, P = 0.0031; recurrence-free survival, P = 0.0245). Cox regression multivariate analysis indicated that high PTP4A3/PRL-3 expression was an independent, unfavorable prognostic factor for overall survival (hazard ratio 0.542; P = 0.048).

Conclusions

PTP4A3/PRL-3 might be closely associated with HCC progression, invasion, and metastasis. Its high expression had a negative impact on the prognosis of HCC patients. This strongly suggests that PTP4A3/PRL-3 should be considered as a prognostic factor. Further analysis should be pursued to evaluate it as a novel prognostic target.     

PIK3CA Exon 20 Mutation is Independently Associated with a Poor Prognosis in Breast Cancer Patients

Background Prognostic factors that could select breast cancer patients with poor survival, and influence clinical trials of targeted therapy, are needed. However, the reported observations regarding the impact of PI3KCA mutation on breast cancers are controversial. Methods We analyzed exons 4, 7, 9, and 20 of PI3KCA on a series of 158 patients. Clinicopathological characteristics were correlated with the mutation data. Results Among 152 patients who were available for follow-up (median follow-up time, 6.57 years), 26% had PIK3CA mutations, more than half of which occurred in exon 20. The five-year survival rate of patients with exon 20 mutations (46%) was significantly lower than that of patients without (75%) (p = 0.0054). Multivariate analysis showed that PIK3CA exon 20 mutations and nodal involvement were independent risk factors for overall survival. The relative risk of death in patients with PIK3CA exon 20 mutations was 2.881 (95% CI, 1.406–5.900; p = 0.0038). Conclusions PIK3CA mutations are common in invasive ductal carcinomas of the breast. Our result suggests that PIK3CA exon 20 mutation is an independent risk factor for poor prognosis in breast cancer patients, indicating that differences in patient numbers with PIK3CA exon 20 mutations in study and control arms should be avoided in clinical trials of PI3K inhibitors.