Lin28 对肝癌HepG2 细胞5-Fu 敏感性的影响及其分子机制

目的：探讨RNA结合蛋白Lin28 对肝癌HepG2细胞5-氟尿嘧啶（5-Fu）敏感性的影响及其机制。方法：应用pcDNA3.1-Lin28 或si-Lin28 转染HepG2 细胞，采用qPCR及WB法检测转染后HepG2 细胞Lin28 的表达水平，CCK-8 法检测5-Fu 作用后转染细胞增殖活性变化并计算5-Fu 对细胞作用的IC50，流式细胞术检测5-Fu 处理后细胞凋亡情况、WB法检测凋亡相关蛋白的表达变化，qPCR法检测转染后耐药相关miRNA（let-7a 和let-7b）及肿瘤干细胞标记物（Oct4、Nanog和Sox2）的mRNA表达水平。结果：与空载对照组（HepG2/Vector）相比，HepG2/Lin28 组细胞中Lin28 mRNA和蛋白的表达水平均显著升高（P<0.05 或P<0.01），过表达Lin28 可显著抑制HepG2 细胞对5-Fu 作用的敏感性（IC50明显升高，P<0.05）和增强细胞增殖活性、使细胞凋亡率和凋亡相关蛋白caspase-3 表达明显降低（均P<0.01）。与阴性对照组（si-control）相比，si-Lin28 细胞中Lin28 表达水平显著下降（P<0.01）；敲减Lin28 可使细胞增殖活性及5-Fu 的IC50显著降低（均P<0.01），凋亡率及凋亡蛋白表达明显增加（P<0.01）。与HepG2/Vector 组比较，HepG2/Lin28 细胞中let-7a、let-7b 及肿瘤干细胞标记物（Oct4、Nanog 和Sox2）的表达水平明显上调（均P<0.01），而si-Lin28 细胞中let-7a、let-7b 及Oct4、Nanog、Sox2 的表达水平较si-control 组明显下调（均P<0.01）。结论：Lin28 可通过调节miRNAs的表达及肿瘤干细胞的形成从而调节肝癌HepG2细胞对化疗的敏感性，靶向调节Lin28 表达有望成为提高肝癌化疗疗效的手段。     

Oct4基因及其与肿瘤的研究进展

Oct4是POU转录因子家族中一员,其在维持胚胎干细胞的多能性及自我更新状态方面发挥重要作用.此外,Oct4是产生诱导多能干细胞的必要因子.近来,有研究发现,Oct4在多种肿瘤中高表达,例如胚胎生殖细胞肿瘤及其它成体细胞肿瘤中,提示Oct4可能参与恶性肿瘤的发生发展过程.本文对Oct4的结构、功能及其与肿瘤的研究做一综述.     

miR-21与舌鳞癌SCC9细胞干细胞样特性及顺铂敏感性的实验研究

目的 观察舌鳞癌SCC9细胞干细胞样特性、顺铂敏感性以及miR-21表达对其影响。方法 选取人舌鳞癌SCC9细胞分别通过贴壁、悬浮培养法培养获得SCC9a和SCC9f细胞;体外转染法将miR-21 mimics、miR-21 inhibitor分别转染至SCC9f细胞,设为SCC9m和SCC9i细胞。实时荧光定量PCR（QPCR）法检测干细胞相关转录因子（Oct3／4、Sox2、Nanog）及miR-21表达水平,ALDEFLOR kit检测ALDH阳性细胞的比例。MTT法、Annexin V／PI双染法分别检测顺铂对舌鳞癌细胞增殖及凋亡的影响。结果 SCC9f细胞中Oct3／4、Sox2和Nanog表达以及ALDH阳性比例高于SCC9a细胞（P＜0.05）。顺铂对SCC9f细胞的增殖抑制率、凋亡率均低于SCC9a细胞。miR-21表达在SCC9f细胞中高于SCC9a细胞（P＜0.05）,而在SCC9m、SCC9i细胞中表达分别较SCC9f细胞明显升高与降低（均P＜0.05）。Oct3／4、Sox2和Nanog在SCC9m细胞中表达高于SCC9f细胞,其中Oct3／4上调差异具有统计学意义（P＜0.001）;而Oct3／4、Sox2和Nanog在SCC9i细胞中表达均较SCC9f细胞呈显著下调（P＜0.05）。SCC9a、SCC9m和SCC9i细胞中ALDH阳性率分别与SCC9f细胞中ALDH阳性率比较,差异均有统计学意义（P＜0.05）。SCC9m、SCC9i细胞的凋亡率分别较SCC9f减低与增高（均P＜0.05）。结论 舌鳞癌SCC9细胞采用悬浮培养法培养获得的SCC9f细胞具有干细胞特性,对顺铂更为耐受;抑制miR-21表达可增强SCC9细胞对顺铂的敏感性,可能与miR-21调节其肿瘤干细胞相关转录因子表达有关。     

人滑膜肉瘤肿瘤干样细胞分离与鉴定

目的:分离鉴定滑膜肉瘤（SW982）肿瘤干样细胞。方法:体外培养人滑膜肉瘤细胞系SW982,用CD133标记方法检测SW982中肿瘤干细胞的数量,用免疫磁珠分选法进行分选,分选后所得CD133+和CD133-细胞群分别进行以下实验。通过无血清悬浮培养基培养获得CD133+悬浮细胞球,并将其进行重新贴壁、重新成球以检测其自我更新能力。顺铂（cisplatin,CDDP）和阿霉素（doxorubicin,DXR）分别处理CD133+与CD133-贴壁细胞,CD133+贴壁细胞与CD133+悬浮细胞球,MTS检测每两种细胞药物敏感性。Real-time PCR及Western blotting 检测CD133+和CD133-贴壁细胞干细胞相关基因ABCG2、Bmi1、c-Myc、Nanog、Oct3/4、Sox2表达情况。将CD133+和CD133-贴壁细胞分别接种于6~8周雌性BALB/c裸鼠,观察成瘤情况,并将接种后所得肿瘤进行免疫组化,观察接种瘤含CD133情况。结果:SW982细胞系中CD133含量为8.59%,CDDP、DXR对CD133+和CD133-贴壁细胞抑制具有浓度依赖性。CD133+和CD133-细胞均表达干细胞相关基因ABCG2、Bmi1、c-Myc、Nanog、Oct3/4、Sox2。相对于CD133-细胞,CD133+细胞ABCG2、Nanog、Oct3/4、Sox2表达明显升高。CD133+细胞裸鼠体内成瘤率高于CD133-细胞,CD133+接种瘤含CD133较CD133-接种瘤多。结论:SW982细胞系CD133+细胞具有高自我更新能力、高耐药性、高表达干细胞相关基因、高成瘤性,是肿瘤干细胞。     

NK/T细胞淋巴瘤p53和β-catenin基因突变的探讨

目的:探讨p53和β-catenin基因在鼻型NK/T细胞淋巴瘤的突变情况.方法:用PCR-SSCP和基因测序的方法检测20例鼻型NK/T细胞淋巴瘤p53基因外显子4～8,β-catenin外显子3的突变情况.结果:8例p53基因发生突变,突变方式主要为错义突变,G:C→A:T转换多见;6例β-catenin基因发生突变,均为错义突变.结论:p53基因突变可能是鼻型NK/T细胞淋巴瘤发生的生物学机制之一,但突变位点并不集中,无明显的突变热点;错义突变是鼻型NK/T细胞淋巴瘤p53和β-catenin基因突变的主要方式.     

多发性内分泌腺瘤病2A型家系RET基因突变筛查的临床意义

摘 要：［目的］ 探讨多发性内分泌腺瘤病2A的临床诊治特点及RET原癌基因检测的意义。［方法］ 对1个MEN2A家系进行家系调查,并提取外周血进行RET原癌基因测序和降钙素检测,并绘制家系图。［结果］ 10名家族成员均存在RET原癌基因第11外显子第634位点TGC→TAC杂合错义突变,即p.C634Y突变,其中病理确诊MEN2A患者7例,另3名成员为该基因突变携带者。家系分析显示Ⅲ、Ⅳ、Ⅴ代中均有基因突变者,并符合单基因显性遗传病的传递规律。经随访发现,接受手术4例患者术后降钙素及CEA升高,其中3例B超示甲状腺肿块,1例甲状腺残留。3名基因突变携带者中,2名降钙素升高,1名经B超示双侧甲状腺小结节,但降钙素水平正常。［结论］ 对MEN2A家系进行RET基因测序和降钙素检测,有利于早期诊断以改善预后;对无症状的RET基因突变携带者,应根据其降钙素水平,实施个体化的预防性甲状腺全切除除或严密随访观察。     

胃肠道间质瘤中c-kit和PDGFRA基因的突变及其意义

背景与目的:胃肠道间质瘤(GIST)是最常见的胃肠道间叶源性肿瘤,GIST中c-kit和PDGFRA基因突变及突变位点与甲磺酸伊马替尼(格列卫)治疗疗效有关,而突变与生物学行为的关系一直存有争议.本研究探讨GIST中c-kit和PDGFRA基因的突变情况及意义.方法:用PCR扩增和直接测序方法检测50例GIST中c-kit外显子9、11、13、17及PDGFRA基因外显子12、18的突变.结果:50例GIST中仅检测出c-kit外显子11突变,突变率为54%(27/50),均为杂合性突变.其中缺失性突变16例(59%)、点突变10例(37%),伴有点突变的缺失性突变1例.突变位点几乎均集中在5'端"热点"区(96%).CD117阴性的2例GIST中1例检测到c-kit外显子11突变.外显子11突变在不同组织学类型之间比较,差异有显著性(P＜0.05),而在不同原发部位及不同侵袭危险性之间比较,差异均无显著性(P＞0.05).结论:c-kit突变是GIST的普遍现象,突变位点有集中趋势,以梭形细胞型突变最常见.突变检测不能作为生物学行为判断参考指标.     

莱菔硫烷抑制肺癌干细胞增殖的体外实验

目的 研究莱菔硫烷（sulforaphane, SFN）对肺癌干细胞增殖的影响及其机制。方法 采用MTT法分析SFN对H460细胞增殖的影响;应用流式细胞术（FACS）检测SFN对细胞凋亡和侧群细胞比例的影响;肿瘤球培养法分析SFN对肿瘤球生长的影响;使用shRNA慢病毒载体构建β-catenin低表达细胞株,并应用蛋白电泳法检测在β-catenin正常或低表达情况下莱菔硫烷对β-catenin、Oct4、Sox2、c-Myc、Nanog等基因表达的影响。结果 SFN有效抑制H460细胞增殖,IC50为11.2 μmol/L。SFN处理72 h后,细胞凋亡呈剂量依赖性增高。SFN有效抑制原代肿瘤球和2代肿瘤球的生长,在较低浓度（1.0 μmol/L）下即有明显的抑制作用。FACS检测提示侧群细胞比例随SFN浓度增高而减少。SFN浓度依赖性地抑制β-catenin、Oct4、Sox2、c-Myc和Nanog等蛋白表达。在低表达β-catenin情况下,Oct4、Sox2、c-Myc、Nanog等基因表达水平与SFN浓度相关。结论 SFN通过β-catenin和干性相关基因（Sox2、c-Myc、Nanog和Oct4）特异地抑制肺癌干细胞的增殖。     

骨髓增生异常综合征ASXL1基因突变的研究

目的 研究骨髓增生异常综合征(MDS)患者中表观遗传调节因子ASXL1基因的突变情况.方法 在DNA水平采用聚合酶链反应(PCR)扩增产物片段直接测序分析法检测53例初发MDS患者及20名健康人ASXL1基因第12外显子突变情况,比较ASXL1突变患者与野生型患者临床及实验室特征;反转录聚合酶链反应(RT-PCR)扩增产物直接测序分析法检测ASXL1突变在mRNA水平的表达.结果 在53例MDS患者中,9例(16.9%)ASXL1基因突变.共检测出6种突变类型,包括4种移码突变(2例p.Glu635ArgfsX15、3例p.Gly646TrpfsX12、1例p.Ala640GlyfsX14、1例p.Gly790TrpfsX10)和2种无义突变(1例p.Gln1063X和1例p.Gln695X).所有突变类型均为杂合突变,其中p.Gly790TrpfsX10和p.Gln695X为新发现突变类型.此外还检测到一种单核苷酸多态性(SNP)位点(4例p.Gly652Ser).20名健康人中检测出pGly652Ser SNP5名和p.Leu1173Leu SNP1名.RT-PCR扩增产物片段直接测序可在mRNA水平检测出移码突变(p.Gly646TrpfsX12).ASXL1突变患者初发时外周血白细胞计数、红细胞计数、血小板计数、血红蛋白、网织红细胞、中性粒细胞绝对值、外周血淋巴细胞比例、T细胞亚群、骨髓原始细胞比例、MDS分型和染色体核型分布与ASXL1野生型患者相比,差异均无统计学意义(均P>0.05).结论 ASXL1基因在MDS患者中的突变频率较高,且可在mRNA水平检测到ASXL1基因突变.     

Low pH reprograms somatic murine cells into pluripotent stem cells: A novel technique with therapeutic implications

Induced pluripotent stem cells (iPSCs) are somatic cells that are reprogrammed into a state resembling embryonic stem cells (ESCs). iPSCs represent a promising technology with applications in cancer research, yet current methods used to generate iPSCs limit their translation to clinical use. In a recent Nature article, Obokata et al. detail a novel technique to generate pluripotent murine cells called stimulus-triggered acquisition of pluripotency (STAP). STAP eliminates the need for exogenous expression of reprogramming factors used in previous iPSC technologies, instead transforming somatic cells to pluripotency using physical and chemical stimuli. The authors found that STAP cells are generated at a 10-fold higher efficiency than prior iPSC technologies. STAP cells display several features of pluripotency, namely the expression of pluripotency-related genes (Oct4, Nanog, Sox2, Ecat1, Esg1, and Dax1), the ability to form teratomas in vivo, and the ability to produce viable, fertile mice in blastocyst complementation assays. Here, we review these findings on STAP and contrast it to previous iPSC technologies, while noting the potential of this method to generate autologous anti-tumor immune cells for cancer therapy.     

HBx drives alpha fetoprotein expression to promote initiation of liver cancer stem cells through activating PI3K/AKT signal pathway

Hepatitis B virus (HBV)‐X protein (HBx) plays critical role in inducing the malignant transformation of liver cells. Alpha fetoprotein (AFP) expression is closely related to hepatocarcinogenesis. We report that Oct4, Klf4, Sox2 and c‐myc expression positively associated with AFP(+)/HBV(+) hepatocellular carcinoma(HCC) tissues, and the expression of the stemness markers CD44, CD133 and EpCAM was significantly higher in AFP(+)/HBV(+) HCC tissues compared to normal liver tissues or AFP (?)/HBV(?) HCC tissues. AFP expression turned on prior to expression of Oct4, Klf4, Sox2 and c‐myc, and the stemness markers CD44, CD133 and EpCAM in the normal human liver L‐02 cell line or CHL cell lines upon transfection with MCV‐HBx vectors. Stem‐like cells generated more tumour colonies compared to primary cells, and xenografts induced tumourigenesis in nude mice. Expression of reprogramming‐related proteins was significantly enhanced in HLE cells while transfected with pcDNA3.1‐afp vectors. The specific PI3K inhibitor Ly294002 inhibited the effects of pcDNA3.1‐afp vectors. AFP‐siRNA vectors were able to inhibit tumour colony formation and reprogramming‐related gene expression. Altogether, HBx stimulates AFP expression to induce natural reprogramming of liver cells, and AFP plays a critical role in promoting the initiation of HCC progenitor/stem cells. AFP may be a potential novel biotarget for combating HBV‐induced hepatocarcinogenesis.     

Klf4 transcription factor is expressed in the cytoplasm of prostate cancer cells

BackgroundCancer initiation and progression might be driven by small populations of cells endowed with stem cell-like properties. Here we comparatively addressed the expression of genes encoding putative stemness regulators including c-Myc, Klf4, Nanog, Oct4A and Sox2 genes in benign prostatic hyperplasia (BPH) and prostate cancer (PCA).MethodsFifty-eight PCA and thirty-nine BPH tissues samples were used for gene expression analysis, as evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of specific Klf4 isoforms was tested by conventional PCR. Klf4 specific antibodies were used for protein detection in a tissue microarray including 404 prostate samples.ResultsNanog, Oct4A and Sox2 genes were comparably expressed in BPH and PCA samples, whereas c-Myc and Klf4 genes were expressed to significantly higher extents in PCA than in BPH specimens. Immunohistochemical studies revealed that Klf4 protein is detectable in a large majority of epithelial prostatic cells, irrespective of malignant transformation. However, in PCA, a predominantly cytoplasmic location was observed, consistent with the expression of a differentially spliced Klf4α isoform.ConclusionKlf4 is highly expressed at gene and protein level in BPH and PCA tissues but a cytoplasmic location of the specific gene product is predominantly detectable in malignant cells. Klf4 location might be of critical relevance to steer its functions during oncogenesis.     

Novel histone demethylase LSD1 inhibitors selectively target cancer cells with pluripotent stem cell properties

Histone modification determines epigenetic patterns of gene expression with methylation of histone H3 at lysine 4 (H3K4) often associated with active promoters. LSD1/KDM1 is a histone demethylase that suppresses gene expression by converting dimethylated H3K4 to mono- and unmethylated H3K4. LSD1 is essential for metazoan development, but its pathophysiologic functions in cancer remain mainly uncharacterized. In this study, we developed specific bioactive small inhibitors of LSD1 that enhance H3K4 methylation and derepress epigenetically suppressed genes in vivo. Strikingly, these compounds inhibited the proliferation of pluripotent cancer cells including teratocarcinoma, embryonic carcinoma, and seminoma or embryonic stem cells that express the stem cell markers Oct4 and Sox2 while displaying minimum growth-inhibitory effects on non-pluripotent cancer or normal somatic cells. RNA interference-mediated knockdown of LSD1 expression phenocopied these effects, confirming the specificity of small molecules and further establishing the high degree of sensitivity and selectivity of pluripotent cancer cells to LSD1 ablation. In support of these results, we found that LSD1 protein level is highly elevated in pluripotent cancer cells and in human testicular seminoma tissues that express Oct4. Using these novel chemical inhibitors as probes, our findings establish LSD1 and histone H3K4 methylation as essential cancer-selective epigenetic targets in cancer cells that have pluripotent stem cell properties.     

A study of embryonic stem cell‐related proteins in human astrocytomas: Identification of Nanog as a predictor of survival

Recent studies suggest that the regulatory networks controlling the functions of stem cells during development may be abnormally active in human cancers. An embryonic stem cell (ESC) gene signature was found to correlate with a more undifferentiated phenotype of several human cancer types including gliomas, and associated with poor prognosis in breast cancer. In the present study, we used tissue microarrays of 80 low‐grade (WHO Grade II) and 98 high‐grade human gliomas (WHO Grades III and IV) to investigate the presence of the ESC‐related proteins Nanog, Klf4, Oct4, Sox2 and c‐Myc by immunohistochemistry. While similar patterns of co‐expressed proteins between low‐ and high‐grade gliomas were present, we found up‐regulated protein levels of Nanog, Klf4, Oct4 and Sox2 in high‐grade gliomas. Survival analysis by Kaplan‐Meier analysis revealed a significant shorter survival in the subgroups of low‐grade astrocytomas (n = 42) with high levels of Nanog protein (p = 0.0067) and of Klf4 protein (p = 0.0368), in high‐grade astrocytomas (n = 85) with high levels of Nanog (p = 0.0042), Klf4 (p = 0.0447), and c‐Myc (p = 0.0078) and in glioblastomas only (n = 71) with high levels of Nanog (p = 0.0422) and of c‐Myc (p = 0.0256). In the multivariate model, Nanog was identified as an independent prognostic factor in the subgroups of low‐grade astrocytomas (p = 0.0039), high‐grade astrocytomas (p = 0.0124) and glioblastomas only (p = 0.0544), together with established clinical variables in these tumors. These findings provide further evidence for the joint regulatory pathways of ESC‐related proteins in gliomas and identify Nanog as one of the key players in determining clinical outcome of human astrocytomas.