Nanog 基因在卵巢癌和卵巢肿瘤干细胞中的表达及意义

目的:探讨胚胎干细胞关键因子Nanog基因mRNA及其蛋白在卵巢癌和卵巢癌肿瘤干细胞中的表达及意义。方法:选取10例正常卵巢上皮组织、10例卵巢良性肿瘤及60例卵巢癌组织,采用逆转录酶-聚合酶链反应(RT-PCR)方法和免疫组织化学PV-6000两步法检测Nanog mRNA和蛋白表达水平;采用无血清悬浮培养法从SKOV-3卵巢癌细胞株中分离培养肿瘤干细胞,流式细胞术鉴定肿瘤干细胞CD117表达,采用RT-PCR和Western Blot方法检测SKOV-3卵巢癌细胞及肿瘤干细胞中NanogmRNA及其蛋白的表达水平。结果:Nanog mRNA在卵巢癌组织中的表达水平均高于正常卵巢组织和卵巢良性肿瘤组织(P<0.05);Nanog mRNA在不同分化程度及临床分期的卵巢癌组织中表达水平不同,低分化组高于高分化组(P<0.05);III-IV期高于I-II期(P<0.05);免疫组化结果同RT-PCR。从SKOV-3卵巢癌细胞株中成功分离出肿瘤干细胞,SKOV-3卵巢癌细胞和肿瘤干细胞Nanog mRNA相对含量分别为0.6044±0.0368,0.8736±0.0537,差异具有统计学意义(P<0.05),两种细胞Nanog蛋白相对含量分别为0.6364±0.0169 1.2788±0.0314,差别具有统计学意义(P<0.05)。结论:Nanog基因在卵巢癌组织和SKOV-3细胞系中均高表达,其在组织中的表达强度与临床分期及病理分级关系密切,且在肿瘤干细胞中表达高于一般卵巢癌细胞,其与卵巢癌的发生发展关系密切,可能是卵巢癌干细胞的表面标志物,有望成为新的标志物。     

MicroRNA 218 Acts as a Tumor Suppressor by Targeting Multiple Cancer Phenotype-associated Genes in Medulloblastoma

Aberrant expression of microRNAs has been implicated in many cancers. We recently demonstrated differential expression of several microRNAs in medulloblastoma. In this study, the regulation and function of microRNA 218 (miR-218), which is significantly underexpressed in medulloblastoma, was evaluated. Re-expression of miR-218 resulted in a significant decrease in medulloblastoma cell growth, cell colony formation, cell migration, invasion, and tumor sphere size. We used C17.2 neural stem cells as a model to show that increased miR-218 expression results in increased cell differentiation and also decreased malignant transformation when transfected with the oncogene REST. These results suggest that miR-218 acts as a tumor suppressor in medulloblastoma. MicroRNAs function by down-regulating translation of target mRNAs. Targets are determined by imperfect base pairing of the microRNA to the 3′-UTR of the mRNA. To comprehensively identify actual miR-218 targets, medulloblastoma cells overexpressing miR-218 and control cells were subjected to high throughput sequencing of RNA isolated by cross-linking immunoprecipitation, a technique that identifies the mRNAs bound to the RNA-induced silencing complex component protein Argonaute 2. High throughput sequencing of mRNAs identified 618 genes as targets of miR-218 and included both previously validated targets and many targets not predicted computationally. Additional work further confirmed CDK6, RICTOR, and CTSB (cathepsin B) as targets of miR-218 and examined the functional role of one of these targets, CDK6, in medulloblastoma.     

维甲酸对人成骨肉瘤MG-63细胞增殖和相关基因表达的影响

目的:研究维甲酸对人成骨肉瘤MG-63细胞增殖和相关基因表达的影响,以探索其对成骨肉瘤细胞的生物学效应.方法:以1μmol/L维甲酸处理人成骨肉瘤MG-63细胞,生长曲线测定,流式细胞仪分析、光镜观察和免疫细胞化学检测等研究维甲酸对MG-63细胞的生长曲线、细胞周期和相关癌基因、抑癌基因表达的影响,并对其作用机理进行初步分析.结果:维甲酸处理7天后,MG-63细胞生长抑制率达到42.2%,G0/G1期比例达到61.8%,细胞形态铺展,排列趋于规则,癌基因c-myc、c-fos的表达降低.而抑癌基因Rb、p27表达上调.结论:1μmol/L维甲酸可以有效抑制细胞的增殖活动,改变细胞恶性形态特征,下调癌基因c-myc、c-fos和上调抑癌基因Rb、p27的表达,从而对人成骨肉瘤细胞分化具有诱导作用.     

Diindolylmethane-mediated Gli1 Protein Suppression Induces Anoikis in Ovarian Cancer Cells in Vitro and Blocks Tumor Formation Ability in Vivo

Anoikis is a cell death that occurs due to detachment of a cell from the extracellular matrix (ECM). Resistance to anoikis is a primary feature of a cell that undergoes metastasis. In this study for the first time, we demonstrated the potential role of Gli1 in anoikis resistance. Treatment of various ovarian cancer cells by different concentrations of diindolylmethane (DIM), an active ingredient of cruciferous vegetables, reduced the anoikis resistance in a concentration-dependent manner. Reduction in anoikis resistance was associated with a decrease in the expression of Gli1 and an increase in the cleavage of poly(ADP-ribose) polymerase (PARP). Sonic hedgehog (Shh) treatment not only increased the expression of Gli1, but also blocked anoikis induced by DIM and abrogated the change in the expression of Gli1 and cleaved PARP by DIM. To confirm the role of Gli1, hedgehog inhibitor cyclopamine, Gli1 siRNA and Gli1(-/-) mouse embryonic fibroblasts (MEFs) were used. Cyclopamine treatment alone significantly reduced anoikis resistance in A2780 and OVCAR-429 cells. Cyclopamine-mediated reduction in anoikis resistance was associated with reduced expression of Gli1 and induction of cleaved PARP. Shh treatment blocked cyclopamine-induced anoikis. Silencing Gli1 expression induced anoikis and cleavage of PARP in A2780 and OVCAR-429 cells. Furthermore, Gli1(-/-) MEFs were more sensitive to anoikis compared with Gli1(+/+) MEFs. Our in vivo studies established that DIM- or cyclopamine-treated ovarian cancer cells under suspension culture conditions drastically lost their ability of tumor formation in vivo in mice. Taken together, our results establish that Gli1 is a critical player in anoikis resistance in ovarian cancer.     

The Tumor Suppressor pVHL Down-regulates Never-in-Mitosis A-related Kinase 8 via Hypoxia-inducible Factors to Maintain Cilia in Human Renal Cancer Cells

NEK8 (never in mitosis gene A (NIMA)-related kinase 8) is involved in cytoskeleton, cilia, and DNA damage response/repair. Abnormal expression and/or dysfunction of NEK8 are related to cancer development and progression. However, the mechanisms that regulate NEK8 are not well declared. We demonstrated here that pVHL may be involved in regulating NEK8. We found that CAK-I cells with wild-type vhl expressed a lower level of NEK8 than the cells loss of vhl, such as 786-O, 769-P, and A-498 cells. Moreover, pVHL overexpression down-regulated the NEK8 protein in 786-O cells, whereas pVHL knockdown up-regulated NEK8 in CAK-I cells. In addition, we found that the positive hypoxia response elements (HREs) are located in the promoter of the nek8 sequence and hypoxia could induce nek8 expression in different cell types. Consistent with this, down-regulation of hypoxia-inducible factors α (HIF-1α or HIF-2α) by isoform-specific siRNA reduced the ability of hypoxia inducing nek8 expression. In vivo, NEK8 and HIF-1α expression were increased in kidneys of rats subjected to an experimental hypoxia model of ischemia and reperfusion. Furthermore, NEK8 siRNA transfection significantly blocked pVHL-knockdown-induced cilia disassembling, through impairing the pVHL-knockdown-up-regulated NEK8 expression. These results support that nek8 may be a novel hypoxia-inducible gene. In conclusion, our findings show that nek8 may be a new HIF target gene and pVHL can down-regulate NEK8 via HIFs to maintain the primary cilia structure in human renal cancer cells.