【作者】 赵世斗；

【导师】 郝爱军； 刘凯；

【作者基本信息】 山东大学 ， 人体解剖与组织胚胎学， 2011， 博士

【摘要】 八聚体结合转录因子4(octamer-binding transcription factor 4,OCT4),是POU转录因子家族的一员,对于维持胚胎干细胞(embryonic stem cells, ES细胞)的多能性和自我更新具有十分重要的作用,并且OCT4是目前产生诱导多能干细胞(induced pluripotent stem cells)的一个必需因子。近年来,OCT4在人类肿瘤中的表达和意义也日益成为研究的热点。现已证实,OCT4基因在多能性生殖细胞肿瘤中有表达,并且其表达与多能性细胞的致瘤性有关。但是,体细胞肿瘤中是否表达OCT4仍有较大争议。许多研究报道,OCT4表达于成体干细胞和多种体细胞肿瘤中,而且肿瘤中的OCT4阳性细胞很可能就是肿瘤干细胞(cancer stem cells, CSCs),甚至有人报道,OCT4的表达对于维持干细胞样肿瘤细胞的存活和自我更新都是必需的。尽管如此,也有大量研究表明,OCT4在多种体细胞肿瘤和肿瘤细胞系中不表达。因此,为了更好的阐明肿瘤发生的分子机制,首先应该确定OCT4在人类体细胞肿瘤中是否有表达。一般来说,肿瘤细胞具有无限增殖、相对未分化和侵袭性等特征,这些都与早期胚胎细胞非常相似。但是,大多数肿瘤中的细胞群体都是异质性的,既有非常幼稚的细胞,也有几乎完全分化的细胞。随着干细胞理论在肿瘤研究中的应用,肿瘤中的未分化细胞即被称为CSCs。CSCs具有自我更新能力并且能够分化产生非致瘤性肿瘤细胞,正是肿瘤中存在的一小部分CSCs促进了肿瘤的形成和生长。到目前为止,许多研究者已经从多种肿瘤组织中成功分离培养出CSCs,包括胶质瘤和乳腺癌。最近有学者提出,CSCs可能来源于自我更新失调的正常干细胞或者重新获得自我更新能力的祖细胞或已分化细胞。不管怎样,自我更新机制的异常激活是肿瘤发生中的一个重要事件。因此,研究肿瘤中包括OCT4在内的参与干细胞自我更新机制的基因的表达和意义就变得十分迫切和重要。人类OCT4基因位于6号染色体,包含5个外显子和4个内含子。OCT4基因通过转录后的选择性剪接形成两种变异体OCT4A和OCT4B。它们翻译形成的蛋白质具有相同的POU和C端结构域,而N端结构域则不同。OCT4A定位于细胞核内,具有维持ES细胞自我更新的功能；而OCT4B主要位于细胞浆中,并且不具有维持ES细胞自我更新的功能。通常所说的OCT4指的是OCT4A。另有研究发现,人类基因组中存在6个OCT4假基因(pseudogenes),它们与OCT4基因有很高的序列同源性。有人报道,OCT4B表达于多种体细胞肿瘤细胞系中,而两个OCT4假基因——OCT4-pgl和OCT4-pg5在多种体细胞肿瘤组织和细胞系中都有转录。OCT4基因剪接异构体和假基因的存在和表达都有可能导致OCT4检测的假阳性实验结果。因此,检测OCT4基因的表达时,需要将真正的OCT4与其剪接变异体和各种假基因区分开来。虽然已有报道表明,可能是OCT4剪接变异体和假基因的存在导致了目前肿瘤和干细胞研究中OCT4是否表达的相悖结果,但是这一问题还没有引起人们足够的重视。在本研究中,我们利用RT-PCR和DNA测序分析的方法检测OCT4在两种体细胞肿瘤——胶质瘤和乳腺癌中的表达状况。我们的实验结果显示,这两种肿瘤中没有检测到真正的OCT4基因的表达,检测出的是三种OCT4假基因——OCT4-pgl、OCT4-pg3和OCT4-pg4。我们进一步研究了它们的蛋白表达情况,并对这些蛋白产物的功能和活性进行了探讨。第一部分胶质瘤和乳腺癌中OCT4假基因的克隆与测序分析为了检测OCT4在人胶质瘤和乳腺癌组织中的表达情况,我们共收集了42例胶质瘤和45例乳腺癌的临床标本。所有的肿瘤标本均经过最后的病理诊断证实,胶质瘤按照世界卫生组织(WHO)制定的标准进行分级,而乳腺癌依据雌激素受体(ER)、孕激素受体(PR)和人类表皮生长因子受体2(HER2)的表达情况进行分型。作为正常对照的4例成人脑组织和5例乳腺组织均取自于外伤病人。在RT-PCR过程中,我们利用两对引物来检测OCT4 mRNA的表达。OCT4A特异性引物能够特异性扩增OCT4A,而OCT4全长引物能够扩增OCT4A的全长编码序列。当用OCT4A特异性引物进行扩增时,除阳性对照精原细胞瘤外,各级别各类胶质瘤、各类型乳腺癌、正常脑组织和乳腺组织中都没有检测到OCT4 mRNA (496bp)的表达；而当用OCT4全长引物进行扩增时,各级别各类胶质瘤和各类型乳腺癌中都能检测到阳性条带(1086bp)。此外,我们还检测了OCT4 mRNA在多种人胶质瘤和乳腺癌细胞系中的表达情况。当用OCT4A特异性引物进行扩增时,除阳性对照人多能性胚胎癌细胞系NTera-2外,人胶质瘤细胞系U251和U87,乳腺癌细胞系MCF7、MDA-MB-231、BT474和SK-BR-3中都没有检测到OCT4 mRNA (496bp)的表达；而当用OCT4全长引物进行扩增时,这几种肿瘤细胞系中都能检测到阳性条带(1086bp)。下一步,我们利用DNA克隆和测序分析来确定胶质瘤和乳腺癌中是否有真正OCT4基因的表达。首先,我们从琼脂糖凝胶中回收由OCT4全长引物扩增得到的DNA片段(1086bp),克隆入pGEM-T easy载体中形成环状质粒,质粒扩增后进行酶切鉴定,插入片段大小正确的克隆再进行测序分析。测序结果显示,OCT4基因的转录本只在阳性对照精原细胞瘤和NTera-2细胞中被检测到,而在本研究收集的各种肿瘤组织和肿瘤细胞系中未检测到真正OCT4基因的表达,检测到的是三种OCT4假基因,即OCT4-pgl、OCT4-pg3和OCT4-pg4,它们分别位于人类染色体8q24、12p13和1q22。我们的测序结果表明,当用OCT4全长引物进行扩增时,正是肿瘤组织和细胞系中存在的这些OCT4假基因的转录本导致了RT-PCR的阳性结果。第二部分OCT4假基因蛋白的表达与定位研究从理论上来说,OCT4-pg1、OCT4-pg3和OCT4-pg4的转录本能够分别翻译生成由359、186和286个氨基酸组成的蛋白产物。尽管存在着氨基酸缺失或替代,这三种OCT4假基因的蛋白产物都有与OCT4蛋白相似的N端结构域。为了研究OCT4假基因的蛋白表达和细胞内定位,我们构建了它们的真核表达质粒,并使它们的C端带有HA标签,以便于检测。将它们分别转染小鼠胚胎成纤维细胞NIH 3T3后,进行免疫细胞化学染色。结果显示,与OCT4相似,OCT4-pg1和OCT4-pg4主要位于细胞核内,而OCT4-pg3主要位于细胞浆中。OCT4-pg1和OCT4-pg3还能被两种OCT4抗体——ab18976和sc-5279所识别,而OCT4-pg4不能被这两种抗体所识别。转染胶质瘤细胞系U251和乳腺癌细胞系MCF7得到了与NIH 3T3相似的结果,不同的是OCT4-pg3在这两种肿瘤细胞的细胞核和细胞浆中都有分布。Western blot结果显示,OCT4-pg1、OCT4-pg3、OCT4-pg4和OCT4蛋白产物所对应的条带分别位于50、30、30和50 KDa。与免疫细胞化学结果一致,细胞核浆分离后进行的Western blot检测结果显示,OCT4-pg1、OCT4-pg4和OCT4位于NIH 3T3的细胞核中,而OCT4-pg3位于细胞浆中。另外,Western blot结果显示,OCT4-pg1和OCT4-pg3还能被OCT4抗体sc-5279所识别。两种OCT4抗体都不能识别OCT4-pg4,可能与OCT4-pg4蛋白的N端存在较多的氨基酸缺失或替代有关。下一步,我们利用免疫化学染色检测了OCT4假基因在肿瘤细胞中的内源性表达。当用OCT4抗体ab18976进行免疫染色时,与NTera-2细胞中明显的核着色不同,荧光信号在几乎所有U251和MCF7细胞的细胞核和细胞浆中都能检测到,说明这些肿瘤细胞中表达OCT4假基因。同样,与精原细胞瘤中广泛的核着色相比,人胶质瘤和乳腺癌组织中只有一小部分细胞能着色,并且一些细胞的细胞核和细胞浆中都有阳性着色,说明这些肿瘤组织中有OCT4假基因的表达。在胶质瘤中,能被ab18976阳性染色的细胞也能同时表达肿瘤干细胞标志物CD133或者星形胶质细胞标志物GFAP,说明胶质瘤中OCT4假基因的表达并不局限于CSCs中。本实验结果表明,当检测肿瘤组织中OCT4的表达时,OCT4假基因蛋白的表达在免疫化学染色和Western blot中都可能产生假阳性实验结果。这可能也是导致目前关于肿瘤和干细胞中OCT4是否表达的相悖研究结果的原因。第三部分OCT4假基因蛋白的活性与功能研究为了研究OCT4假基因的生物学功能,我们用OCT4及其假基因的表达质粒转染胶质瘤细胞系U251、U87和乳腺癌细胞系MCF7、MDA-MB-231。转染后各个时间点的细胞活力用MTT实验来检测。结果显示,OCT4转染组肿瘤细胞的细胞活力明显高于对照组,而各OCT4假基因转染组的细胞活力与对照组相比没有明显差别。细胞克隆形成实验结果显示,OCT4转染组肿瘤细胞的克隆形成率明显高于对照组,而各OCT4假基因转染组细胞的克隆形成率与对照组相比没有明显差别。OCT4假基因不能增加肿瘤细胞的细胞活力,也不能促进肿瘤细胞的克降形成率,说明OCT4假基因对肿瘤细胞的增殖无明显作用。最后,我们利用荧光素酶报告基因检测的方法检测了这三种OCT4假基因的转录激活功能。OCT4能够明显激活三种OCT4依赖的荧光素酶报告基因载体(6×W、PORE和MORE),而三种OCT4假基因与空载体PCDNA3相比无明显差别,说明OCT4-pg1、OCT4-pg3和OCT4-pg4对这三种OCT4依赖的荧光素酶报告基因载体无明显的转录激活功能。这三种OCT4假基因与真正OCT4基因的高度同源性提示它们可能具有相似的生物学功能,但本研究结果显示,OCT4-pg1、OCT4-pg3和OCT4-pg4不具有类似OCT4的功能。OCT4假基因在肿瘤中表达的意义以及它们在肿瘤发生中的作用有待于进一步研究。总之,通过本研究我们发现,人类胶质瘤和乳腺癌中表达三种OCT4假基因——OCT4-pg1、OCT4-pg3和OCT4-pg4,而没有真正OCT4基因的表达,说明OCT4的表达在人类体细胞肿瘤发生过程中可能不是必需的。我们进一步通过免疫化学染色和Western blot检测了这三种OCT4假基因的蛋白表达情况,这些假基因的表达可能导致了目前人类体细胞肿瘤中OCT4表达的假阳性研究结果。本研究还发现OCT4假基因不具有类似OCT4的功能。我们的研究结果表明,当研究OCT4在肿瘤和干细胞中的表达时,必需联合采用多种实验方法并设立合适的对照组以排除假阳性结果,从而保证实验结果的可靠性。更多还原

【Abstract】 Octamer-binding transcription factor 4 (OCT4) is a POU family transcription factor, notable for its maintaining the pluripotency and self-renewal of embryonic stem (ES) cells and for generating induced pluripotent stem (iPS) cells, as a uniquely essential reprogramming factor to date. Recently, it becomes a hot topic to study the expression and function of OCT4 in human tumours. Although OCT4 is putatively expressed in and associated with germ cell tumours with pluripotent potential, its expression in human somatic tumours remains controversial. Several studies showed that OCT4 is expressed in adult stem cells and somatic cancers, and OCT4-positive cells identified in cancers are likely to represent cancer stem cells (CSCs) and, further, OCT4 expression is required for maintaining the survival and self-renewal property of cancer stem-like cells. However, there is ample evidence suggesting that OCT4 is not expressed in somatic tumours and tumour cell lines. In view of this and to fully clarify the molecular mechanism of tumour biology, it would be vital to first determine whether OCT4 is expressed in human somatic cancers.In general, cancer cells, which are immortal, relatively undifferentiated, and invasive, are very similar to early embryonic cells. However, the cancer cell populations in most cancers are intrinsically heterogeneous, encompassing cells ranging from very immature to nearly differentiated. With stem cell theory being applied to cancer study, the undifferentiated cells in cancer may be considered to be CSCs, which have the exclusive ability to self-renew and give rise to nontumourigenic cancer cells. It is the small population of CSCs that drives tumour formation and growth. CSCs have been isolated from many somatic cancers including glioma and breast cancer and cultured in vitro. Recently, CSCs were proposed to be derived either from normal stem cells with dysregulated self-renewal or from restricted progenitors or differentiated cells regaining the capacity to self-renew. Therefore, it is likely that aberrant activation of the self-renewal pathway is a key event in carcinogenesis, and it becomes imperative and important to examine the expression and significance of genes involved in the regulation of stem cell self-renewal in cancers, including OCT4.The human OCT4 gene is located on chromosome 6 and comprises five exons and four introns. There are two isoforms generated by alternative splicing designated as OCT4A and OCT4B. They have identical POU DNA binding and C-terminal domains but differ in their N termini. OCT4A is localized in the nucleus, whereas OCT4B is mainly localized in the cytoplasm and, unlike OCT4A, it can not maintain the self-renewal of ES cells. OCT4 is commonly referred to as OCT4A and the function of OCT4 to maintain the self-renewal of ES cells should be attributed to OCT4A. In addition, there are six pseudogenes for human OCT4, which are highly homologous to human OCT4 gene. While OCT4B could be detected in various non-pluripotent somatic cancer cells, two OCT4 pseudogenes, OCT4-pg1 and OCT4-pg5, were found to be transcribed in somatic cancers and cancer cell lines. The existence and expression of both OCT4 alternative splicing variant and various pseudogenes are likely to yield undesirable false positive results. Therefore, when addressing OCT4 expression, it is necessary to distinguish the bona fide OCT4 from its alternative splicing variant and various pseudogenes.Although it was reported that the existence of OCT4 splicing variants and pseudogenes might have led to some confusion in cancer and stem cell research, the issue has not received much attention in many recent studies. In the present study, we used RT-PCR and sequencing analysis to detect OCT4 expression in two types of human somatic tumours, glioma and breast carcinoma, from which CSCs have already been isolated and cultured in vitro. Our results showed that three OCT4 pseudogenes, viz. OCT4-pg1, OCT4-pg3 and OCT4-pg4 rather than OCT4 gene, are expressed in these two human solid tumours. We further demonstrated their protein expression and explored the function and activity of their protein products.Part I Cloning and sequencing analysis of OCT4 pseudogenes from gliomas and breast carcinomasTo detect OCT4 expression in glioma and breast carcinoma, we collected 42 cases of gliomas and 45 cases of breast carcinomas. All tumour specimens were verified by pathological analysis. Gliomas were classified according to the WHO classification standard, whereas breast cancer subtypes were defined according to the status of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2). The adult normal brain and breast tissues were obtained from surgical resections of four and five trauma patients, respectively. In RT-PCR assays, we used two pairs of primers to detect OCT4 mRNA expression. OCT4A-specific primer set is able to specifically amplify OCT4A, while OCT4-full length primers were designed to amplify the entire coding sequence of OCT4A. When using OCT4A-specific primers, except for seminoma, which served as a positive control,OCT4A mRNA (496bp) was not detected in any tumour and normal tissues. But, when using OCT4-full length primers, besides seminoma, the predicated 1086bp PCR products were detected in gliomas of different grades and breast cancers of various subtypes. In addition, we detected OCT4 mRNA expression in many human glioma cell lines and breast cancer cell lines. When using OCT4A-specific primers, except for human pluripotent embryonal carcinoma cells NTcra-2, which served as a positive control, OCT4A mRNA (496bp) was not detected in U251, U87, MCF7, MDA-MB-231, BT474 and SK-BR-3 cells. But, when using OCT4-full length primers, besides NTera-2 cells, the predicated 1086bp PCR products were detected in these tumour cell lines.Next, we used DNA cloning and sequencing analysis to detect OCT4 expression in glioma and breast carcinoma. First, all the 1086bp DNA fragments were extracted from the agarose gel, cloned into pGEM-T easy vector and amplified. Plasmids with an insert of correct size were sequenced. Sequencing results showed that while the transcripts of OCT4 gene were only found in samples of positive control, the seminoma and NTera-2 cells, transcripts of three OCT4 pseudogenes, OCT4-pg1, OCT4-pg3 and OCT4-pg4, which are mapped to chromosomal bands 8q24,12p13 and 1q22, respectively, were found in these cancer tissues and cancer cell lines. Our sequencing results confirmed that it was the transcripts of OCT4 pseudogenes that contributed to the RT-PCR products in the cancer tissues and cancer cell lines when using OCT4-full length primers for PCR amplification.PartⅡProtein expression and localization of OCT4 pseudogenesTheoretically, the transcripts from OCT4-pg1, OCT4-pg3 and OCT4-pg4 can translate into protein products of 359,186 and 286 amino acids, respectively. Although there are amino acid deletions or substitutions, all these three putative proteins contain N-terminal domains similar to that of human OCT4.In order to investigate the expression and subcellular localization of these OCT4 pseudogene products, we constructed their expression plasmids with HA tag at their C termini. We then transfected them into NIH 3T3 cells and carried out immunocytochemistry staining. Our results showed that OCT4-pg1 and OCT4-pg4, as well as OCT4, were mainly localized in the nuclei, whereas OCT4-pg3 was mainly localized in the cytoplasm. Besides OCT4, OCT4-pg1 and OCT4-pg3 could also be detected by two anti-OCT4 antibodies, ab18976 and sc-5279. By contrast, OCT4-pg4 could not be detected by these two antibodies. Similar results were obtained in U251 and MCF7 cells transfected with the plasmids, except that OCT4-pg3 was located in both the nucleus and cytoplasm. Furthermore, Western blotting showed that OCT4-pg1, OCT4-pg3, OCT4-pg4 and OCT4 migrated at 50 kDa,30 kDa,30 kDa, and 50 kDa, respectively. In agreement with the immunocytochemistry results, subcellular fractionation analysis confirmed that OCT4-pgl, OCT4-pg4 and OCT4 were localized in the nuclei, while OCT4-pg3 was localized in the cytoplasm of NIH 3T3 cells. Besides OCT4, OCT4-pg1 and OCT4-pg3 could also be stained by sc-5279 in Western blotting. The two antibodies, ab18976 and sc-5279, could not recognize OCT4-pg4, possibly due to amino acid deletion or substitution. We further used immunochemistry to detect the endogenous expression of OCT4 pseudogenes in cancer cells. As expected, the fluorescence signal was strongly detected in the nucleus of NTera-2 cells when using the polyclonal ab18976 antibody. As to U251 glioma cells and MCF7 breast cancer cells, the fluorescence signals were detected in both the nucleus and cytoplasm of almost all cells, indicating the expression of OCT4 pseudogenes in these cancer cells. In contrast to extensive and nuclear staining of OCT4 in seminoma, only a few cells in human glioma and breast cancer were immunostained by ab18976, with positive staining in both the nucleus and cytoplasm of some cells, indicating the expression of OCT4 pseudogenes in these cancer tissues. In addition, the cells that were positively stained by ab 18976 in glioma also expressed the cancer stem cell marker CD 133 or the astroglial marker GFAP, indicating that expression of OCT4 pseudogenes is not restricted to CSCs in gliomas.These results suggest that, when detecting OCT4 expression in cancers, the expression of OCT4 pseudogenes might lead to false detection for OCT4 in both immunochemistry and Western blotting. This might also contribute to the misinterpretation of OCT4 expression in cancers and stem cells.Part III Protein activity and function of OCT4 pseudogenesTo determine the biological activities of these OCT4 pseudogenes, U251, U87, MCF7 and MDA-MB-231 cells were transfected with expression plasmids of OCT4 or its pseudogenes. The cell viability was measured by MTT assay. There was a significant increase in cell viability in cancer cells transfected with OCT4 compared to the control group, but there was no significant difference between cell groups transfected with OCT4 pseudogenes and the mock vector. The colony formation assay showed that the colony formation rate of cancer cells transfected with OCT4 was significantly higher than that of the control group cells, while there was no significant difference between cell groups transfected with OCT4 pseudogenes and the mock vector. These results showed that the OCT4 pseudogenes could not increase cell viability in MTT analysis and did not promote colony formation rate of cancer cells, indicating that the OCT4 pseudogenes have no effect on the proliferation of cancer cells. Finally, the transcriptional activation potentials of OCT4 pseudogenes were detected by luciferase reporter assay. OCT4 strongly transactivated three OCT4 reporter constructs (6×W, PORE and MORE), but there was no significant difference between OCT4 pseudogenes and the mock vector, suggesting that OCT4-pgl, OCT4-pg3 and OCT4-pg4 have no significant transcriptional activity toward these three OCT4 luciferase reporter constructs.The high degree of homology between OCT4 and its pseudogenes suggests that they might have similar functions, but our results showed that OCT4-pg1, OCT4-pg3 and OCT4-pg4 did not possess OCT4-like activities. The functional implication of OCT4 pseudogenes expression in cancers and roles of these genes playing in carcinogenesis remain to be further investigated.In conclusion, we have demonstrated the expression of three OCT4 pseudogenes, OCT4-pgl, OCT4-pg3, and OCT4-pg4 with an apparent lack of OCT4 in human glioma and breast carcinoma, indicating that OCT4 may not be essential for somaic tumourigenesis. We further demonstrated their protein expression by using immunochemistry and Western blotting. Our results suggest that the protein expression of these pseudogenes might have produced the false detection of OCT4 in human somatic cancers. Remarkably, we have revealed that the OCT4 pseudogenes did not display OCT4-like activities. We suggest that, in order to obtain reliable results, combined approaches with appropriate controls must be followed to exclude possibility of false-positive results in investigations of OCT4 expression in cancers and stem cells.更多还原