生殖干细胞与肿瘤干细胞多能性及分裂机制的研究
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摘要
干细胞( stem cells )是生命科学研究的重要工具。一方面可用于研究增殖、分化、生长、发育、衰老、死亡等最基本的生命现象,另一方面经体外诱导可用于临床细胞治疗和组织工程材料研究,为一些目前难治性疾病的治疗带来新的希望。随着对干细胞生理特性认识的深入,研究者们发现干细胞与肿瘤细胞有很多共同特点,认为肿瘤很可能是干细胞在长期的自我更新过程中,由于多基因突变导致干细胞生长失去正常调控,而停止在分化的某一阶段无限制增殖所形成的,肿瘤组织内存在干细胞-肿瘤干细胞。本研究讨论人生殖嵴干细胞多能性及其不对称分裂机制,以及胚胎干细胞多能性因子在肿瘤发生中的作用。
本研究的第一章内容讨论人生殖嵴干细胞多能性及其不对称分裂机制。
胚胎干细胞在再生医学领域有着十分诱人的应用前景。胚性多能性干细胞具有体外分化方向不确定性,分化为所有细胞类型的潜能。因此,多能性干细胞在疾病治疗和组织工程中是一种重要的资源。多能性干细胞也是体外研究胚胎发育的一种重要工具。目前,人多能性干细胞包括三种类型:人胚胎干细胞(ESC)、人胚胎生殖细胞(EGC)和人诱导多能性干细胞(iPS)。人胚胎干细胞(ESC)起源于囊胚的内细胞团,而人胚胎生殖细胞(EGC)由发育后期的原始生殖细胞(PGC)发育而来。人诱导多能性干细胞(iPS)来源于重编程的成体细胞,这种成体细胞的重编程主要是通过人为导入一些胚胎多能性干细胞因子:如OCT4, SOX2, NANOG等使细胞获得多能性。
毫无疑问,人类胚胎干细胞(ESC)在组织重建、分化和基因功能等多方面的研究中吸引了公众的眼球,同时,人诱导多能性干细胞(iPS)也在2007年涌现在世人面前。然而,到目前为止,仅有少数的几个实验室对人胚胎生殖细胞(EGC)进行了了研究报道。生殖细胞系担负着遗传信息的世代传递,因此自然不同于ESC和iPS。体外建立和诱导EGC分化为研究再生工程分子机制提供了一个范例。因此,给予EGC研究更多的观注就显得尤其重要。
不同于ESCs和iPS,研究人员发现hEGCs在培养过程中生长速度缓慢,并且很难培养大量的未分化hEGCs,这将较大的限制了hEGCs的基础研究和临床应用。一些研究者发现人生殖干细胞(hEGCs)可能进行的是不对称分裂。Numb是一个重要的细胞命运决定子,在细胞不对称分裂中通过负调控Notch1信号通路来调节细胞的分裂。本研究通过免疫荧光化学等方法检测发现,Numb在原始生殖细胞hPGCs和人胚胎生殖细胞hEGCs中的表达和不对称分布。在培养的hEGCs单个细胞中,Numb的不对称分布被观察到,Numb呈月牙形分布,这一现象和其它神经细胞进行不对称分裂时,Numb的分布相似。
本研究进一步检测了Numb功能被抑制之后对hEGCs细胞增殖的影响。为此,构建了一DOX诱导的siRNA干扰系统:慢病毒载体pLKO-Tet-On-TRE U6-puro sh Numb。通过Numb RNA干扰的方法可以使hEGCs的生长增殖方式改变,由缓慢的增殖转变为旺盛增殖,细胞的分裂方式可能从不对称分裂转变为对称分裂。这一结果进一步提示Numb蛋白参与了hEGCs细胞分裂的调控。
Numb蛋白是Notch1信号通路的一个负调控因子,那么hEGCs的分裂是否也是受Numb/Notch1信号通路的调控呢?本文通过免疫荧光化学和流式细胞技术等进行了验证:在SiN-hEGCs克隆中,由于Numb的表达受到抑制,Notch1的表达水平显著上调。在hEGCs细胞中Numb/Notch1信号通路被激活,并且介导了hEGCs的细胞分裂。因此,也进一步揭示Numb调控hEGCs细胞不对称分裂是通过负调节Notch1信号通路。
同时,通过这种方法得到的EG细胞,保持了胚胎生殖干细胞的特征,表达一系列的干细胞标志分子,如碱性磷酸酶AP,SSEA-1, SSEA-4, Oct-4, Nanog, Pum2, C-kit和Vasa等分子;同时维持了胚胎干细胞的多能性:在体外培养时,能构形成类胚体EBs (embryoid bodies)。
本研究结果显示:发现不对称分裂的细胞命运决定子Numb在人PGCs和EGCs中表达,并且Numb在子代细胞中呈不对称分布从而导致EG细胞的不对称分裂。这种不称分裂可以通过RNA干扰Numb的表达而逆转,使EG细胞转变为旺盛生长。进一步的研究表明,Numb蛋白的表达下调后促进EG的增殖是因为Numb蛋白的表达下调激活了Notch1信号通路。
本研究的第二章内容主要讨论胚胎干细胞多能性因子在肿瘤发生中的作用。尽管在过去的几十年里人类对于恶性肿瘤的诊断和治疗已经取得了很大的进步,但是从实际的治疗情况来看肿瘤病人仍没有得到令人满意的治疗效果,对于大多数的恶性肿瘤我们依然束手无策。成功的根除这类疾病需要我们更加深入的了解肿瘤的起源和发展过程。目前认为肿瘤干细胞样细胞(CSCLC)在肿瘤的起源和发展过程中起关键作用,所以应用干细胞生物学方法研究靶向这些恶性肿瘤干细胞治疗方法成为治愈肿瘤的希望。
目前,越来越多的研究资料显示CSCLC具有正常干细胞的形态和性质,而且可能通过一些特异调节正常干细胞行为的信号通路来维持CSCLC的生存。调控干细胞行为的机制有两个重要的特点:多能性和潜在的无限增殖特性。正常干细胞一般有两种状态:一是处于一种未分化状态,此时它们可以通过自我更新来重建自我并且能够无限增殖;或者在一定条件下迅速分化,这时它们就会丧失自我更新的能力。干细胞多能性的维持主要是通过以Octamer 4 (Oct4), Nanog和BMPs等一系列信号分子为起始的信号通路,其中Oct4研究的最为广泛。Oct4基因是POU转录因子家族的一个成员,在胚胎干细胞和成人干细胞上均有表达。最近的研究表明Oct4不但可以维持胚胎干(ES)细胞的多能性,而且可以控制其无限增殖的特性。Oct4/Tcl1/Akt1信号通路已被证实参与ES细胞的增殖调控,主要是通过抑制ES细胞的凋亡来维持其增殖。有意思的是,研究者在生殖细胞肿瘤和一些体细胞肿瘤如肝癌、乳腺癌、膀胱癌和黑色素瘤(B16F10)中也同样发现了Oct4。尽管许多肿瘤细胞都表达Oct4,但是它在这些肿瘤中所起的作用我们仍然知之甚少。此外,许多肿瘤细胞都表达Oct4分子从而表现出一些干细胞的特性,所以Oct4可以看作是CSCLC的重要标志。因此阐明Oct4的表达是否参与了维持CSCLC的存活状态这一问题具有重要意义。
在本研究中通过体内、体外实验,应用RNA干扰技术成功抑制两种体细胞肿瘤模型中Oct4基因的表达(小鼠3LL细胞和人乳腺癌细胞MCF7)。研究证实减少Oct4分子在体细胞肿瘤中的表达将会使肿瘤细胞凋亡进而抑制肿瘤的生长。
为了进行靶向肿瘤干细胞中的Oct4RNA干扰研究,首先检测鉴定了3LL肿瘤细胞中Oct4的表达。为此,3LL肿瘤细胞和人乳腺癌细胞MCF7被Oct4抗体标记,并进行流式细胞分析结果显示所有的3LL肿瘤细胞和MCF7都高表达Oct4。Oct4RNA干扰实验显示,体外培养的3LL和MCF7肿瘤细胞高水平表达胚胎干细胞多能性细胞因子Oct4,并且采用siRNA干扰技术下调Oct4的表达将最终诱导细胞的凋亡。同时,在体内SiRNA干扰Oct4能成功减少肿瘤干细胞样细胞(CSCLC)数量并显著抑制肿瘤生长。
最近的研究报道表明: Oct4/Tcl1/ Akt1信号通路不仅直接调控ES细胞的分化,而且还调控细胞的增殖。那么这个信号通路是否参与了肿瘤细胞的增殖调控?本实验结果揭示,Oct4/Tcl1/ Akt1信号通路的激活可能介导了肿瘤细胞的凋亡。也就是说Oct4可能是通过阻止细胞的凋亡来维持肿瘤细胞的增殖。
总之,本研究证实通过应用RNA干扰技术下调Oct4分子的表达能够成功诱导CSCLC的凋亡以及抑制肿瘤体的生长,并且通过实验肯定了Oct4/Tcl1/Akt1通路在此过程中发挥了部分作用。因而本实验结果强烈提示这种靶向抑制干细胞的信号传导通路的策略能够成功的应用到肿瘤的治疗中去,为临床治疗肿瘤奠定了基础。
Pluripotent embryonic stem cells can be expanded seemingly indefinitely in vitro and have the potential to differentiate into any cell type in the body. Thus, they represent an valuable resource for the repair of diseased or damaged tissues. These cells also provide powerful tools for the study of early human embryonic development in vitro. Currently, there are three types of human pluripotent stem cells available for study: embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced pluripotent stem cells (iPS). ESCs are derived from the inner cell mass of pre-implantation blastocysts, whereas embryonic germ cells (EGCs) are derived from the primordial germ cells (PGCs) of embryos in later stages of development. Human iPS cell lines are derived from somatic cells that have been“reprogrammed”by the introduction of key stem cell identity factors such as OCT4, SOX2, NANOG, and others。
Research on human ESCs has caught public attention, and many studies have begun to elucidate the mechanisms of establishment and differentiation. iPS, which only emerged in 2007 have also served to stimulate interest in stem cell biology. However, only a handful of laboratories currently perform research on hEGCs. Germline cells have some key differences from ESCs and iPS cells, as they are responsible for transmitting genetic information from generation to generation. Understanding the establishment and differentiation of hEGCs in vitro will be an important step towards successful germline gene therapies, thus hEGCs deserve more study.
Unlike ESCs and iPS, hEGCs expand very slowly in culture. It is difficult to generate large quantities of undifferentiated hEGCs, which limits further basic and clinical applications of these cells. We have investigated hEGC cell division as a potential step at which hEGC proliferation might be controlled in the lab. Cell division can occur in one of two patterns, symmetric or asymmetric. Several studies have indicated that hEGCs might undergo asymmetric division.
Numb has been shown to play a determinant role in generating asymmetric cell divisions by negatively regulating Notch1 signaling. In order to test our hypothesis that Numb is involved in controlling hEGC division, we first investigated the expression of Numb in hPGCs in vivo. we confirmed that hPGCs and cultured hEGCs both express Numb at high levels. In the 24 h culture, hEGCs displayed a crescent of Numb staining, similar to other cells that undergo asymmetric division.
We next investigated whether cell proliferation was affected when Numb function is suppressed. To this end, we used a Dox-induced siRNA system with the lentiviral vector pLKO-Tet-On-TRE U6-puro sh Numb (Tet-on Numb shRNA). These results show that hEGC cultures could be converted from poorly proliferating to vigorously proliferating cells by Numb RNAi, suggesting that Numb expression is involved in hEGC division.
These results indicate that the Numb/Notch1 pathway is activated in, and plays a role in mediating, hEGC division. This, in turn, suggests that Numb might be responsible for maintaining asymmetric cell division potential by negatively regulating Notch1 protein levels.
SiN-hEGCs expressed characteristic markers of hEG cells and were able to differentiate into cell types of all three primary germ layers. The results presented here suggest that specifically targeted inhibition of Numb expression could be used to facilitate hEGC culture in vitro. These encouraging findings provide a starting point for basic and clinical study of hEGCs.
Octamer 4 (Oct4), a member of the POU family of transcription factors, plays a key role in the maintenance of pluripotency and proliferation potential of embryonic stem cells. Cancer stem cell–like cells (CSCLC) are reported to be a minor population in tumors or even in tumor cell lines which also express Oct4.
The role of Oct4 in CSCLCs still remains to be defined. In our study, we show that, in vitro, almost all murine Lewis lung carcinoma 3LL cells and human breast cancer MCF7 cells express Oct4 at high levels. This expression of Oct4 is successfully reduced by small interfering RNA, which eventually results in cell apoptosis. The signal pathway Oct4/Tcl1/Akt1 has been observed to be involved in this event. The repression of Oct4 reduces Tcl1 expression and further downregulates the level of p-Ser.473-Akt1. In vivo, only 5% of tumor cells were detected to express Oct4 in established 3LL and MCF7 tumor models, respectively. Small interfering RNA against Oct4 successfully decreases the CSCLCs and markedly inhibits tumor growth. In summary, we show that Oct4 might maintain the survival of CSCLCs partly through Oct4/Tcl1/ Akt1 by inhibiting apoptosis.
In summary, we show here for the first time that reduction of Oct4 expression in CSCLCs induces apoptosis and the inhibition of tumor growth partly through the Oct4/Tcl1/Akt1 pathway. The strategy described here strongly suggests that specific targeted inhibition of stem cell signaling pathways could be applied to cancer therapy.
本研究的第一章内容讨论人生殖嵴干细胞多能性及其不对称分裂机制。
胚胎干细胞在再生医学领域有着十分诱人的应用前景。胚性多能性干细胞具有体外分化方向不确定性,分化为所有细胞类型的潜能。因此,多能性干细胞在疾病治疗和组织工程中是一种重要的资源。多能性干细胞也是体外研究胚胎发育的一种重要工具。目前,人多能性干细胞包括三种类型:人胚胎干细胞(ESC)、人胚胎生殖细胞(EGC)和人诱导多能性干细胞(iPS)。人胚胎干细胞(ESC)起源于囊胚的内细胞团,而人胚胎生殖细胞(EGC)由发育后期的原始生殖细胞(PGC)发育而来。人诱导多能性干细胞(iPS)来源于重编程的成体细胞,这种成体细胞的重编程主要是通过人为导入一些胚胎多能性干细胞因子:如OCT4, SOX2, NANOG等使细胞获得多能性。
毫无疑问,人类胚胎干细胞(ESC)在组织重建、分化和基因功能等多方面的研究中吸引了公众的眼球,同时,人诱导多能性干细胞(iPS)也在2007年涌现在世人面前。然而,到目前为止,仅有少数的几个实验室对人胚胎生殖细胞(EGC)进行了了研究报道。生殖细胞系担负着遗传信息的世代传递,因此自然不同于ESC和iPS。体外建立和诱导EGC分化为研究再生工程分子机制提供了一个范例。因此,给予EGC研究更多的观注就显得尤其重要。
不同于ESCs和iPS,研究人员发现hEGCs在培养过程中生长速度缓慢,并且很难培养大量的未分化hEGCs,这将较大的限制了hEGCs的基础研究和临床应用。一些研究者发现人生殖干细胞(hEGCs)可能进行的是不对称分裂。Numb是一个重要的细胞命运决定子,在细胞不对称分裂中通过负调控Notch1信号通路来调节细胞的分裂。本研究通过免疫荧光化学等方法检测发现,Numb在原始生殖细胞hPGCs和人胚胎生殖细胞hEGCs中的表达和不对称分布。在培养的hEGCs单个细胞中,Numb的不对称分布被观察到,Numb呈月牙形分布,这一现象和其它神经细胞进行不对称分裂时,Numb的分布相似。
本研究进一步检测了Numb功能被抑制之后对hEGCs细胞增殖的影响。为此,构建了一DOX诱导的siRNA干扰系统:慢病毒载体pLKO-Tet-On-TRE U6-puro sh Numb。通过Numb RNA干扰的方法可以使hEGCs的生长增殖方式改变,由缓慢的增殖转变为旺盛增殖,细胞的分裂方式可能从不对称分裂转变为对称分裂。这一结果进一步提示Numb蛋白参与了hEGCs细胞分裂的调控。
Numb蛋白是Notch1信号通路的一个负调控因子,那么hEGCs的分裂是否也是受Numb/Notch1信号通路的调控呢?本文通过免疫荧光化学和流式细胞技术等进行了验证:在SiN-hEGCs克隆中,由于Numb的表达受到抑制,Notch1的表达水平显著上调。在hEGCs细胞中Numb/Notch1信号通路被激活,并且介导了hEGCs的细胞分裂。因此,也进一步揭示Numb调控hEGCs细胞不对称分裂是通过负调节Notch1信号通路。
同时,通过这种方法得到的EG细胞,保持了胚胎生殖干细胞的特征,表达一系列的干细胞标志分子,如碱性磷酸酶AP,SSEA-1, SSEA-4, Oct-4, Nanog, Pum2, C-kit和Vasa等分子;同时维持了胚胎干细胞的多能性:在体外培养时,能构形成类胚体EBs (embryoid bodies)。
本研究结果显示:发现不对称分裂的细胞命运决定子Numb在人PGCs和EGCs中表达,并且Numb在子代细胞中呈不对称分布从而导致EG细胞的不对称分裂。这种不称分裂可以通过RNA干扰Numb的表达而逆转,使EG细胞转变为旺盛生长。进一步的研究表明,Numb蛋白的表达下调后促进EG的增殖是因为Numb蛋白的表达下调激活了Notch1信号通路。
本研究的第二章内容主要讨论胚胎干细胞多能性因子在肿瘤发生中的作用。尽管在过去的几十年里人类对于恶性肿瘤的诊断和治疗已经取得了很大的进步,但是从实际的治疗情况来看肿瘤病人仍没有得到令人满意的治疗效果,对于大多数的恶性肿瘤我们依然束手无策。成功的根除这类疾病需要我们更加深入的了解肿瘤的起源和发展过程。目前认为肿瘤干细胞样细胞(CSCLC)在肿瘤的起源和发展过程中起关键作用,所以应用干细胞生物学方法研究靶向这些恶性肿瘤干细胞治疗方法成为治愈肿瘤的希望。
目前,越来越多的研究资料显示CSCLC具有正常干细胞的形态和性质,而且可能通过一些特异调节正常干细胞行为的信号通路来维持CSCLC的生存。调控干细胞行为的机制有两个重要的特点:多能性和潜在的无限增殖特性。正常干细胞一般有两种状态:一是处于一种未分化状态,此时它们可以通过自我更新来重建自我并且能够无限增殖;或者在一定条件下迅速分化,这时它们就会丧失自我更新的能力。干细胞多能性的维持主要是通过以Octamer 4 (Oct4), Nanog和BMPs等一系列信号分子为起始的信号通路,其中Oct4研究的最为广泛。Oct4基因是POU转录因子家族的一个成员,在胚胎干细胞和成人干细胞上均有表达。最近的研究表明Oct4不但可以维持胚胎干(ES)细胞的多能性,而且可以控制其无限增殖的特性。Oct4/Tcl1/Akt1信号通路已被证实参与ES细胞的增殖调控,主要是通过抑制ES细胞的凋亡来维持其增殖。有意思的是,研究者在生殖细胞肿瘤和一些体细胞肿瘤如肝癌、乳腺癌、膀胱癌和黑色素瘤(B16F10)中也同样发现了Oct4。尽管许多肿瘤细胞都表达Oct4,但是它在这些肿瘤中所起的作用我们仍然知之甚少。此外,许多肿瘤细胞都表达Oct4分子从而表现出一些干细胞的特性,所以Oct4可以看作是CSCLC的重要标志。因此阐明Oct4的表达是否参与了维持CSCLC的存活状态这一问题具有重要意义。
在本研究中通过体内、体外实验,应用RNA干扰技术成功抑制两种体细胞肿瘤模型中Oct4基因的表达(小鼠3LL细胞和人乳腺癌细胞MCF7)。研究证实减少Oct4分子在体细胞肿瘤中的表达将会使肿瘤细胞凋亡进而抑制肿瘤的生长。
为了进行靶向肿瘤干细胞中的Oct4RNA干扰研究,首先检测鉴定了3LL肿瘤细胞中Oct4的表达。为此,3LL肿瘤细胞和人乳腺癌细胞MCF7被Oct4抗体标记,并进行流式细胞分析结果显示所有的3LL肿瘤细胞和MCF7都高表达Oct4。Oct4RNA干扰实验显示,体外培养的3LL和MCF7肿瘤细胞高水平表达胚胎干细胞多能性细胞因子Oct4,并且采用siRNA干扰技术下调Oct4的表达将最终诱导细胞的凋亡。同时,在体内SiRNA干扰Oct4能成功减少肿瘤干细胞样细胞(CSCLC)数量并显著抑制肿瘤生长。
最近的研究报道表明: Oct4/Tcl1/ Akt1信号通路不仅直接调控ES细胞的分化,而且还调控细胞的增殖。那么这个信号通路是否参与了肿瘤细胞的增殖调控?本实验结果揭示,Oct4/Tcl1/ Akt1信号通路的激活可能介导了肿瘤细胞的凋亡。也就是说Oct4可能是通过阻止细胞的凋亡来维持肿瘤细胞的增殖。
总之,本研究证实通过应用RNA干扰技术下调Oct4分子的表达能够成功诱导CSCLC的凋亡以及抑制肿瘤体的生长,并且通过实验肯定了Oct4/Tcl1/Akt1通路在此过程中发挥了部分作用。因而本实验结果强烈提示这种靶向抑制干细胞的信号传导通路的策略能够成功的应用到肿瘤的治疗中去,为临床治疗肿瘤奠定了基础。
Pluripotent embryonic stem cells can be expanded seemingly indefinitely in vitro and have the potential to differentiate into any cell type in the body. Thus, they represent an valuable resource for the repair of diseased or damaged tissues. These cells also provide powerful tools for the study of early human embryonic development in vitro. Currently, there are three types of human pluripotent stem cells available for study: embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced pluripotent stem cells (iPS). ESCs are derived from the inner cell mass of pre-implantation blastocysts, whereas embryonic germ cells (EGCs) are derived from the primordial germ cells (PGCs) of embryos in later stages of development. Human iPS cell lines are derived from somatic cells that have been“reprogrammed”by the introduction of key stem cell identity factors such as OCT4, SOX2, NANOG, and others。
Research on human ESCs has caught public attention, and many studies have begun to elucidate the mechanisms of establishment and differentiation. iPS, which only emerged in 2007 have also served to stimulate interest in stem cell biology. However, only a handful of laboratories currently perform research on hEGCs. Germline cells have some key differences from ESCs and iPS cells, as they are responsible for transmitting genetic information from generation to generation. Understanding the establishment and differentiation of hEGCs in vitro will be an important step towards successful germline gene therapies, thus hEGCs deserve more study.
Unlike ESCs and iPS, hEGCs expand very slowly in culture. It is difficult to generate large quantities of undifferentiated hEGCs, which limits further basic and clinical applications of these cells. We have investigated hEGC cell division as a potential step at which hEGC proliferation might be controlled in the lab. Cell division can occur in one of two patterns, symmetric or asymmetric. Several studies have indicated that hEGCs might undergo asymmetric division.
Numb has been shown to play a determinant role in generating asymmetric cell divisions by negatively regulating Notch1 signaling. In order to test our hypothesis that Numb is involved in controlling hEGC division, we first investigated the expression of Numb in hPGCs in vivo. we confirmed that hPGCs and cultured hEGCs both express Numb at high levels. In the 24 h culture, hEGCs displayed a crescent of Numb staining, similar to other cells that undergo asymmetric division.
We next investigated whether cell proliferation was affected when Numb function is suppressed. To this end, we used a Dox-induced siRNA system with the lentiviral vector pLKO-Tet-On-TRE U6-puro sh Numb (Tet-on Numb shRNA). These results show that hEGC cultures could be converted from poorly proliferating to vigorously proliferating cells by Numb RNAi, suggesting that Numb expression is involved in hEGC division.
These results indicate that the Numb/Notch1 pathway is activated in, and plays a role in mediating, hEGC division. This, in turn, suggests that Numb might be responsible for maintaining asymmetric cell division potential by negatively regulating Notch1 protein levels.
SiN-hEGCs expressed characteristic markers of hEG cells and were able to differentiate into cell types of all three primary germ layers. The results presented here suggest that specifically targeted inhibition of Numb expression could be used to facilitate hEGC culture in vitro. These encouraging findings provide a starting point for basic and clinical study of hEGCs.
Octamer 4 (Oct4), a member of the POU family of transcription factors, plays a key role in the maintenance of pluripotency and proliferation potential of embryonic stem cells. Cancer stem cell–like cells (CSCLC) are reported to be a minor population in tumors or even in tumor cell lines which also express Oct4.
The role of Oct4 in CSCLCs still remains to be defined. In our study, we show that, in vitro, almost all murine Lewis lung carcinoma 3LL cells and human breast cancer MCF7 cells express Oct4 at high levels. This expression of Oct4 is successfully reduced by small interfering RNA, which eventually results in cell apoptosis. The signal pathway Oct4/Tcl1/Akt1 has been observed to be involved in this event. The repression of Oct4 reduces Tcl1 expression and further downregulates the level of p-Ser.473-Akt1. In vivo, only 5% of tumor cells were detected to express Oct4 in established 3LL and MCF7 tumor models, respectively. Small interfering RNA against Oct4 successfully decreases the CSCLCs and markedly inhibits tumor growth. In summary, we show that Oct4 might maintain the survival of CSCLCs partly through Oct4/Tcl1/ Akt1 by inhibiting apoptosis.
In summary, we show here for the first time that reduction of Oct4 expression in CSCLCs induces apoptosis and the inhibition of tumor growth partly through the Oct4/Tcl1/Akt1 pathway. The strategy described here strongly suggests that specific targeted inhibition of stem cell signaling pathways could be applied to cancer therapy.
引文
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