调控性RNAi系统构建及靶向诱导肿瘤干细胞凋亡对抗肿瘤免疫机制影响研究
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摘要
Burnet提出的“免疫监视”理论认为机体的免疫系统能够通过细胞免疫机制识别并清除癌变的异常细胞。研究表明机体免疫系统确实对肿瘤存在特异性和非特异性应答。然而,在机体如此严密的免疫监视下,肿瘤仍能够产生、发展。且随着其进展,机体的免疫系统受到抑制,免疫功能下降。这说明肿瘤具有一系列的机制对抗机体的免疫系统。现已明确的机制包括肿瘤细胞多种表面分子表达改变(MHC-I分子表达降低或缺失、非经典MHC-I分子表达、共刺激因子表达缺失及Fas/FasL表达改变等)导致免疫系统无法识别肿瘤细胞,阻止免疫系统对肿瘤细胞的识别、清除,造成肿瘤细胞免疫逃避；肿瘤细胞能够分泌多种细胞因子(IL4、IL10、TGF-β、PGE2等)诱导免疫细胞分化或凋亡,抑制免疫系统的抗肿瘤效应。
有研究报道在动物实验模型中,有效的抗肿瘤免疫反应能够明显导致肿瘤细胞减少,抑制肿瘤的生长。提示增强或者恢复机体抗肿瘤免疫反应有助于肿瘤的治疗。因此深入了肿瘤发生过程中对免疫系统相互对抗的机理,有助于对肿瘤生物学特性的全面了解,进而为肿瘤治疗提供理论依据。
研究表明在肿瘤组织中只占少数比例(不超过5%)的肿瘤干细胞在肿瘤发生／发展中具有重要作用。肿瘤干细胞是在肿瘤细胞群中具有自我更新,多分化潜能及无限增殖潜能等干细胞特性的小部分细胞。利用各种不同的分子标记,已经在血癌、脑瘤、肺癌、乳腺癌、卵巢癌、前列腺癌等多种肿瘤中发现肿瘤干细胞的存在。在肿瘤治疗中,不能有效清除掉肿瘤干细胞会造成肿瘤复发及转移发生。肿瘤干细胞的作用可能存在下面两种机制：一方面干细胞特性能够不断产生新生肿瘤细胞,促进肿瘤生长；另一方面,肿瘤干细胞产生某些具有抑制免疫细胞活化或诱导免疫细胞凋亡功能的细胞因子,抑制机体的抗肿瘤免疫反应。由于肿瘤干细胞对药物及放疗的抗性,现在广泛应用的肿瘤治疗方法治疗效果不理想。根据肿瘤干细胞的特性找到特异性针对肿瘤干细胞的有效治疗方法显得至关重要。
Oct4具有保持干细胞细胞多能性作用的干细胞多能因子。现在研究报道在胚胎干细胞、成体干细胞及肿瘤干细胞中都能检测到Oct4的表达,并确定Oct4在保持干细胞的多能性和无限增殖潜能方面具有重要作用。我们前面的研究发现使用Oct4siRNA干扰Oct4促进肿瘤细胞凋亡,肿瘤组织中肿瘤干细胞减少,延缓小鼠皮下肿瘤生长。并发现其调控作用是通过Oct4／Tcl 1／Akt1信号通路实现。我们的研究说明Oct4在肿瘤干细胞抗凋亡活性、促进肿瘤发生／发展方面具有重要作用。通过RNAi技术抑制Oct4的表达能够特异性减少肿瘤组织中肿瘤干细胞数量,延缓肿瘤生长。这提示特异靶向肿瘤干细胞的肿瘤治疗方法是可行的。
RNA干扰(RNAi)是一种通过特定寡核苷酸序列靶向降解细胞中互补mRNA,进而抑制基因在体内和体外的表达的技术。现在广泛应用的质粒或病毒载体表达siRNA的方法,无法控制基因表达的空间及时间的特异性。这导致了各种调控表达系统的构建,应用最广泛的是Tet调控表达系统。基于以上基础,我们进行了以下研究：
第一部分,改建tet调控表达系统。根据购自Clontech公司的Tet-on调控表达系统,在pTet-On-Advanced Vector、pTRE-tight vector、pEGFP-N1 (AgeI位点突变缺失)及pLKO1.-puro质粒的基础上构建单一质粒的四环素调控表达质粒pEGFP-Tet-on-TRE-sh.利用此质粒进行了后续的靶基因Oct4的RNAi干扰实验,并筛选了稳定转染细胞株。提高了体内实验过程中RNAi的干扰效率。
第二部分在实验室前期工作的基础上,检测RNAi技术靶向Oct4诱导肿瘤干细胞凋亡后,免疫状态的改变。在试验中我们发现,利用RNAi技术干扰Oct4表达致使肿瘤干细胞数目减少后,肿瘤微环境中IL4,IL10和TGF-β减少。为了进一步确认这是否导致肿瘤微环境中肿瘤免疫反应增强,利用流式技术检测肿瘤浸润DC、CD4+T及CD8+T细胞情况。发现肿瘤干细胞减少后,肿瘤浸润的DC中mature DC的比例增加,CD4+CD25+T降低,CD4+CD25-T升高, CD8+CD62L-T升高。说明肿瘤干细胞减少以后,肿瘤微环境中免疫反应增强。
本研究发现在肿瘤微环境中肿瘤细胞通过某些免疫抑制性细胞因子抑制肿瘤浸润的免疫细胞发挥作用,靶向清除肿瘤干细胞可以增强机体抗肿瘤反应,抑制肿瘤的生长。.这提示靶向肿瘤干细胞联合增强抗肿瘤免疫的方式治疗肿瘤的可行性。
Host anti-tumor immunity has been proved to play a key role during tumor initiation and progression. However, a large numbers of previous studies indicate that tumor cells can escape from the host immune surveillance. Several mechanisms have been identified to affect this progression, such as the secretion of suppressor cytokines, inhibition of dendritic cells (DC) maturation, infiltration of CD4+CD25+regulatory T cells (Treg), which can all suppress effector T cells function and tumor-specific T-cell response in tumor site. In this regard, recovery of anti-tumor immunity and protection of immune cells from tumor-induced suppression may be a helpful procedure for tumor therapy. Although there are numerous investigations indicating that effective anti-tumor immunity was developed by depleting Treg, few ones focus on the role of cancer stem cell-like cells (CSCLCs) in tumor immunity, which is the main purpose of our current study.
CSCLCs, a small subgroup of cancer cells, which have the same functional properties with normal stem cells as following:self-renewal, pluripotency, and extensive proliferation potential, have been found to initiate tumor and decrease the effectiveness of widely applied treatment used to eliminate tumor. It has been proved that current clinical therapies are unable to eliminate CSCLCs. In fact, because of the resistance to chemotherapy and radiotherapy like normal stem cells, CSCLCs can not be killed by widely used therapies, which function against the bulk population of tumor cells and surely shrink tumors, effectively. So, it is absolutely important to find out effective therapeutic strategies against CSCLCs. And there are already several researches focusing on CSCLCs special treatment.
Oct4, a member of the POU family of transcription factors, first reported to have an important role in keeping pluripotency and proliferation potential of stem cells, is also detected in various tumors. Because of the ability to keep proliferation of CSCLCs, Oct4 seem to be an important target to deplete CSCLCs. Our previously research indicates that expression of Oct4 turned off by Oct4 small interfering RNA cause CSCLCs apoptosis and tumor growth inhibition in vivo. We also found Oct4/Tcll/Aktl signal pathway, involving in ES proliferation by inhibiting the apoptosis of ES cells, also affects tumor cells apoptosis. Those studies provided an effective procedure to reduce CSCLCs in vivo.
Aiming to demonstrate the anti-tumor immunity recovery after reduction of CSCLCs, in the present study, we employed Oct4 small interfering RNA to induce CSCLCs apoptosis and then investigated the immunity changes happening in vitro and in vivo.
RNAi is a specific, potent, and highly successful approach for loss-of-function studies in virtually all eukaryotic organisms in vivo or in vitro. There are several appropriate tools to induce RNAi-including chemically synthesized siRNA、shRNA or siRNA-encoding plasmid or viral vectors. However, it is impossible to tightly regulate gene expression by this way. So, variety of controlled gene expression systems were developed, among which Tetracycline (Tet)-inducible expression system is one of the most prominent and widely-accepted inducible systems so far. The ideal controlled system should permit the investigators to rapidly and reversibly switch the transgene expression on and off, exclusively in the desired cells or tissue(s) at any time point during development.
Based on pTet-On-Advanced vector、pTRE-tight vector pEGFP-N1 (mutation in AgeⅠsite) and pLKO.1-puro, pEGFP-Tet-on-TRE-sh, a new tet-on plasmid which could express siRNA targeted Oct4 induced by DOX was constructed.
Then, we investigated if anti-tumor immunity in tumor site recovery, when Oct4 siRNA was injected into tumor. Firstly, immunosuppressive cytokines, such as IL4、IL10 and TGF-β, were tested in mouse hepatoma cell line hepal-6(in vitro) and mouse tumor model(in vivo), before and after cells(or tumors) were treated with Oct4 siRNA. We found reduction of cytokines when Oct4 siRNA was applied. The same phenomenon was found from experiments in vitro carried out in human hepatoma cell line Huh7. We also found that mature DC、activated CD4+T cells and CD8+T cells infiltrated in tumor issue increased after Oct4 siRNA treatment. Our results indicate that Oct4 RNA interference could enhance immunoreactions in situ and this procedure may be applied in tumor therapy.
有研究报道在动物实验模型中,有效的抗肿瘤免疫反应能够明显导致肿瘤细胞减少,抑制肿瘤的生长。提示增强或者恢复机体抗肿瘤免疫反应有助于肿瘤的治疗。因此深入了肿瘤发生过程中对免疫系统相互对抗的机理,有助于对肿瘤生物学特性的全面了解,进而为肿瘤治疗提供理论依据。
研究表明在肿瘤组织中只占少数比例(不超过5%)的肿瘤干细胞在肿瘤发生／发展中具有重要作用。肿瘤干细胞是在肿瘤细胞群中具有自我更新,多分化潜能及无限增殖潜能等干细胞特性的小部分细胞。利用各种不同的分子标记,已经在血癌、脑瘤、肺癌、乳腺癌、卵巢癌、前列腺癌等多种肿瘤中发现肿瘤干细胞的存在。在肿瘤治疗中,不能有效清除掉肿瘤干细胞会造成肿瘤复发及转移发生。肿瘤干细胞的作用可能存在下面两种机制：一方面干细胞特性能够不断产生新生肿瘤细胞,促进肿瘤生长；另一方面,肿瘤干细胞产生某些具有抑制免疫细胞活化或诱导免疫细胞凋亡功能的细胞因子,抑制机体的抗肿瘤免疫反应。由于肿瘤干细胞对药物及放疗的抗性,现在广泛应用的肿瘤治疗方法治疗效果不理想。根据肿瘤干细胞的特性找到特异性针对肿瘤干细胞的有效治疗方法显得至关重要。
Oct4具有保持干细胞细胞多能性作用的干细胞多能因子。现在研究报道在胚胎干细胞、成体干细胞及肿瘤干细胞中都能检测到Oct4的表达,并确定Oct4在保持干细胞的多能性和无限增殖潜能方面具有重要作用。我们前面的研究发现使用Oct4siRNA干扰Oct4促进肿瘤细胞凋亡,肿瘤组织中肿瘤干细胞减少,延缓小鼠皮下肿瘤生长。并发现其调控作用是通过Oct4／Tcl 1／Akt1信号通路实现。我们的研究说明Oct4在肿瘤干细胞抗凋亡活性、促进肿瘤发生／发展方面具有重要作用。通过RNAi技术抑制Oct4的表达能够特异性减少肿瘤组织中肿瘤干细胞数量,延缓肿瘤生长。这提示特异靶向肿瘤干细胞的肿瘤治疗方法是可行的。
RNA干扰(RNAi)是一种通过特定寡核苷酸序列靶向降解细胞中互补mRNA,进而抑制基因在体内和体外的表达的技术。现在广泛应用的质粒或病毒载体表达siRNA的方法,无法控制基因表达的空间及时间的特异性。这导致了各种调控表达系统的构建,应用最广泛的是Tet调控表达系统。基于以上基础,我们进行了以下研究：
第一部分,改建tet调控表达系统。根据购自Clontech公司的Tet-on调控表达系统,在pTet-On-Advanced Vector、pTRE-tight vector、pEGFP-N1 (AgeI位点突变缺失)及pLKO1.-puro质粒的基础上构建单一质粒的四环素调控表达质粒pEGFP-Tet-on-TRE-sh.利用此质粒进行了后续的靶基因Oct4的RNAi干扰实验,并筛选了稳定转染细胞株。提高了体内实验过程中RNAi的干扰效率。
第二部分在实验室前期工作的基础上,检测RNAi技术靶向Oct4诱导肿瘤干细胞凋亡后,免疫状态的改变。在试验中我们发现,利用RNAi技术干扰Oct4表达致使肿瘤干细胞数目减少后,肿瘤微环境中IL4,IL10和TGF-β减少。为了进一步确认这是否导致肿瘤微环境中肿瘤免疫反应增强,利用流式技术检测肿瘤浸润DC、CD4+T及CD8+T细胞情况。发现肿瘤干细胞减少后,肿瘤浸润的DC中mature DC的比例增加,CD4+CD25+T降低,CD4+CD25-T升高, CD8+CD62L-T升高。说明肿瘤干细胞减少以后,肿瘤微环境中免疫反应增强。
本研究发现在肿瘤微环境中肿瘤细胞通过某些免疫抑制性细胞因子抑制肿瘤浸润的免疫细胞发挥作用,靶向清除肿瘤干细胞可以增强机体抗肿瘤反应,抑制肿瘤的生长。.这提示靶向肿瘤干细胞联合增强抗肿瘤免疫的方式治疗肿瘤的可行性。
Host anti-tumor immunity has been proved to play a key role during tumor initiation and progression. However, a large numbers of previous studies indicate that tumor cells can escape from the host immune surveillance. Several mechanisms have been identified to affect this progression, such as the secretion of suppressor cytokines, inhibition of dendritic cells (DC) maturation, infiltration of CD4+CD25+regulatory T cells (Treg), which can all suppress effector T cells function and tumor-specific T-cell response in tumor site. In this regard, recovery of anti-tumor immunity and protection of immune cells from tumor-induced suppression may be a helpful procedure for tumor therapy. Although there are numerous investigations indicating that effective anti-tumor immunity was developed by depleting Treg, few ones focus on the role of cancer stem cell-like cells (CSCLCs) in tumor immunity, which is the main purpose of our current study.
CSCLCs, a small subgroup of cancer cells, which have the same functional properties with normal stem cells as following:self-renewal, pluripotency, and extensive proliferation potential, have been found to initiate tumor and decrease the effectiveness of widely applied treatment used to eliminate tumor. It has been proved that current clinical therapies are unable to eliminate CSCLCs. In fact, because of the resistance to chemotherapy and radiotherapy like normal stem cells, CSCLCs can not be killed by widely used therapies, which function against the bulk population of tumor cells and surely shrink tumors, effectively. So, it is absolutely important to find out effective therapeutic strategies against CSCLCs. And there are already several researches focusing on CSCLCs special treatment.
Oct4, a member of the POU family of transcription factors, first reported to have an important role in keeping pluripotency and proliferation potential of stem cells, is also detected in various tumors. Because of the ability to keep proliferation of CSCLCs, Oct4 seem to be an important target to deplete CSCLCs. Our previously research indicates that expression of Oct4 turned off by Oct4 small interfering RNA cause CSCLCs apoptosis and tumor growth inhibition in vivo. We also found Oct4/Tcll/Aktl signal pathway, involving in ES proliferation by inhibiting the apoptosis of ES cells, also affects tumor cells apoptosis. Those studies provided an effective procedure to reduce CSCLCs in vivo.
Aiming to demonstrate the anti-tumor immunity recovery after reduction of CSCLCs, in the present study, we employed Oct4 small interfering RNA to induce CSCLCs apoptosis and then investigated the immunity changes happening in vitro and in vivo.
RNAi is a specific, potent, and highly successful approach for loss-of-function studies in virtually all eukaryotic organisms in vivo or in vitro. There are several appropriate tools to induce RNAi-including chemically synthesized siRNA、shRNA or siRNA-encoding plasmid or viral vectors. However, it is impossible to tightly regulate gene expression by this way. So, variety of controlled gene expression systems were developed, among which Tetracycline (Tet)-inducible expression system is one of the most prominent and widely-accepted inducible systems so far. The ideal controlled system should permit the investigators to rapidly and reversibly switch the transgene expression on and off, exclusively in the desired cells or tissue(s) at any time point during development.
Based on pTet-On-Advanced vector、pTRE-tight vector pEGFP-N1 (mutation in AgeⅠsite) and pLKO.1-puro, pEGFP-Tet-on-TRE-sh, a new tet-on plasmid which could express siRNA targeted Oct4 induced by DOX was constructed.
Then, we investigated if anti-tumor immunity in tumor site recovery, when Oct4 siRNA was injected into tumor. Firstly, immunosuppressive cytokines, such as IL4、IL10 and TGF-β, were tested in mouse hepatoma cell line hepal-6(in vitro) and mouse tumor model(in vivo), before and after cells(or tumors) were treated with Oct4 siRNA. We found reduction of cytokines when Oct4 siRNA was applied. The same phenomenon was found from experiments in vitro carried out in human hepatoma cell line Huh7. We also found that mature DC、activated CD4+T cells and CD8+T cells infiltrated in tumor issue increased after Oct4 siRNA treatment. Our results indicate that Oct4 RNA interference could enhance immunoreactions in situ and this procedure may be applied in tumor therapy.
引文
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