MEK1通过SIRT1调控肝癌干细胞自我更新的机制研究
发布时间：2018-08-02 17:48
【摘要】：肝癌(Hepatocellular carcinoma,HCC)在中国是死亡率高居第三的恶性肿瘤。尽管,当前我们已经把手术切除确立为了治疗早期肝癌的一线治疗手段,并且辅助结合其他治疗方式帮助患者。但是肝癌患者们不仅饱受肝癌耐药之苦,同时还承担着较高的术后复发风险,以至于最终大多都以差预后和短生存期而结束。然而根据最新研究,人们把癌症的这种高复发率归咎于一群叫做肿瘤干细胞(cancer stem cells,CSCs)的罪魁祸首。由于这群细胞不仅拥有肿瘤形成的始动能力和类似于胚胎干细胞的自我更新特性,还拥有高度的分化潜能,使得它们在肿瘤的耐药已经放疗耐受中发挥重要作用。先前的研究已表明:肿瘤干细胞与肿瘤的形成、转移、耐药以及术后复发密切相关。同时,伴随着关于肿瘤干细胞的研究越来越多,更多揭示了它的作用机制,一方面可以加深我们对于恶性肿瘤的研究和了解,另一方面也为我们战胜癌症提供了新的希望。我们课题组先前的研究已经证明了,转录因子Nanog是肝癌干细胞的一个干性指标,它可以在体内或者体外条件下,通过IGF-1信号通路来调控肝癌干细胞的自我更新能力。同时我们也发现这群Nanog阳性的肝癌干细胞是肝癌患者不良预后的重要病因,它们不仅能够维持肝癌的自我更新,还在肝癌形成以及分化中发挥重要作用。然而实际上,我们无法阐明肝癌干细胞在肝癌的发生发展以及复发中作用的详细机制。去乙酰化酶sirtuin家族是一群高度依赖于烟酰胺腺嘌呤二核苷酸(NAD+)的酶。该家族包含SIRT1-7 一共七个成员,已有研究表明它们参与调控了各种细胞生物学功能,例如:细胞周期、细胞代谢、细胞增殖、细胞分化、基因组稳定性以及肿瘤形成。同时,SIRT1也被证实在肝癌的维持以及发展中发挥关键作用。尽管人们发现在肝癌中microRNA (miRNA)-34a、miRNA-29c和c-MYC的表达能够有效调控致癌基因SIRT1的功能,但是SIRT1如何通过肝癌干细胞在肝癌中发挥作用,这个具体的调控机制我们依旧不得而知。MEK1和MEK2属于丝裂原活化蛋白激酶激酶(MAPKKs)家族的成员,这种激酶能够特异性的磷酸化苏氨酸和酪氨酸残基进而激活丝裂原活化蛋白激酶(MAPK)基质。MEK1异常调控会引发很多疾病包括肿瘤。而且,研究发现MEK1在很多肿瘤形成过程中会呈现上调趋势。另一方面,MEK1信号通路通常被认为是肝癌维持和发展的一个重要因素。可是,关于MEK1信号通路如何调控肝癌干细胞自我更新的具体机制还有待于进一步探索。我们的此次研究便揭示了一个MEK1失活时在体内外条件下,能通过促进SIRT1泛素化来加速其蛋白降解,进而抑制肝癌干细胞肿瘤形成能力的重要作用机制。为了达到此项研究目的,我们将这个课题分为四步来完成。首先,我们证明了 MEK1信号通路在肝癌干细胞自我更新和增殖中发挥重要作用:然后,我们又发现了在肝癌患者中MEK1的表达水平与SIRT1正相关,同时我们在肝癌干细胞中抑制或者敲除MEK1之后会导致SIRT1的明显下调;接下来我们又阐明了 MEK1对SIRT1的调控是通过维持其蛋白水平降解而达到的,也就证明了 MEK1信号通路是通过维持SIRT1蛋白水平来调控肝癌干细胞的自我更新的;最后,我们还惊喜的发现了 MEK1和SIRT1蛋白的共表达水平与患者的预后密切相关。本篇文章的主要结果及结论如下所示:1.在肝癌干细胞中抑制MEK1的活性可以有效地减慢其细胞增殖能力(1)我们将Huh7-Nanogpos和PLC/PRF/5-Nanogpos的肝癌干细胞以1×103每孔的密度铺在96孔板中,并用不同浓度的U0126进行持续处理6天,然后进行CCk-8检测。我们发现U0126能够显著抑制肝癌干细胞的增殖,而且这种抑制效果表现出浓度依赖的模式。同时,细胞生存曲线排除了这些抑制浓度对于肿瘤干细胞的药物毒性杀伤作用。(2)我们将 Huh7-Nanogpos 和 PLC/PRF/5-Nanogpos 肝癌干细胞与 U0126 或者二甲基亚砜(DMSO)共培养48小时之后,免疫荧光(Immunofluorescence, IF)实验表明比起对照组U0126处理后的肝癌干细胞Ki-67表达水平显著下降。(3)将丁酸钠同步化处理后的Huh7-和PLC/PRF/5-Nanogpos肝癌干细胞用U0126或者二甲基亚砜(阴性对照)处理之后,细胞周期实验发现U0126处理,诱发了大量肝癌干细胞将细胞周期停滞在G0/G1期,同时还伴随着S期和G2/M期细胞的显著减少。2. MEK1抑制剂U0126能够明显地降低肝癌干细胞的自我更新能力(1)我们发现在肝癌干细胞中抑制MEK1活性后能够显著下调其克隆形成及成球能力,且此结论已在两个细胞系中得到重复验证。而与肝癌干细胞相反的是,低浓度的U0126处理并不能降低非肝癌干细胞的克隆形成及成球能力。(2)免疫印迹实验表明在肝癌干细胞中抑制MEK1活性后能够显著抑制干性指标例如SOX2、OCT4以及Nanog的表达。(3)当我们把U0126处理后的肝癌干细胞以不同浓度梯度1×102、1×103,、1×104皮下注入NOD-SCID小鼠后,发现相对于对照组其肿瘤形成及生长能力被显著抑制。3.敲除MEK1能显著抑制肝癌干细胞的自我更新和增殖能力(1)蛋白水平检测发现在肝癌干细胞中敲除MEK1后能显著下调MEK1及磷酸化ERK1/2的蛋白表达水平,但是ERK1/2的表达水平没有明显差异。(2)在肝癌干细胞中用两种不同的MEK1干扰慢病毒敲除MEK1后,发现其增殖能力被明显抑制。(3)在肝癌干细胞中敲除MEK1后能显著抑制肝癌干细胞的成球和克隆形成能力。(4)免疫印迹实验表明在肝癌干细胞中敲除MEK1后干性相关蛋白的表达例如Nanog、OCT4、c-Myc和SOX2都被明显抑制。4.抑制或敲除MEK1能显著降低SIRT1的表达(1)通过检测我们发现sirtuin家族中,只有SIRT1和SIRT7在肝癌干细胞中的表达水平高于非肝癌干细胞,然而用U0126抑制MEK1活性之后只有SIRT1的表达水平被降低了。(2)正如免疫印迹实验结果所示:U0126对于肝癌干细胞中SIRT1表达水平的下调作用不仅与药物作用时间正相关,还与药物作用浓度正相关。(3)另一种MEK1抑制剂PD98059同样证实了在肝癌干细胞中抑制MEK1活性能有效地降低SIRT1蛋白表达。(4)在肝癌干细胞中敲除MEK1后能有效地降低SIRT1蛋白表达水平。5. MEK1信号通路通过调控组蛋白去乙酰化酶SIRT1来促进肝癌干细胞的自我更新和肿瘤形成能力(1)在非肝癌干细胞中过表达SIRT1后能显著地提高其克隆形成及成球能力,但是通过抑制MEK1信号通路的活性能够有效地逆转这种激活效果。(2)在肝癌干细胞中抑制MEK1活性后,这群细胞的克隆形成及成球能力被显著抑制,然后通过过表达SIRT1蛋白水平能够恢复它们的克隆形成和成球能力。(3)在体内肿瘤形成模型中,注射了 MEK1活性抑制的肝癌干细胞的NOD-SCID小鼠其肿瘤形成能力比起阳性对照组差很多。同时,我们还发现通过过表达SIRT1水平能够部分恢复肝癌干细胞在NOD-SCID小鼠体内的肿瘤形成能力。6. MEK1信号通路是通过抑制SIRT1的泛素化水平来抑制蛋白酶体的降解作用,从而维持SIRT1蛋白的表达水平(1)我们将肝癌干细胞分为四组:一组为阴性对照即与二甲基亚砜共培养,一组为阳性对照即与蛋白酶体抑制剂MG-132共培养,一组与MEK1抑制剂U0126共培养,一组为MEK1敲除的肝癌干细胞。这四组细胞培养48小时后,同时加入放线菌酮(CHX)处理不同时间,最后检测SIRT1蛋白水平发现:相对于阴性对照,MEK1抑制或者敲除后,肝癌干细胞中SIRT1蛋白的降解速度明显加快,而抑制蛋白酶体后SIRT1蛋白的降解速度被显著减慢。(2)在肝癌干细胞中抑制或者敲除MEK1后,会激活蛋白酶体活性从而有效地促进SIRT1蛋白的降解。(3)免疫共沉淀实验(Co-IP)结果显示:在肝癌干细胞中抑制或者敲除MEK1后,SIRT1的泛素化状态被极大地提高,导致了SIRT1蛋白的降解。7.在肝癌患者中MEK1/SIRT1的高水平表达与其不良预后呈现显著的正相关关系(1)我们在148例肝癌患者的组织标本中,通过免疫组化(Immunohistochemistry,IHC)检测了 p-MEK1、SIRT1和Nanog蛋白的表达水平。其中同时高水平表达p-MEK1/SIRT1的患者有48例,而同时低水平表达p-MEK1/SIRT1的患者有39例。通过卡方检验(Person chi-square test)统计分析发现P值为0.035,也就是说在肝癌患者中MEK1与SIRT1的表达水平是相关的。同样的方法也证实了 Nanog的表达水平与MEK1/SIRT1的表达在肝癌患者中是相关的,其中两个P值分别是0.013和0.038。(2)我们将回访搜集到的148例肝癌患者的临床病理信息进行统计和分析发现:MEK1/SIRT1的共表达高低状态与肝癌患者的年龄、性别、血清甲胎蛋白(AFP)水平、肿瘤间质增生、肿瘤坏死及肿瘤复发没有统计学关联;但是MEK1/SIRT1同时表达水平的高低与肝癌患者的肿瘤大小(p=0.012)、血管癌栓(p0.001)、包膜浸润(p=p=0.048)和临床肿瘤分期(p0.001)都具有显著的统计学关系。(3)我们将患者生存周期与预后联合MEK1和SIRT1的表达水平进行分析发现两者存在显著关联,同时高水平表达MEK1和SIRT1的肝癌患者比起低水平的患者往往预后更差。总而言之,我们的研究结果表明:1.在肝癌患者中MEK1表达水平与SIRT1表达水平显著相关,同时高水平表达MEK1/SIRT1的肝癌患者提示预后不佳和较短的生存期。2.激活MEK1信号通路能促进肝癌干细胞增殖、自我更新和肿瘤形成特征,敲除或者抑制MEK1能显著降低干性标记物的表达水平。3. MEK1信号通路是通过SIRT1蛋白表达来调控肝癌干细胞的增殖、肿瘤形成和自我更新。4. MEK1通过泛素化SIRT1控制蛋白酶体降解来维持SIRT1蛋白的表达水平。5.可以将MEK1/SIRT1作为判断肝癌患者情况的指标,同时MEK1信号通路可以作为一个治疗肝癌的潜在靶标。综上所述,本研究证明了无论在体内还是体外环境下,抑制或者干扰MEK1能够有效地减缓肝癌干细胞的肿瘤形成速度。同时,我们还证实了 MEK1信号通路是通过维持SIRT1蛋白水平来促进肝癌干细胞的自我更新和肿瘤形成。从机制上阐明了MEK1信号通路通过抑制SIRT1的泛素化水平来抑制蛋白酶体的降解,从而维持较高的SIRT1蛋白表达水平。肝癌患者临床预后数据分析表明:高表达MEK1和SIRT1的肝癌患者与预后不良相关。我们的研究提示MEK1-SIRT1可以作为一个重要的诊断指标,同时抑制MEK1可能是靶向肝癌干细胞治疗的一个潜在作用靶点。
[Abstract]:Hepatocellular carcinoma (HCC) is the third highest death rate in China. Although we have now established surgical excision to treat early liver cancer as a first-line treatment and assist patients with other treatment methods, patients with liver cancer are not only suffering from the resistance of liver cancer, but also in the same way. The high risk of postoperative recurrence ends up mostly in poor prognosis and short life. However, according to the latest research, the high recurrence rate of cancer is blamed for a group of cancer stem cells, CSCs, because the cells do not only have the ability to start the tumor and similar to the embryo. The self renewal characteristics of stem cells also have high differentiation potential that make them play an important role in the tolerance of cancer to radiotherapy. Previous studies have shown that cancer stem cells are closely related to tumor formation, metastasis, drug resistance, and postoperative recurrence, and more and more research on cancer stem cells is accompanied by more and more research. On the one hand, it can deepen our research and understanding of malignant tumors, and on the other hand, it provides new hope for us to overcome cancer. Our previous research group has shown that the transcription factor Nanog is a dry indicator of liver cancer stem cells, which can be used in vivo or in vitro. IGF-1 signaling pathway is used to regulate the self-renewal capacity of liver cancer stem cells. We also found that this group of Nanog positive liver cancer stem cells is an important cause of poor prognosis in the patients with liver cancer. They not only maintain the self renewal of liver cancer, but also play an important role in the formation and differentiation of liver cancer. However, we can not explain the fact. A detailed mechanism for the role of liver cancer stem cells in the development and recurrence of liver cancer. The deacetylase sirtuin family is a group of enzymes highly dependent on nicotinamide adenine dinucleotide (NAD+). The family contains seven members of the SIRT1-7, which has been studied to show that they are involved in the regulation of various cellular biological functions, such as cell cycle. Cell metabolism, cell proliferation, cell differentiation, genomic stability and tumor formation, and SIRT1 also proved to play a key role in the maintenance and development of liver cancer. Although it is found that the expression of microRNA (miRNA) -34a, miRNA-29c and c-MYC can effectively regulate the function of the oncogene SIRT1 in liver cancer, but how SIRT1 passes through it Liver cancer stem cells play a role in liver cancer. This specific regulatory mechanism remains unknown that.MEK1 and MEK2 belong to the members of the mitogen activated protein kinase kinase (MAPKKs) family. This kinase can specifically phosphorylate threonine and tyrosine residues and then activate the abnormality of the matrix.MEK1 of the mitogen activated protein kinase (MAPK) matrix. In addition, the MEK1 signaling pathway is often considered to be an important factor in the maintenance and development of liver cancer. However, the specific mechanism of how the MEK1 signaling pathway regulates the self-renewal of liver cancer stem cells remains to be found in the MEK1 signaling pathway. Further exploration. Our present study reveals the important mechanism of a MEK1 inactivation in vivo and in vitro, which can accelerate its protein degradation by promoting SIRT1 ubiquitination, and then inhibit the ability of liver cancer stem cell tumor formation. In order to achieve this goal, we divide the subject into four steps. First, we The MEK1 signaling pathway plays an important role in the self renewal and proliferation of liver cancer stem cells. Then, we also found that the expression level of MEK1 in liver cancer patients is positively related to SIRT1, and our inhibition or knockout of MEK1 in liver cancer stem cells will lead to the obvious downregulation of SIRT1; then we also elucidate MEK1 to SIRT1. The regulation is achieved by maintaining protein level degradation, and it is also proved that the MEK1 signaling pathway regulates the self renewal of liver cancer stem cells by maintaining the SIRT1 protein level. Finally, we also surprised to find that the co expression level of MEK1 and SIRT1 proteins is closely related to the patient's preconditioning. The main results of this article are as follows The conclusions are as follows: 1. inhibition of MEK1 activity in liver cancer stem cells can effectively slow down its cell proliferation ability (1) we spread the Huh7-Nanogpos and PLC/PRF/5-Nanogpos liver cancer stem cells in 96 orifice with the density of 1 x 103 per pore, and carry on the continuous treatment for 6 days with different concentrations of U0126, and then carry out CCk-8 detection. We found U0126 It can significantly inhibit the proliferation of liver cancer stem cells, and this inhibitory effect shows a concentration dependent pattern. At the same time, the cell survival curve excludes the toxic effects of these inhibitory concentrations on tumor stem cells. (2) we put Huh7-Nanogpos and PLC/PRF/5-Nanogpos liver cancer stem cells with U0126 or two methyl sulfoxide (DMS). O) after 48 hours co culture, the immunofluorescence (Immunofluorescence, IF) experiment showed that the Ki-67 expression level of liver cancer stem cells decreased significantly compared with the control group after U0126 treatment. (3) Huh7- and PLC/PRF/5-Nanogpos liver cancer stem cells treated with sodium butyrate were treated with U0126 or two methyl sulfoxide (negative control), and the cell cycle was true. It was found that U0126 treatment induced a large number of liver cancer stem cells to stagnate the cell cycle in the G0/G1 phase, and also accompanied by the significant reduction of.2. MEK1 inhibitor U0126 in S and G2/M phase cells (1) the self-renewal capacity of liver cancer stem cells was significantly reduced (1) we found that after the inhibition of MEK1 activity in the stem cells of the liver cancer, the clones could be significantly reduced. The formation and formation of ball forming ability, and this conclusion has been repeated in two cell lines. Contrary to liver cancer stem cells, low concentration of U0126 treatment does not reduce the cloning and formation of non hepatoma stem cells. (2) immunoblotting experiments showed that the inhibition of MEK1 activity in liver cancer stem cells could significantly inhibit the dry index. Such as the expression of SOX2, OCT4 and Nanog. (3) when U0126 treated liver cancer stem cells were injected with different concentrations of 1 x 102,1 x 103 and 1 x 104 subcutaneous injected into NOD-SCID mice, it was found that the tumor formation and growth ability of the cancer stem cells compared with the control group was significantly inhibited by.3. knockout MEK1 can significantly inhibit the self renewal and proliferation of liver cancer stem cells (1) Protein level detection showed that after knockout MEK1 in liver cancer stem cells, the protein expression level of MEK1 and phosphorylated ERK1/2 was significantly reduced, but the expression level of ERK1/2 was not significantly different. (2) the proliferation ability was obviously suppressed after two different MEK1 interfering lentivirus knockout MEK1 in liver cancer stem cells. (3) in liver cancer stem cells Knockout MEK1 significantly inhibited the formation of ball and clonogenic ability of liver cancer stem cells. (4) immunoblotting experiments showed that the expression of dry related proteins after knockout MEK1 in liver cancer stem cells such as Nanog, OCT4, c-Myc and SOX2 were significantly inhibited by.4. inhibition or knockout MEK1 can significantly reduce the expression of SIRT1 (1) through detection we found sirtuin home. The expression level of only SIRT1 and SIRT7 in HCC stem cells was higher than that of non hepatoma stem cells. However, only SIRT1 expression level was reduced after U0126 inhibition of MEK1 activity. (2) the downregulation of U0126 to the level of SIRT1 expression in liver cancer stem cells is not only positively related to the time of drug action. There is also a positive correlation with drug concentration. (3) another MEK1 inhibitor PD98059 also confirms that the inhibition of MEK1 activity in liver cancer stem cells can effectively reduce the expression of SIRT1 protein. (4) after the knockout of MEK1 in the liver cancer stem cells, the SIRT1 protein expression level is effectively reduced by the.5. MEK1 signaling pathway by regulating the histone deacetylase SIRT1 Promoting self renewal and tumorigenesis of liver cancer stem cells (1) can significantly increase their cloning and formation after overexpressing SIRT1 in non hepatoma stem cells, but it can effectively reverse the activation effect by inhibiting the activity of MEK1 signaling pathway. (2) cloning of this group of cells after the inhibition of MEK1 activity in liver cancer stem cells The ability to form and form the ball was significantly suppressed, and then their cloning and forming ability could be restored through the expression of SIRT1 protein. (3) in the tumor formation model of the body, the NOD-SCID mice injected with MEK1 active liver cancer stem cells were much less capable of tumorigenesis than the positive control group. Overexpression of SIRT1 level can partially restore the tumor formation ability of liver cancer stem cells in NOD-SCID mice. The.6. MEK1 signaling pathway inhibits the degradation of proteasome by inhibiting the ubiquitination level of SIRT1, thus maintaining the expression level of SIRT1 protein (1) we divide the liver cancer stem cells into four groups: a group of negative controls Two methyl sulfoxide co culture, a group of positive control and proteasome inhibitor MG-132 co culture, a group of MEK1 inhibitor U0126 co culture, a group of MEK1 knockout liver cancer stem cells. These four groups of cell culture 48 hours after the addition of actinomycone (CHX) treatment at the same time, the final detection of SIRT1 protein levels: relative to negative pairs After MEK1 inhibition or knockout, the degradation rate of SIRT1 protein in liver cancer stem cells accelerated significantly, and the degradation rate of SIRT1 protein was significantly slowed down after the inhibition of proteasome. (2) proteasome activity could be activated to effectively promote the degradation of SIRT1 protein in the stem cells of liver cancer. (3) immunoprecipitation experiment (Co) -IP) the results showed that after the suppression or knockout of MEK1 in the liver cancer stem cells, the ubiquitination of SIRT1 was greatly improved, resulting in the degradation of SIRT1 protein in the liver cancer patients, the high expression of MEK1/SIRT1 was positively correlated with the poor prognosis (1) we used immunohistochemistry in the tissue specimens of 148 patients with liver cancer. (Immunohistochemistry, IHC) detected the expression level of p-MEK1, SIRT1 and Nanog protein. At the same time, there were 48 patients with high level of p-MEK1/SIRT1 and 39 patients with low-level expression of p-MEK1/SIRT1. The statistical analysis of Person chi-square test (Person chi-square test) found that P was 0.035, that is to say, MEK1 and in the patients with liver cancer. The expression level of SIRT1 is related. The same method also confirms that the expression level of Nanog is associated with the expression of MEK1/SIRT1 in the patients with liver cancer, of which two P values are 0.013 and 0.038. (2). We will return the clinicopathological information of the 148 patients with liver cancer and find that the co expression of MEK1/SIRT1 is high and low. Status was not associated with age, sex, serum alpha fetoprotein (AFP) level, tumor interstitial hyperplasia, tumor necrosis and tumor recurrence, but the level of MEK1/SIRT1 simultaneous expression was associated with tumor size (p=0.012), vascular tumor thrombus (p0.001), membrane infiltration (p=p=0.048) and clinical tumor staging (p0.001) in patients with liver cancer. There was a significant statistical relationship. (3) we found that there was a significant association between the MEK1 and SIRT1 expression levels associated with the patient's survival cycle and prognosis, and the high level of MEK1 and SIRT1 patients with liver cancer tended to have worse prognosis than those with lower levels. All in all, our results showed that 1. in patients with liver cancer, MEK1 The expression level is significantly related to the level of SIRT1 expression, while patients with high level of MEK1/SIRT1 expression of liver cancer suggest that poor prognosis and shorter survival time.2. activation MEK1 signaling pathway can promote the proliferation of liver cancer stem cells, self renewal and tumor formation characteristics, knockout or inhibition of MEK1 can significantly reduce the expression level of.3. MEK1 letters of dry markers. The signal transduction pathway regulates the proliferation of liver cancer stem cells through the expression of SIRT1 protein. Tumor formation and self-renewal.4. MEK1 control the proteasome degradation through ubiquitination of SIRT1 to maintain the expression level of SIRT1 protein,.5. can be used as an indicator to determine the condition of liver cancer patients, and MEK1 signaling pathway can be used as a treatment for liver cancer. In summary, this study demonstrates that inhibition or interference with MEK1 can effectively slow down both in vivo and in vitro
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7
本文编号：2160204
[Abstract]:Hepatocellular carcinoma (HCC) is the third highest death rate in China. Although we have now established surgical excision to treat early liver cancer as a first-line treatment and assist patients with other treatment methods, patients with liver cancer are not only suffering from the resistance of liver cancer, but also in the same way. The high risk of postoperative recurrence ends up mostly in poor prognosis and short life. However, according to the latest research, the high recurrence rate of cancer is blamed for a group of cancer stem cells, CSCs, because the cells do not only have the ability to start the tumor and similar to the embryo. The self renewal characteristics of stem cells also have high differentiation potential that make them play an important role in the tolerance of cancer to radiotherapy. Previous studies have shown that cancer stem cells are closely related to tumor formation, metastasis, drug resistance, and postoperative recurrence, and more and more research on cancer stem cells is accompanied by more and more research. On the one hand, it can deepen our research and understanding of malignant tumors, and on the other hand, it provides new hope for us to overcome cancer. Our previous research group has shown that the transcription factor Nanog is a dry indicator of liver cancer stem cells, which can be used in vivo or in vitro. IGF-1 signaling pathway is used to regulate the self-renewal capacity of liver cancer stem cells. We also found that this group of Nanog positive liver cancer stem cells is an important cause of poor prognosis in the patients with liver cancer. They not only maintain the self renewal of liver cancer, but also play an important role in the formation and differentiation of liver cancer. However, we can not explain the fact. A detailed mechanism for the role of liver cancer stem cells in the development and recurrence of liver cancer. The deacetylase sirtuin family is a group of enzymes highly dependent on nicotinamide adenine dinucleotide (NAD+). The family contains seven members of the SIRT1-7, which has been studied to show that they are involved in the regulation of various cellular biological functions, such as cell cycle. Cell metabolism, cell proliferation, cell differentiation, genomic stability and tumor formation, and SIRT1 also proved to play a key role in the maintenance and development of liver cancer. Although it is found that the expression of microRNA (miRNA) -34a, miRNA-29c and c-MYC can effectively regulate the function of the oncogene SIRT1 in liver cancer, but how SIRT1 passes through it Liver cancer stem cells play a role in liver cancer. This specific regulatory mechanism remains unknown that.MEK1 and MEK2 belong to the members of the mitogen activated protein kinase kinase (MAPKKs) family. This kinase can specifically phosphorylate threonine and tyrosine residues and then activate the abnormality of the matrix.MEK1 of the mitogen activated protein kinase (MAPK) matrix. In addition, the MEK1 signaling pathway is often considered to be an important factor in the maintenance and development of liver cancer. However, the specific mechanism of how the MEK1 signaling pathway regulates the self-renewal of liver cancer stem cells remains to be found in the MEK1 signaling pathway. Further exploration. Our present study reveals the important mechanism of a MEK1 inactivation in vivo and in vitro, which can accelerate its protein degradation by promoting SIRT1 ubiquitination, and then inhibit the ability of liver cancer stem cell tumor formation. In order to achieve this goal, we divide the subject into four steps. First, we The MEK1 signaling pathway plays an important role in the self renewal and proliferation of liver cancer stem cells. Then, we also found that the expression level of MEK1 in liver cancer patients is positively related to SIRT1, and our inhibition or knockout of MEK1 in liver cancer stem cells will lead to the obvious downregulation of SIRT1; then we also elucidate MEK1 to SIRT1. The regulation is achieved by maintaining protein level degradation, and it is also proved that the MEK1 signaling pathway regulates the self renewal of liver cancer stem cells by maintaining the SIRT1 protein level. Finally, we also surprised to find that the co expression level of MEK1 and SIRT1 proteins is closely related to the patient's preconditioning. The main results of this article are as follows The conclusions are as follows: 1. inhibition of MEK1 activity in liver cancer stem cells can effectively slow down its cell proliferation ability (1) we spread the Huh7-Nanogpos and PLC/PRF/5-Nanogpos liver cancer stem cells in 96 orifice with the density of 1 x 103 per pore, and carry on the continuous treatment for 6 days with different concentrations of U0126, and then carry out CCk-8 detection. We found U0126 It can significantly inhibit the proliferation of liver cancer stem cells, and this inhibitory effect shows a concentration dependent pattern. At the same time, the cell survival curve excludes the toxic effects of these inhibitory concentrations on tumor stem cells. (2) we put Huh7-Nanogpos and PLC/PRF/5-Nanogpos liver cancer stem cells with U0126 or two methyl sulfoxide (DMS). O) after 48 hours co culture, the immunofluorescence (Immunofluorescence, IF) experiment showed that the Ki-67 expression level of liver cancer stem cells decreased significantly compared with the control group after U0126 treatment. (3) Huh7- and PLC/PRF/5-Nanogpos liver cancer stem cells treated with sodium butyrate were treated with U0126 or two methyl sulfoxide (negative control), and the cell cycle was true. It was found that U0126 treatment induced a large number of liver cancer stem cells to stagnate the cell cycle in the G0/G1 phase, and also accompanied by the significant reduction of.2. MEK1 inhibitor U0126 in S and G2/M phase cells (1) the self-renewal capacity of liver cancer stem cells was significantly reduced (1) we found that after the inhibition of MEK1 activity in the stem cells of the liver cancer, the clones could be significantly reduced. The formation and formation of ball forming ability, and this conclusion has been repeated in two cell lines. Contrary to liver cancer stem cells, low concentration of U0126 treatment does not reduce the cloning and formation of non hepatoma stem cells. (2) immunoblotting experiments showed that the inhibition of MEK1 activity in liver cancer stem cells could significantly inhibit the dry index. Such as the expression of SOX2, OCT4 and Nanog. (3) when U0126 treated liver cancer stem cells were injected with different concentrations of 1 x 102,1 x 103 and 1 x 104 subcutaneous injected into NOD-SCID mice, it was found that the tumor formation and growth ability of the cancer stem cells compared with the control group was significantly inhibited by.3. knockout MEK1 can significantly inhibit the self renewal and proliferation of liver cancer stem cells (1) Protein level detection showed that after knockout MEK1 in liver cancer stem cells, the protein expression level of MEK1 and phosphorylated ERK1/2 was significantly reduced, but the expression level of ERK1/2 was not significantly different. (2) the proliferation ability was obviously suppressed after two different MEK1 interfering lentivirus knockout MEK1 in liver cancer stem cells. (3) in liver cancer stem cells Knockout MEK1 significantly inhibited the formation of ball and clonogenic ability of liver cancer stem cells. (4) immunoblotting experiments showed that the expression of dry related proteins after knockout MEK1 in liver cancer stem cells such as Nanog, OCT4, c-Myc and SOX2 were significantly inhibited by.4. inhibition or knockout MEK1 can significantly reduce the expression of SIRT1 (1) through detection we found sirtuin home. The expression level of only SIRT1 and SIRT7 in HCC stem cells was higher than that of non hepatoma stem cells. However, only SIRT1 expression level was reduced after U0126 inhibition of MEK1 activity. (2) the downregulation of U0126 to the level of SIRT1 expression in liver cancer stem cells is not only positively related to the time of drug action. There is also a positive correlation with drug concentration. (3) another MEK1 inhibitor PD98059 also confirms that the inhibition of MEK1 activity in liver cancer stem cells can effectively reduce the expression of SIRT1 protein. (4) after the knockout of MEK1 in the liver cancer stem cells, the SIRT1 protein expression level is effectively reduced by the.5. MEK1 signaling pathway by regulating the histone deacetylase SIRT1 Promoting self renewal and tumorigenesis of liver cancer stem cells (1) can significantly increase their cloning and formation after overexpressing SIRT1 in non hepatoma stem cells, but it can effectively reverse the activation effect by inhibiting the activity of MEK1 signaling pathway. (2) cloning of this group of cells after the inhibition of MEK1 activity in liver cancer stem cells The ability to form and form the ball was significantly suppressed, and then their cloning and forming ability could be restored through the expression of SIRT1 protein. (3) in the tumor formation model of the body, the NOD-SCID mice injected with MEK1 active liver cancer stem cells were much less capable of tumorigenesis than the positive control group. Overexpression of SIRT1 level can partially restore the tumor formation ability of liver cancer stem cells in NOD-SCID mice. The.6. MEK1 signaling pathway inhibits the degradation of proteasome by inhibiting the ubiquitination level of SIRT1, thus maintaining the expression level of SIRT1 protein (1) we divide the liver cancer stem cells into four groups: a group of negative controls Two methyl sulfoxide co culture, a group of positive control and proteasome inhibitor MG-132 co culture, a group of MEK1 inhibitor U0126 co culture, a group of MEK1 knockout liver cancer stem cells. These four groups of cell culture 48 hours after the addition of actinomycone (CHX) treatment at the same time, the final detection of SIRT1 protein levels: relative to negative pairs After MEK1 inhibition or knockout, the degradation rate of SIRT1 protein in liver cancer stem cells accelerated significantly, and the degradation rate of SIRT1 protein was significantly slowed down after the inhibition of proteasome. (2) proteasome activity could be activated to effectively promote the degradation of SIRT1 protein in the stem cells of liver cancer. (3) immunoprecipitation experiment (Co) -IP) the results showed that after the suppression or knockout of MEK1 in the liver cancer stem cells, the ubiquitination of SIRT1 was greatly improved, resulting in the degradation of SIRT1 protein in the liver cancer patients, the high expression of MEK1/SIRT1 was positively correlated with the poor prognosis (1) we used immunohistochemistry in the tissue specimens of 148 patients with liver cancer. (Immunohistochemistry, IHC) detected the expression level of p-MEK1, SIRT1 and Nanog protein. At the same time, there were 48 patients with high level of p-MEK1/SIRT1 and 39 patients with low-level expression of p-MEK1/SIRT1. The statistical analysis of Person chi-square test (Person chi-square test) found that P was 0.035, that is to say, MEK1 and in the patients with liver cancer. The expression level of SIRT1 is related. The same method also confirms that the expression level of Nanog is associated with the expression of MEK1/SIRT1 in the patients with liver cancer, of which two P values are 0.013 and 0.038. (2). We will return the clinicopathological information of the 148 patients with liver cancer and find that the co expression of MEK1/SIRT1 is high and low. Status was not associated with age, sex, serum alpha fetoprotein (AFP) level, tumor interstitial hyperplasia, tumor necrosis and tumor recurrence, but the level of MEK1/SIRT1 simultaneous expression was associated with tumor size (p=0.012), vascular tumor thrombus (p0.001), membrane infiltration (p=p=0.048) and clinical tumor staging (p0.001) in patients with liver cancer. There was a significant statistical relationship. (3) we found that there was a significant association between the MEK1 and SIRT1 expression levels associated with the patient's survival cycle and prognosis, and the high level of MEK1 and SIRT1 patients with liver cancer tended to have worse prognosis than those with lower levels. All in all, our results showed that 1. in patients with liver cancer, MEK1 The expression level is significantly related to the level of SIRT1 expression, while patients with high level of MEK1/SIRT1 expression of liver cancer suggest that poor prognosis and shorter survival time.2. activation MEK1 signaling pathway can promote the proliferation of liver cancer stem cells, self renewal and tumor formation characteristics, knockout or inhibition of MEK1 can significantly reduce the expression level of.3. MEK1 letters of dry markers. The signal transduction pathway regulates the proliferation of liver cancer stem cells through the expression of SIRT1 protein. Tumor formation and self-renewal.4. MEK1 control the proteasome degradation through ubiquitination of SIRT1 to maintain the expression level of SIRT1 protein,.5. can be used as an indicator to determine the condition of liver cancer patients, and MEK1 signaling pathway can be used as a treatment for liver cancer. In summary, this study demonstrates that inhibition or interference with MEK1 can effectively slow down both in vivo and in vitro
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7
【参考文献】
相关期刊论文 前3条
1 Kerstin Schütte;Christian Schulz;Alexander Link;Peter Malfertheiner;;Current biomarkers for hepatocellular carcinoma: Surveillance, diagnosis and prediction of prognosis[J];World Journal of Hepatology;2015年02期
2 李扩;许秋然;刘欣;刘青光;王茂德;;miR-204通过下调Bcl-2和Sirt1表达抑制肝癌细胞生长[J];细胞与分子免疫学杂志;2015年02期
3 Wanqing Chen;Rongshou Zheng;Siwei Zhang;Ping Zhao;Hongmei Zeng;Xiaonong Zou;Jie He;;Annual report on status of cancer in China,2010[J];Chinese Journal of Cancer Research;2014年01期
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