CYLD通过调节自噬逆转膀胱癌化疗耐药性的机制研究

发布时间：2018-06-16 07:27

本文选题：RT112-GR细胞株 + RT112细胞株　；参考：《山东大学》2016年博士论文

【摘要】：第一部分细胞自噬对膀胱癌化疗耐药性的影响研究背景膀胱癌是泌尿系统最常见的恶性肿瘤,在我国其发病率位于泌尿系统肿瘤的首位。大约25%的膀胱癌初发患者是肌层浸润性膀胱癌,尽管另外75%的膀胱癌患者为非肌层浸润性膀胱癌,但大部分也会出现复发或侵犯肌层。因此,早期手术,术后常规进行膀胱灌注化疗是治疗膀胱癌的关键,这样可以有效降低膀胱癌术后的复发率,改善患者的预后并提高其生存率。但是仍有2/3的患者会出现复发,并且复发患者中15%～20%会进展为更高的病理分期和/或伴有分级升高。大部分膀胱癌相关死亡是因为肌层浸润性膀胱癌导致的局部浸润或远处转移。较高的死亡率亟待泌尿外科医师寻找新的更有效的治疗方案来治疗膀胱癌。近年来,尽管吉西他滨(Gemcitabine)和顺铂(Cisplatin)的新型GC联合方案作为一线晚期膀胱癌的化疗措施疗效显著,但耐药的发生大大的限制了化疗药物的远期疗效。吉西他滨广泛应用于胰腺癌、卵巢癌、乳腺癌、膀胱癌、非小细胞肺癌等肿瘤的化疗。然而,越来越多的泌尿外科医师意识到了吉西他滨耐药带来的危害。尽管膀胱癌患者最初的化疗效果显著,但60～70%有效的患者5年内出现复发,平均生存期仅有12～14月,这要归因于在治疗过程中出现的耐药现象。许多研究表明,化疗药物能诱导肿瘤细胞的凋亡而发挥治疗作用,但是化疗药物也能诱导肿瘤细胞产生自噬,这种条件下诱导的自噬往作为一种保护性机制来抑制细胞的凋亡,而这种保护性机制又可以使肿瘤细胞对抗各种化疗药物治疗,从而使肿瘤细胞具有耐药性,不利于肿瘤的治疗。已有研究表明,肿瘤细胞在接受化疗后,再利用RNAi技术干扰自噬相关基因的表达可以增加其自噬死亡。另外,有学者把3-甲基腺嘌呤、氯喹等一些自噬抑制剂与抗肿瘤化疗药物联用,发现在自噬抑制剂与化疗药物对肿瘤细胞凋亡具有协同作用。所以,我们假想在肿瘤化疗过程中,我们通过抑制肿瘤细胞化疗中由自噬引起的耐药性,那么可能会激活肿瘤相关的凋亡活性,可能会增加细胞凋亡过程,从而增加化疗药物的效应。如果能通过某种方式抑制由肿瘤细胞自噬引起的化疗耐药性,则可以启动凋亡相关信号通路,通过促进肿瘤细胞的凋亡而提高疗效。目的探讨细胞自噬(cell autophagy)对膀胱癌吉西他滨化疗耐药性的影响。方法吉西他滨浓度梯度加药进行反复筛选、构建膀胱癌RT112吉西他滨耐药株(RT112-GR),并且通过MTT细胞毒性实验检测细胞的耐药性。利用RT-PCR及Western boltting等方法研究RT112及RT112-GR两组细胞株中自噬标记蛋白(LC-3)的mRNA水平及蛋白水平的表达差异；利用电镜检测RT112及RT112-GR两组细胞株中自噬小体水平的差异。利用MTT细胞活力实验及CCK8细胞毒性实验检测氯喹(CQ)抑制RT112-GR自噬水平后,RT112-GR细胞株对吉西他滨耐药性的变化。结果MTT实验结果显示,RT112-GR细胞株的吉西他滨半数抑制浓度(IC50)为4.2 umol/L,是其亲代细胞RT1 12的350倍(RT112 IC50值为12 nmol/L),并且对吉西他滨耐药的RT112-GR细胞株对阿霉素及顺铂并无交叉耐药性。当加用自噬抑制剂CQ处理耐药细胞株后,MTT实验结果显示其IC50值为10.9 nmol/L,与普通膀胱癌细胞比较,统计学无显著性差异(NS)。45 nM的吉西他滨处理RT112和RT112-GR两组细胞株,RT-PCR结果显示RT112-GR组细胞株的LC-3的mRNA表达水平明显高于普通RT112细胞组(P=0.023)。Western boltting结果显示膀胱癌耐药细胞株LC3的蛋白表达明显高于普通RT112细胞组(P0.05)。电镜照片中可见,45 nM吉西他滨处理两组细胞株后,RT112-GR胞浆中的自噬小体明显增多(P0.05)。MTT细胞毒性实验检测,加入CQ后吉西他滨对耐药组的毒性恢复。结论本研究中,我们成功构建了膀胱癌吉西他滨耐药株(RT112-GR)。等同治疗量45 nM的吉西他滨处理RT112和RT112-GR两组细胞株后,RT112-GR的自噬水平明显高于亲代RT112细胞,表明保护性自噬的产生,而当加用自噬抑制剂CQ处理上述吉西他滨耐药细胞后,自噬水平降低,且耐药性缺失。第二部分圆柱瘤基因转染细胞构建及相关功能学实验研究背景肿瘤抑制因子圆柱瘤基因(Cylindromatosis, CYLD)编码一种去泛素化酶,CYLD基因突变或者缺失导致圆柱瘤,并与多种肿瘤的发生、发展密切关联。CYLD通过自身去泛素化酶活性移除特定底物的K63连接的泛素链,并调控包括NF-κB、JNK及AKT等在内的多条信号通路。CYLD在肿瘤中与自噬的关系,目前国内外尚无研究。有研究提出,成年大鼠脑后突触中,CYLD可能通过影响lysine48及lysine6的关联泛素化,影响细胞自噬,而自噬已经明确与多种肿瘤化疗耐药性有关,CYLD是否通过调节自噬影响膀胱癌生长、凋亡及膀胱癌化疗敏感性的能力亟待我们研究进一步明确。我们前期的研究结果表明：通过腺病毒感染的方法降低大鼠血管平滑肌细胞内CYLD表达水平可以提高NF-κB、JNK及Akt传导通路活化水平,以上三条通路的激活正是自噬激活的关键信号通路。李丹丹等的研究证实,化疗药物正是通过激活JNK,磷酸化下游底物c-Jun,上调Beclinl基因的表达,介导肿瘤细胞Hep3B、CNE2发生自噬性死亡。而高俊玲等人的研究则指出：阻断JNK的传导通路可以有效的抑制脑创伤后神经元的自噬,具有细胞保护作用。Wang W等人的研究证实对NF-k,B的调节可以影响自噬对细胞的保护作用。除此,我们最近的研究结果表明,使用腺病毒感染的方法上调大鼠血管平滑肌细胞内CYLD表达水平后,可以显著降低自噬蛋白LC3的表达；而下调节大鼠血管平滑肌细胞内CYLD表达水平后,可以显著提高自噬蛋白LC3的表达,这说明CYLD表达水平与自噬密切相关。综上所述,以往研究已经证实自噬可以影响肿瘤生长、凋亡及化疗耐药性。而细胞自噬可能由CYLD调控,其调控机制很可能与NF-κB、JN、Akt传导通路的活化水平相关。综合我们前期的研究结果,我们提出研究假设：CYLD在膀胱癌细胞中表达上调,影响自噬关键信号转导通路NF-κB、JNK、Akt传导通路的活化水平下调,抑制自噬,从而诱导自噬性细胞死亡或者凋亡,逆转膀胱癌化疗敏感性。目的探讨圆柱瘤基因(CYLD)与膀胱癌自噬的关系。方法利用免疫组化技术检测,收集我院病例库中的膀胱癌患者55例,手术切取患者的癌组织,以10例良性膀胱组织作为对照,构建组织芯片。应用免疫组化染色法对芯片组织进行CYLD免疫组化染色,同时应用免疫组化染色法对芯片组织进行LC-3染色。应用Pearson相关性分析研究LC-3与CYLD表达之间是否存在相关性；构建敲除CYLD的膀胱癌细胞,利用Western Blot和RT-PCR技术检测实验组(敲除CYLD, Ad-Cyld shRNA沉默腺病毒)、CYLD过表达组(Ad-Cyld上调腺病毒)、空病毒转染组及空白对照组的CYLD表达；利用Western Blot和RT-PCR技术检测敲除CYLD的膀胱癌细胞、过表达组、空病毒转染组及普通细胞的LC-3表达。结果免疫组化染色结果提示,与对照组(正常膀胱组织)相比较,膀胱癌组织中CYLD的表达明显降低(P0.05),差异有统计学意义。LC-3在膀胱癌组织中的表达与CYLD的表达呈负相关,Pearson相关系数为-0.511(P0.05)。实时定量PCR的研究结果表明：与对照组细胞(RTl12)和空载病毒转染组相比,实验组细胞(敲除CYLD的RT112)内的LC-3的基因水平表达升高,而CYLD的表达显著下调,并且差异具有统计学意义(P0.05)。结论敲除CYLD的普通膀胱癌细胞构建成功；CYLD在膀胱癌中的作用可能与自噬相关。第三部分圆柱瘤对细胞自噬的调节与膀胱癌化疗耐药性的关系研究背景CYLD是一种公认的抑癌基因,当CYLD基因突变或缺失,可能影响肿瘤的发生及发展。微管相关轻链蛋白-3 (Microtubule associated protein light chain3,LC-3)是酵母细胞自噬相关基因ATG8的同源基因。LC-3在多种肿瘤疾病中呈现异常表达。有研究发现LC-3的表达量与肿瘤的浸润深度、淋巴结转移、淋巴结入侵及不良预后等呈负相关。与LC-3低表达相比,LC-3高表达的肿瘤患者整体生存期要好,而且LC-3蛋白是一个较好的自噬相关性的标记物。有研究表明,细胞自噬可能由CYLD调控,其调控机制很可能与NF-κB、JNK、Akt传导通路的活化水平相关。在膀胱癌化疗过程中,CYLD是否与LC-3存在一定的相互作用需要我们进一步的研究。CYLD对膀胱癌自噬的调节作用及其机制,对我们了解自噬调控的精确分子生物学机制,并藉此设计新颖、合理的针对性化疗方案,进一步提高我国膀胱癌的诊疗水平有重要意义。目的研究CYLD对自噬的调节与膀胱癌吉西他滨化疗耐药性之间的关系。方法利用实时定量PCR技术检测实验组(敲除CYLD:RT112-CYLD shRNA)、耐药组(RT112-GR)及普通细胞组(RT112)中CYLD的基因表达；CCK8检测实验组(敲除CYLD)与两组细胞IC50的差异；应用自噬抑制剂CQ作用于实验组(敲除CYLD)后检测IC50与前三组的差异；同等治疗量吉西他滨处理三组细胞后,利用实时定量PCR技术检测三组细胞的LC-3的mRNA表达及应用CCK8实验进行各组细胞毒性的检测,实验组加用CQ,同样利用RT-PCR技术对LC-3的mRNA水平进行检测及用CCK8实验对CQ的细胞毒性进行检测。结果实时定量PCR结果显示：膀胱癌耐药组的CYLD呈现类似敲除实验组的低表达,与普通细胞组比较差异具有统计学意义(P0.05)；敲除CYLD实验组LC-3表达呈现类似膀胱癌耐药组的高表达；敲除CYLD实验组呈现出类似耐药组的吉西他滨耐药性,耐药组的吉西他滨IC50为4.2μmol/L,敲除CYLD实验组IC50约为4.12μmol/L,无显著性差异,即IC50与耐药组比较差异无统计学意义(P0.05)；加入自噬抑制剂CQ后,耐药性反转即IC50降低与普通细胞组相比较差异无统计学意义(P0.05)。结论耐药细胞株中CYLD的低表达所导致的保护性自噬可能是其耐药性产生的原因,同时应用自噬抑制剂CQ作用于细胞后,耐药株恢复对吉西他滨的敏感性。
[Abstract]:Part 1 the effect of autophagy on the chemotherapeutic drug resistance of bladder cancer. Bladder cancer is the most common malignant tumor of the urinary system. In our country the incidence is at the top of the urological tumor. About 25% of the initial patients with bladder cancer are musculocutaneous invasive bladder cancer, although the other 75% of the bladder cancer patients are non muscularis infiltrating bladder. Cancer, but most of the recurrence or invasion of the muscle layer. Therefore, early surgery, postoperative routine bladder perfusion chemotherapy is the key to the treatment of bladder cancer, which can effectively reduce the recurrence rate after bladder cancer surgery, improve the prognosis of patients and improve their survival rate. However, there are still 2/3 patients with recurrence, and 15% to 20% of the recurrent patients. There will be a higher pathological stage and / or higher grade. Most of the bladder cancer related deaths are due to local infiltration or distant metastasis caused by myometrium infiltrating bladder cancer. Higher mortality is urgent for Department of Urology physicians to find new and more effective treatments for bladder cancer. In recent years, although gemcitabine (Gemcitabine) The combination of Cisplatin and cisplatin (cisplatin) (cisplatin), a new combination of GC, is effective as a chemotherapy for advanced bladder cancer, but the occurrence of drug resistance greatly restricts the long-term effect of chemotherapeutic drugs. Doctors are aware of the dangers of gemcitabine resistance. Although the initial chemotherapy effect of bladder cancer patients is significant, 60 to 70% effective patients have a recurrence within 5 years, with an average survival of only 12~14 months. This is due to the phenomenon of drug resistance in the course of treatment. Many studies have shown that chemotherapeutic drugs can induce apoptosis of cancer cells. However, chemotherapy can also induce tumor cells to produce autophagy, which is induced by autophagy as a protective mechanism to inhibit cell apoptosis, and this protective mechanism can also make tumor cells against various chemotherapeutic drugs, thus making the tumor cells resistant to cancer and is not conducive to the treatment of tumors. Therapy. It has been shown that tumor cells can increase autophagy death by using RNAi technology to interfere with autophagy related genes after chemotherapy. In addition, some autophagic inhibitors, such as 3- methyl adenine, chloroquine, and other antitumor drugs, have been combined with anti tumor chemotherapeutic agents. Therefore, we assume that in tumor chemotherapy, we can activate tumor related apoptosis by inhibiting the resistance to autophagy in tumor cell chemotherapy, which may increase the process of apoptosis and increase the effect of chemotherapeutic drugs. For example, the inhibition of autophagy by tumor cells in some way can be achieved. In order to improve the effect of cell autophagy on the chemotherapeutic resistance of gemcitabine in bladder cancer, the effect of cell autophagy (cell autophagy) on the drug resistance of gemcitabine in bladder cancer. 112-GR) and detection of cell resistance by MTT cytotoxicity test. RT-PCR and Western boltting were used to study the difference in the level of mRNA and protein expression of autophagic marker protein (LC-3) in RT112 and RT112-GR two groups, and the difference in the level of autophagic corpuscles in the cell lines of RT112 and RT112-GR two groups was detected by electron microscopy. MTT cell viability test and CCK8 cytotoxicity test were used to detect the change of RT112-GR cell line resistance to gemcitabine after chloroquine (CQ) inhibition of RT112-GR autophagy. Results MTT experimental results showed that the median inhibitory concentration (IC50) of gemcitabine in RT112-GR cell line was 4.2 umol/L, which was 350 times of its parent cell RT1 12 (RT112 IC50 value was 12). Ol/L), and there was no cross resistance to doxorubicin and cisplatin resistant RT112-GR cells resistant to gemcitabine. When the drug resistant cell line was treated with autophagy inhibitor CQ, the MTT test showed that the IC50 value was 10.9 nmol/L, compared with common bladder cancer cells, there was no statistically significant difference (NS).45 nM for RT112 and RT112-GR. Two groups of cell lines, RT-PCR results showed that the mRNA expression level of LC-3 in RT112-GR group was significantly higher than that of normal RT112 cell group (P=0.023).Western boltting results showed that the expression of LC3 in the drug resistant cell line of the bladder cancer cell line was significantly higher than that of the common RT112 cell group (P0.05). The 45 nM gemcitabine treated two groups of cell lines. The autophagic corpuscle in 12-GR cytoplasm increased significantly (P0.05).MTT cytotoxicity test, and the toxicity of gemcitabine was restored to the drug resistant group after CQ. Conclusion we successfully constructed the cystine resistant gemcitabine resistant strain (RT112-GR). The equivalent treatment amount of 45 nM of gemcitabine treatment RT112 and RT112-GR two cell lines, RT112-GR self The level of phagocytosis was significantly higher than that of the parent RT112 cells, indicating the production of protective autophagy, and the decrease of autophagy and the lack of drug resistance when adding the autophagic inhibitor CQ to the above - mentioned gemcitabine resistant cells. Second part of the gene transfection cell construction and related functional study of the tumor suppressor columnoma gene (Cylindr Omatosis, CYLD) encodes a ubiquitin, CYLD gene mutation or deletion that leads to a cylindric tumor, and is closely associated with the occurrence of a variety of tumors, closely related to the ubiquitin chain of K63 connected by.CYLD through its own ubiquitonasase activity to remove specific substrates, and to regulate multiple signaling pathways, including NF- kappa B, JNK, and AKT, in the tumor and in the tumor. There has been no study at home and abroad. It is suggested that CYLD may affect the autophagy of lysine48 and lysine6 in the postsynaptic synapses of adult rats, and autophagy has been clearly related to the chemotherapeutic resistance of various tumors. Whether CYLD affects the growth of bladder cancer, apoptosis and chemotherapy sensitivity of bladder cancer by regulating autophagy. The perceptual ability needs to be further studied. Our previous study showed that the reduction of CYLD expression level in rat vascular smooth muscle cells by adenovirus infection could improve the activation level of NF- kappa B, JNK and Akt conduction pathway. The activation of the above three pathways is the key signal pathway for autophagy activation. The study confirmed that the chemotherapeutic drugs are activating JNK, phosphorylating the downstream substrate c-Jun, up regulating the expression of Beclinl gene, mediating the tumor cell Hep3B, and the autophagic death of CNE2, while Gao Junling et al. Research points out that blocking JNK conduction pathway can effectively inhibit autophagy of neurons after traumatic brain injury and have cellular protective effect.Wan G W and others have confirmed that the regulation of NF-k, B can affect the protective effect of autophagy on cells. In addition to this, our recent study shows that the expression of CYLD can be significantly reduced after the use of adenovirus infection to increase the expression of CYLD in the vascular smooth muscle cells of rats; and the expression of the autophagic protein LC3 can be significantly reduced. The expression level of CYLD can significantly increase the expression of autophagin LC3, which indicates that the level of CYLD expression is closely related to autophagy. To sum up, previous studies have confirmed that autophagy may affect tumor growth, apoptosis and chemotherapeutic resistance. The autophagy may be regulated by CYLD, and its modulation mechanism is likely to be activated by the NF- kappa B, JN, Akt conduction pathway In our previous study, we put forward the hypothesis that the expression of CYLD in bladder cancer cells was up-regulated, and that the activation level of NF- kappa B, JNK, Akt pathway was down regulated, and autophagy inhibited, thus inducing autophagic cell death or apoptosis and reversing chemosensitivity of bladder cancer. The relationship between the CYLD and the autophagy of bladder cancer. Methods 55 patients with bladder cancer in our hospital case library were collected by immunohistochemical technique. The tumor tissues were removed by operation and 10 cases of benign bladder tissue were used as control to construct the tissue microarray. Immunohistochemical staining was used to stain the tissue of the microarray. LC-3 staining was applied to the microarray tissue by immunohistochemical staining. The correlation between LC-3 and CYLD expression was studied by Pearson correlation analysis; the bladder cancer cells that knocked out CYLD were constructed, and Western Blot and RT-PCR technology were used to detect the experimental group (knockout CYLD, Ad-Cyld shRNA silent adenovirus). CYLD expression of adenovirus), empty virus transfection group and blank control group; Western Blot and RT-PCR technique were used to detect bladder cancer cells that knocked out CYLD, overexpressed group, empty virus transfection group and common cell LC-3 expression. Results immunohistochemical staining results showed that the CYLD table in bladder cancer tissue was compared with the control group (normal bladder tissue). The difference was significantly decreased (P0.05). The difference was statistically significant in the expression of.LC-3 in the bladder cancer tissue and the expression of CYLD, and the Pearson correlation coefficient was -0.511 (P0.05). The results of real-time quantitative PCR showed that the LC-3 gene water in the experimental group (RTl12) and the unloaded virus transfected group was in the experimental group (the CYLD RT112). The expression of CYLD increased significantly, while the expression of CYLD was significantly down, and the difference was statistically significant (P0.05). Conclusion the common bladder cancer cells that knocked out CYLD were successfully constructed, and the role of CYLD in bladder cancer may be related to autophagy. The recognized tumor suppressor gene, when the mutation or deletion of CYLD gene, may affect the occurrence and development of tumor. Microtubule related light chain protein -3 (Microtubule associated protein light chain3, LC-3) is the homologous gene.LC-3 of the yeast cell autophagy related gene ATG8, which presents abnormal expression in a variety of tumor diseases. The tumor invasion depth, lymph node metastasis, lymph node invasion and bad prognosis are negatively correlated. Compared with the low expression of LC-3, the overall survival time of the tumor patients with high expression of LC-3 is better, and the LC-3 protein is a better autophagy correlation marker. B, JNK, Akt conduction pathways are associated with activation levels. In the course of chemotherapy for bladder cancer, the interaction between CYLD and LC-3 requires us to further study the regulatory role of.CYLD on autophagy of bladder cancer and its mechanism, to understand the precise molecular mechanism of autophagy, and to design novel and reasonable targeting It is of great significance to further improve the level of diagnosis and treatment of bladder cancer in China. Objective to study the relationship between the regulation of autophagy and the drug resistance of gemcitabine in bladder cancer. Methods using real-time quantitative PCR technique to detect the gene of CYLD in the experimental group (knockout CYLD:RT112-CYLD shRNA), drug resistance group (RT112-GR) and common cell group (RT112). The difference between the experimental group (knockout CYLD) and the two groups of IC50 was detected by CCK8; the use of autophagy inhibitor CQ in the experimental group (knockout CYLD) was used to detect the difference between the IC50 and the first three groups. After the same treatment, gemcitabine was used to detect the mRNA expression of LC-3 in the three groups of cells by real-time quantitative PCR and the application of CCK8 experiment. The test group was tested for cytotoxicity in each group, the experimental group was added with CQ, and the mRNA level of LC-3 was detected by RT-PCR technique and the cytotoxicity of CQ was detected by CCK8 test. The results of real-time quantitative PCR showed that the CYLD of the drug resistance group of bladder cancer was similar to that of the experimental group, and the difference between the group and the common cell group was statistically significant. The LC-3 expression in the CYLD test group was similar to the high expression of the drug resistance group in the bladder cancer group; the knockout CYLD experimental group showed a similar drug resistance in the drug resistant group, and the gemcitabine IC50 was 4.2 u mol/L in the drug resistant group, and the IC50 in the knockout CYLD experimental group was about 4.12 u mol/L, and there was no significant difference between the IC50 and the drug resistant group, that is, there was no difference between the IC50 and the drug resistant group. Statistical significance (P0.05); after adding autophagy inhibitor CQ, there was no statistical difference in drug resistance reversal or IC50 reduction compared with normal cell group.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.14

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