【作者】 李晓燕；

【导师】 田永杰；

【作者基本信息】 山东大学 ， 妇产科学（专业学位）， 2018， 博士

【摘要】 卵巢癌(Ovarian cancer,OC)是女性生殖系统最常见恶性肿瘤之一,因其发病隐匿,早期病变不易发现,故而卵巢癌患者就诊时,多数已是临床晚期。肿瘤细胞减灭术并辅以紫杉醇、顺铂等为主的联合化疗方案是目前治疗卵巢癌的重要手段。而长期困扰卵巢癌化疗的最大的障碍是肿瘤细胞耐药性的产生,卵巢癌患者对铂类药物为基础的联合化疗的初次化疗反应率可达80%,但最终因化疗耐药等原因使得卵巢癌的五年生存率始终徘徊在30%左右。因此,研究卵巢癌耐药的发生机制及可能的逆转途径,有助于采取针对性的措施逆转卵巢癌的化疗耐药性,提高患者对化疗药物的敏感性,从而提高生存率,改善预后。40多年前Christian de Duve首次提出自噬(autophagy)的概念,指细胞在饥饿、缺氧等营养不足和外界压力等应激条件下,在复杂的通路和因子调控下,细胞内形成双层或多层自噬膜结构,包裹变形受损的细胞器、病原微生物、大分子物质等,然后运送至溶酶体并与之结合形成自噬溶酶体,进而对内容物进行消化、降解,相应的产生氨基酸、核苷酸、水等可以进行循环再利用,从而满足细胞自身的能量再利用以及细胞器的更新。自噬是真核生物中的一种保守的分解代谢过程,它通过溶酶体系统降解胞内受损变形及失去功能的蛋白质、细胞器,从而维持细胞内环境稳态及基因组的稳定性。正常情况下自噬以基础水平维持着细胞内环境的平衡,近年来,更多的研究发现自噬功能的异常参与了多种疾病的发生和发展,如糖尿病、免疫系统疾病、神经退行性变以及肿瘤的发生、侵袭等。近年来研究发现,多种化疗药物可以引起肿瘤细胞发生自噬。肿瘤细胞通过自噬清除化疗药物引起的损伤的细胞器以及错误折叠的蛋白质来保持自身的稳定性,进而逃避化疗药物引起的凋亡,促进肿瘤细胞生存,研究证实自噬可作为肿瘤细胞的一种保护机制,降低化疗药物的敏感性,导致肿瘤细胞耐药。多项研究表明,自噬参与了多种肿瘤的化疗耐药过程,应用自噬抑制剂可能成为改善化疗耐药、提高化疗敏感性的重要策略之一。化疗药物是否引起卵巢癌细胞自噬以及自噬在卵巢癌细胞化疗敏感性中的作用目前报道甚少。自噬是由一些特殊基因调控、多条信号通路传导的极复杂的过程,这些调控基因被称为自噬相关基因(ATG),目前已有30多种自噬相关基因被发现。Beclin1是目前研究比较成熟的自噬相关基因,它与ClassIIIP13K结合形成复合物,在自噬体的成核过程中发挥着重要作用,研究显示,Beclin1也与肿瘤的发生及耐药相关。Ambra1(Activating molecule in Beclinl-regulated autophagy1,Beclin1 自噬相关激活分子)是一种新型的ATG基因,作为Beclin1的上游因子,通过调控Beclin1-ClassIIlP13K-Vps15复合体,促进自噬体的形成。在线粒体中,Ambra1通过结合Bcl-2(B淋巴细胞瘤-2)调控Beclin1依赖的自噬和凋亡。所以Ambra1可以通过调控自噬和凋亡来决定肿瘤细胞的命运,但Ambra1在化疗药物处理的卵巢癌细胞中的可能作用尚未见报道。已有研究表明,在结肠癌细胞株SW620中,Ambra1可以结合Beclin1正性调控自噬并同时负性调控凋亡;Ambra1在顺铂处理的乳腺癌细胞中的表达上调并增加了乳腺癌细胞的耐药性。故而课题组推测Ambra1是卵巢癌细胞自噬和凋亡的重要调节器,拟利用顺铂处理卵巢癌OVCAR-3细胞后,通过评估微管相关蛋白1轻链3(LC3)的定位、自噬体的存在、LC3水平以及Ambra]、Beclin1蛋白的表达情况,证实顺铂处理的卵巢癌OVCAR-3细胞的自噬诱导以及发生自噬时Ambra1的表达,确定Ambra1在顺铂处理的卵巢癌细胞中的自噬促进作用;然后通过shRNA转染沉默Ambra1基因表达,重新评估Ambra1基因受到干扰抑制后对顺铂诱导的卵巢癌OVCAR-3细胞自噬的影响,进一步验证Ambra]在顺铂处理的卵巢癌细胞中的自噬促进作用;通过测定Ambra1基因敲除后,顺铂对卵巢癌OVCAR-3细胞生长、细胞活性和凋亡诱导的影响,明确Ambra1基因敲除对OVCAR-3细胞顺铂敏感性的影响,为寻找以Ambra1为靶点的改善卵巢癌化疗敏感性的治疗药物提供理论依据。第一部分:顺铂化疗对卵巢癌OVCAR-3细胞活性、细胞自噬和Ambra1表达影响的研究研究目的1、测定人卵巢癌上皮OVCAR-3细胞系对顺铂的敏感性。2、检测顺铂对卵巢癌OVCAR-3细胞自噬的诱导和自噬相关分子表达的调控。研究方法:1、利用MTT法检测不同浓度的顺铂处理卵巢癌OVCAR-3细胞后,细胞活性的改变和时间依赖性。2、利用顺铂处理卵巢癌OVCAR-3细胞,观察卵巢癌细胞自噬诱导、自噬体形成及自噬关键分子——LC3转换。3、以自噬诱导剂雷帕霉素作为阳性对照,观察顺铂处理卵巢癌OVCAR-3细胞后,自噬相关蛋白Ambra1及Beclin1表达的改变。结果:1、不同浓度的顺铂处理卵巢癌OVCAR-3细胞后,细胞活性明显降低利用0、10、50pM浓度的顺铂分别处理卵巢癌OVCAR-3细胞0、12、24、48小时后,用MTT法检测卵巢癌OVCAR-3细胞的细胞活性,结果显示:用10、50uM的顺铂分别处理卵巢癌OVCAR-3细胞24小时(p<0.005,p<0.01)、48小时(p<0.001,p<0.0001)后,OVCAR-3细胞活性均表现为下降,且下降的差异具有统计学意义,而且在10M、50uM浓度的顺铂处理细胞24(p<0.05)小时和48(p<0.01)小时后,这两个时间差之间的细胞活性均具有显著差异,且差异具有统计学意义。结果详见图1A和1B。2、顺铂诱导卵巢癌OVCAR-3细胞自噬形成用10uM的顺铂处理卵巢癌细胞24小时(实验组)后,与未用顺铂处理的卵巢癌细胞(对照组)相比,给予转染GFP-LC3融合蛋白质粒后,在荧光显微镜下观察到实验组卵巢癌细胞中的自噬囊泡数量明显多于对照组,结果详见图1C、1D,且差异具有统计学意义(p<0.001),结果详见图1E。3、顺铂对卵巢癌OVCAR-3细胞的自噬诱导伴有Ambra1和Beclin1表达升高与未用化疗药物处理的卵巢癌OVCAR-3细胞相比,用100nM的雷帕霉素作为自噬诱导剂处理卵巢癌OVCAR-3细胞后,雷帕霉素可以显著提高LC3-1蛋白转换为LC3-II蛋白的水平(p<0.001)。用10μM、50μM的顺铂处理卵巢癌OVCAR-3细胞24小时后,也可以显著提高LC3-I蛋白转换为LC3-II蛋白的水平(p<0.01、p<0.001),这种转化作用呈计量依赖性(p<0.001),化疗药物浓度越高,由LC3-I蛋白转换为LC3-II蛋白的水平越高,1OOnM的雷帕霉素的这种自噬诱导作用比1OμM、50μM顺铂的诱导作用更强烈,结果详见图2A和2B。与未用化疗药物处理的卵巢癌OVCAR-3细胞相比,用1OOnM的雷帕霉素作为自噬诱导剂处理OVCAR-3细胞后,可以显著提高自噬相关蛋白Ambra1和Beclin1蛋白的表达水平(p均<0.001)。用10μM、50μM的顺铂处理卵巢癌OVCAR-3细胞24小时后,也可以显著提高Ambra1和Beclin1蛋白的表达水平(p<0.05、p<0.01),且呈计量依赖性(p<0.05),Ambra1 和 Beclin1 蛋白在 50μM的顺铂诱导下的表达量比在10μM的顺铂诱导下的表达量要高,差异具有统计学意义(p均<0.05)。Ambra1和Beclin1蛋白在1OOnM的雷帕霉素诱导下的表达量比在50μM的顺铂诱导下的表达量要高,差异具有统计学意义(p均<0.0001)。结果见图2C和2D。结论1、卵巢癌OVCAR-3细胞的细胞活性在顺铂作用下降低,且细胞活性降低呈时间和浓度依赖性。2、顺铂可以诱导卵巢癌OVCAR-3细胞出现自噬。3、顺铂诱导卵巢癌OVCAR-3细胞出现自噬时,自噬相关蛋白Ambra1和Beclin1蛋白表达水平提高,提示Ambra1在顺铂处理的卵巢癌细胞中的自噬促进作用。第二部分:shRNA介导的Ambra1基因敲除对顺铂诱导的卵巢癌OVCAR-3细胞自噬的影响和Beclin1表达的研究研究目的:1、筛选Ambra1基因特异shRNA转染条件并可稳定表达的卵巢癌OVCAR-3细胞株。2、探讨Ambra1基因敲除(干扰抑制)对顺铂在卵巢癌OVCAR-3细胞中自噬诱导的影响。3、探讨Ambra1基因敲除对顺铂处理的卵巢癌OVCAR-3细胞中自噬相关蛋白表达的影响。研究方法:1、细胞转染Ambra1特异shRNA并通过RT-PCR实验测定Ambra1基因敲除后Ambra1 mRNA表达变化。2、利用自噬囊泡形成实验,检测干扰抑制Ambra1基因表达对卵巢癌OVCAR-3细胞自噬囊泡形成的影响。3、利用Western blotting实验,检测干扰抑制Ambra1基因表达对卵巢癌OVCAR-3细胞自噬相关蛋白Ambra1、LC3及Beclin1表达的影响。结果:1、卵巢癌OVCAR-3细胞的转染效率在荧光显微镜下观察到GFP(绿色荧光)即可视为转染成功,于转染后12小时、24小时、36小时、72小时分别进行荧光计数,对荧光细胞的数量进行比较,我们发现:随着时间的延长,GFP的表达在转染后72小时表达量最高,此时卵巢癌OVCAR-3细胞的转染效率达到了 90%以上,可以用来进行后续实验。根据是否转染shRNA-Ambra1,将卵巢癌OVCAR-3细胞分为两组,卵巢癌OVCAR-3(Ambra1-)组和 OVCAR-3(Ctrl)组。2、RT-PCR检测两组细胞目的基因Ambra1 mRNA的表达OVCAR-3(Ambra1-)组的 Ambra1 mRNA 表达水平较 OVCAR-3(Ctrl)组的mRNA水平低,差异具有统计学意义(p均<0.01),结果详见图3A。说明shRNA-Ambra1可以显著抑制目的基因Ambra1的表达水平,达到干扰效果。3、检测基因敲除后对卵巢癌OVCAR-3细胞自噬囊泡形成的影响用10μM的顺铂同样处理两组卵巢癌OVCAR-3细胞后,与卵巢癌OVCAR-3(Ctr1)细胞组相比,GFP阳性的自噬点在OVCAR-3(Ambra1-)细胞组中表达下降,且差异具有统计学意义(p<0.01),结果详见图3C。4、检测基因敲除后对卵巢癌OVCAR-3细胞中Ambra1、LC3-I蛋白、LC3-II蛋白及Beclin 1蛋白表达的影响用10μM浓度的顺铂同样处理两组卵巢癌OVCAR-3细胞12、24小时后,利用Western Blotting实验方法检测发现:顺铂处理两组卵巢癌OVCAR-3细胞12小时和24小时后,Ambra1蛋白在卵巢癌OVCAR-3(Ambra1-)组中的表达较在卵巢癌OVCAR-3(Ctrl)组中的表达下降,差异具有统计学意义(p<0.01,p<0.05)。结果详见图3B。处理时间为12小时和24小时后,在OVCAR-3(Ambra1-)细胞组中,LC3-II/LC3-I蛋白的比值比在卵巢癌OVCAR-3(Ctrl)细胞组中LC3-II/LC3-I蛋白的比值下降,差异具有统计学意义(p<0.01)。说明在卵巢癌OVCAR-3细胞中,Ambra1基因的表达被抑制后,自噬的诱导被抑制。结果详见图3D、3E。处理时间为12小时后,在OVCAR-3(Ambral-)细胞组中,Beclin1蛋白的表达比在卵巢癌OVCAR-3(Ctrl)细胞组中的表达下降,差异具有统计学意义(p<0.05)。同样发现,处理时间为24小时后,在OVCAR-3(Ambra1-)细胞组中,Beclin1蛋白的表达比在卵巢癌OVCAR-3(Ctrl)细胞组中的表达下降,差异具有统计学意义(p<0.01)。说明在卵巢癌OVCAR-3细胞中,Ambra1基因的表达被抑制后,自噬相关基因Beclin1蛋白的表达也受到了抑制。结果详见图3F。结论1、shRNA-Ambra1可以显著抑制目的基因Ambra1的表达水平,达到干扰效果。2、沉默Ambra1基因的表达后,抑制了顺铂诱导的卵巢癌细胞自噬囊泡的形成。3、沉默Ambra1基因的表达后,自噬相关蛋白LC3-II/LC3-I蛋白的比值下降,Ambra1、Beclin 1蛋白的表达下降。4、在顺铂处理的卵巢癌OVCAR-3细胞的自噬诱导中,Ambra1有正性调控作用。第三部分:shRNA介导的Ambra1基因敲除对顺铂处理的卵巢癌OVCAR-3细胞凋亡影响的研究实验目的:通过测定Ambra1基因敲除后,顺铂对卵巢癌OVCAR-3细胞生长、细胞活性和凋亡诱导的影响,明确Ambra1基因敲除对OVCAR-3细胞顺铂敏感性的影响。实验方法:1、利用细胞集落形成实验,检测用10μM的顺铂处理OVCAR-3(Ctrl)和OVCAR-3(Ambra1-)两组细胞后,细胞集落的形成情况。2、利用MTT实验,检测1OμμM的顺铂处理OVCAR-3(Ctrl)和OVCAR-3(Ambral-)细胞后,细胞活性的改变。3、利用流式细胞学实验,检测10μM的顺铂处理OVCAR-3(Ctrl)和OVCAR-3(Ambra1-)细胞后,细胞凋亡率的改变。结果:1、Ambra1基因被敲除后,顺铂处理卵巢癌OVCAR-3细胞集落形成的改变如图4所示:用10μM的顺铂处理卵巢癌细胞可以显著降低OVCAR-3(Ctrl)细胞的集落形成(p<0.01,图4B中的第3栏和第1栏),这种类似的集落形成下降同样出现在OVCAR-3(Ambra1-)细胞中(p<0.01,图4B中的第4栏和第2栏)。更有意义的是,在OVCAR-3(Ctrl)和OVCAR-3(Ambra1-)组中细胞集落形成是不同的,OVCAR-3(Ambra1-)组的细胞集落下降的更为显著(p<0.05,图4B中的第4栏和第3栏)。2、Ambra1基因被敲除后,顺铂处理卵巢癌OVCAR-3细胞后细胞活性的改变OVCAR-3(Ctrl)和OVCAR-3(Ambral-)两组细胞的细胞活性没有差异,如图5A所示。但是用10μM的顺铂处理两组细胞后,OVCAR-3(Ambral-)组的细胞活性比OVCAR-3(Ctrl)组的细胞活性显著下降,差异具有统计学意义(如图5B,处理后24小时时P<0.05,处理后48小时,(P<0.01)。3、Ambra1基因被敲除后,顺铂处理卵巢癌OVCAR-3细胞后细胞凋亡的改变用10μM的顺铂处理两组细胞后,OVCAR-3(Ambra1-)组的细胞凋亡率比OVCAR-3(Ctrl)组的细胞凋亡率显著上升,差异具有统计学意义(如图5C,处理24、48小时后p均<0.01)。结论:1、沉默Ambral基因可以提高OVCAR-3对顺铂的敏感性,增加细胞凋亡率。2、Ambra1可能是保持卵巢癌细胞自噬和凋亡之间平衡的关键调节器,Ambra1靶向抑制可能是卵巢癌细胞对化疗药物敏感的有效方法。更多还原

【Abstract】 Ovarian cancer is one of the common malignant tumors in the female reproductive system.The incidence of the disease was third in gynecologic tumors.However,Most of them are in the late stage of the clinic when they found them.Tumor cell subtraction combined with paclitaxel,cisplatin and other combined chemotherapy is an important method for the treatment of ovarian cancer.The primary response rate of ovarian cancer patients treated with combined chemotherapy based on platinum drugs was up to 80%.But most patients eventually fail in the appearance of chemotherapeutic drug resistance.The five year survival rate of ovarian cancer was around 30%.Drug resistance of ovarian cancer,in particular,the mechanisms and reversals of platinum resistance have become an extremely urgent and important research topic.Therefore,the mechanism and possible reversing pathways of ovarian cancer resistance are need to be studied.It is helpful to take specific measures to reverse chemotherapeutic drug resistance of ovarian cancer and to improve the patient’s sensitivity to chemotherapy drugs.Thus,the survival rate is improved and the prognosis is improved.Autophagy is proposed for the first time by Christian de Duve more than 40 years ago.Autophagy means that a membranous structure that forms a double or multilayered fold within a cell encapsulating a cell or macromolecule that is damaged in a cell under stress conditions such as starvation,hypoxia,and other undernutrition,and outside pressure.The autophagic corpuscle is formed,and the autophagosome is combined with the lysosome.Thus,the contents are digested and degraded,and the corresponding amino acids,nucleotides are circulated and reused.It satisfies the energy reuse of the cell itself and the renewal of the organelles.Autophagy is a conservative metabolic process in eukaryotes.It degrade intracellular damaged and degenerated proteins and organelles through lyase system,thrus maintaining cell homeostasis and genome stability.Under normal circumstances,autophagy maintains the balance of the intracellular environment at a basic level.In recent years,more research found that the function of autophagy may be involved in the occurrence and development of many diseases,such as diabetes,immune system disease,neurodegeneration and cancer invasion.In recent years,a variety of chemotherapeutic drugs have been found to induce autophagy in tumor cells.Tumor cells remove the damaged organelles caused by chemotherapeutic drugs and the wrong folding proteins to maintain their stability by autophagy and then evade the apoptosis induced by chemotherapeutic drugs and promote the survival of tumor cells.So autophagy may be a protective mechanism for cancer cells.The sensitivity of chemotherapeutic drugs was reduced.It leads to the tolerance of tumor cells to chemotherapeutic drugs.A number of studies have shown that autophagy has been involved in the process of chemotherapeutic drug resistance in a variety of tumors.The application of autophagy inhibitors may be one of the most important strategies for improving chemotherapeutic resistance and enhancing chemosensitivity.The effect of chemotherapeutic drugs on the autophagy of ovarian cancer cells and the role of autophagy in the chemosensitivity of ovarian cancer cells is rarely reported.Autophagy is a very complex process regulated by some special genes and multiple signal pathways.These regulatory genes are known as autophagy related genes(ATG).At present,more than 30 autophagy related genes have been found.Beclinl is a relatively mature autophagy related gene.It is combined with Class III P13K to form a complex.It plays an important role in the nucleation process of autophagy.Studies show that Beclin1 is also associated with the occurrence and drug resistance of tumors.Ambral(Activating molecule in Beclinl-regulated autophagy 1,Beclinl autophagy related activation molecule)is a new ATG gene.As the upstream factor of Beclinl,it controls the formation of autophagy by regulating the Beclinl-Class III complex.In mitochondria,Ambral regulates the autophagy and apoptosis of Beclin1 dependent Bcl-2 by combining with B lymphocyoma-2.Therefore,Ambra1 can determine the fate of tumor cells by regulating autophagy and apoptosis,but the role of Ambra1 in cisplatin treated ovarian cancer cells has not been reported.Studies have shown that in colon cancer cell line SW620,Ambral combined with Beclinl can positively regulate autophagy and negatively regulate apoptosis at the same time.The expression of Ambral upregulates in breast cancer cells treated by cisplatin and the drug resistance of breast cancer cells are increased.This study conjectured that Ambral is an important regulator of autophagy and apoptosis in ovarian cancer cell line OVCAR-3,proposed treatment of ovarian cancer OVCAR-3 cells by cisplatin,through the assessment of microtubule associated protein 1 light chain 3(LC3)positioning,autophagy exists,LC3 level and Ambral,Beclinl protein expression,confirmed the expression of Ambral and cisplatin the treatment of ovarian cancer cell OVCAR-3 induced autophagy and autophagy,to confirmed that the autophagy induction and the expression of Ambral in autophagy induced by cisplatin in ovarian cancer OVCAR-3 cells.We determined the autophagy promoting effect of Ambral in cisplatin treated ovarian cancer cells,and then transfected Ambral gene silencing by shRNA.Re evaluate the effect of Ambral gene interference on cisplatin induced autophagy in ovarian cancer OVCAR-3 cells.Further verify the role of Ambral in promoting autophagy in cisplatin treated ovarian cancer cells.The effects of cisplatin on the growth,cell viability and apoptosis induction of ovarian cancer OVCAR-3 cells after Ambral knockout were determined.It is clear that the effect of Ambra1 knockout on the sensitivity of OVCAR-3 cells to cisplatin.It provides a theoretical basis for finding therapeutic agents that improve the chemosensitivity of ovarian cancer based on Ambral.Part I:Cisplatin reduces cellular viability,promotes autophagy and upregulates Ambral in OVCAR-3 cellsObjectives:1.To study the sensitivity of OVCAR-3 cells in ovarian cancer to cisplatin2.To explore the regulation of autophagy and expression of autophagy related molecules in ovarian cancer OVCAR-3 cells by cisplatinMethods:1.MTT assay was used to detect the cell viability and time dependence of OVCAR-3 cells treated with different concentrations of cisplatin.2.Using cisplatin to treat ovarian cancer OVCAR-3 cells,we observed the LC3 transformation of ovarian cancer cells,including autophagy,autophagy formation and the key molecule of autophagy.3.The autophagic inducer,rapamycin as positive control,was used to observe the changes in the expression of autophagy related protein Ambral and Belinl after cisplatin treatment of ovarian cancer OVCAR-3 cells.Results1.Changes of cell activity in ovarian cancer OVCAR-3 cells which werre treated with different concentrations of cisplatinBy 0,10 and 50μM concentrations of cisplatin treating of ovarian cancer cells 0,12,24,48 hours later,MTT was used to detect the ovarian cancer cells,and the results showed as follows:with 10 or 50uM cisplatin treatment of ovarian cancer OVCAR-3 cells(p<0.005,p<0.01)for 24 hours,48 hours(p<0.001,p<0.0001),the activity of OVCAR-3 cells were decreased,and the decreased with difference was statistically significant.Moreover,there was a significant difference in cell survival rates between the two time differences between 10μM and 50μM cisplatin treated cells 24(p<0.05)hours and 48(p<0.01)hours,and the difference was statistically significant.The results are detailed in figures 1A and 1B.2.The effect of cisplatin on the autophagy of ovarian cancer OVCAR-3 cellsOvarian cancer cells were treated with 10μM cisplatin for 24 hours(experimental group).Compared with the ovarian cancer cells(control group)that were not treated with cisplatin,After transfection of GFP-LC3 fusion protein particles,the number of autophagic vesicles in the ovarian cancer cells in the experimental group was more than that in the control group.The results are detailed in figure 1C and 1D.The difference was statistically significant(p<0.001).The results are detailed in figure 1E.3.Cisplatin induced autophagy in ovarian cancer OVCAR-3 cells,accompanied by increased expression of Ambral and Beclin1.Compared with the ovarian cancer OVCAR-3 cells treated with non chemotherapy drugs,rapamycin could significantly improve the level of LC3-I protein conversion to LC3-II protein(p<0.001)after treating ovarian cancer OVCAR-3 cells with 100nM as autophagy as an autophagic inducer.After 24 hours treatment of cisplatin with 10 and 50μM of cisplatin,it can also significantly increase the level of LC3-II protein converted from LC3-I protein(p<0.01,p<0.001)after 24 hours.This transformation is metrological dependence(p<0.001).The higher the concentration of chemotherapeutic drugs,The higher level of LC3-II protein converted form LC3-1 protein.The induction of autophagy of rapamycin in 100nM was stronger than that of 10,and 50μM cisplatin.The results are detailed in figures 2A and 2B.Compared with the ovarian cancer OVCAR-3 cells treated with untreated chemotherapy drugs,After treating OVCAR-3 cells with rapamycin in 100nM as autophagic inducer,the expression level of Ambra 1 and Beclinl protein was significantly increased(P<0.001).After treating ovarian cancer OVCAR-3 cells with 10 and 50μM cisplatin for 24 hours,the expression level of Ambral and Beclin1 protein can also be significantly increased(p<0.05,p<0.01).And the measurement dependence(p<0.05),The expression of Ambral and Beclinl protein induced by cisplatin at 50μM was higher than that induced by cisplatin at 10μM.The difference was statistically significant(P<0.05).The expression of Ambra1 and Beclin1 protein in rapamycin induced by 100nM was higher than that induced by cisplatin at 50μM.The difference was statistically significant(P<0.0001).The results are shown in figures 2C and 2D.Conclusion:1.The cell activity of ovarian cancer OVCAR-3 cells decreased with cisplatin in time and concentration dependent.2.Cisplatin can induce autophagy in ovarian cancer OVCAR-3 cells.3.When cisplatin induced autophagy in ovarian cancer OVCAR-3 cells,the expression of autophagy related protein Ambral and Beclinl protein was increased,suggesting that Ambral can promote autophagy in ovarian cancer cells treated with cisplatinPart Ⅱ The study of shRNA mediated Ambral gene silencing on autophagy induced by cisplatin and the Beclinl expression in ovarian cancer OVCAR-3 cellsObjective:1.Screening Ambral gene transfected and stable expression of ovarian cancer OVCAR-3 cell line.2.we investigated the effect of Ambral knockout(interference suppression)on the induction of autophagy in cisplatin in ovarian cancer OVCAR-3 cells.3.To investigate the effect of interference inhibition of Ambral gene on the expression of autophagy related protein in ovarian cancer OVCAR-3 cells induced by cisplatin.Methods:1 LCells transfected with Ambral specific shRNA and RT-PCR mRNA assay were used to detect the expression of Ambral mRNA after Ambral knockout2.The effects of Ambral gene expression on the formation of autophagic vesicles in ovarian cancer OVCAR-3 cells were detected by autophagic vesicles.3.The effect of interference inhibition of Ambral gene expression on the expression of autophagic protein in ovarian cancer OVCAR-3 cells was detected by Western Blotting.Results:1.Transfection efficiency of OVCAR-3 cells in ovarian cancerIt is observed that GFP(green fluorescence)can be regarded as transfection work under the fluorescence microscope.Fluorescence counts were carried out at 12 hours,24 hours,36 hours and 72 hours after transfection.Comparing the number of fluorescent cells,We find that as time goes on,the expression of GFP was highest at 72 hours after transfection and the transfection efficiency of ovarian cancer OVCAR-3 cells reached more than 90%.It can be used for the follow-up experiments.According to whether transfection of shRNA-Ambral,ovarian cancer OVCAR-3 cells were divided into two groups,ovarian cancer OVCAR-3(Ambral-)group and OVCAR-3(Ctrl)group.2.RT-PCR was used to detect the expression of Ambral mRNA in two groups of cells.The expression level of Ambra1 mRNA in the OVCAR-3(Ambra1-)group was lower than that of the OVCAR-3(Ctrl)group.The difference was statistically significant(P<0.01),and the result was shown in figure 3A.It indicates that shRNA-Ambra1 can significantly inhibit the expression level of target gene Ambral and achieve the interference effect.3.The effect of Ambra 1 knockout on autophagic vesicle formation in ovarian cancer OVCAR-3 cells was detected.After the same treatment of two groups of ovarian cancer cells with 10 M cisplatin,the expression of GFP positive autophagy was decreased in the OVCAR-3(Ambral-)cell group compared with the ovarian cancer OVCAR-3(Ctrl)cell group,and the difference was statistically significant(p<0.01).The results were detailed in figure 3C.4.Detect the effect of Ambra 1 knockout on the expression of Ambra 1,LC3-I protein,LC3-Ⅱ protein and Beclin 1 protein in ovarian cancer OVCAR-3 cells.Cisplatin in 10 μM were used to treat two groups of ovarian OVCAR-3 cells for 12 and 24 hours,the Western Blotting method was used to detect and find:After cisplatin treatment of two groups of ovarian cancer OVCAR-3 cells for 12 hours and 24 hours,the expression of Ambral protein in the OVCAR-3(Ambra1-)group of ovarian cancer was lower than that in the OVCAR-3(Ctrl)group of ovarian cancer,and the difference was statistically significant(p<0.01,p<0.05).The results are detailed in figure 3B.After 12 and 24 hours of treatment,the ratio of LC3-II/LC3-I protein in the OVCAR-3(Ambral-)cell group decreased with the ratio of LC3-II/LC3-I protein in the OVCAR-3(Ctrl)cell group of ovarian cancer,and the difference was statistically significant(p<0.01,p<0.01).It is indicated that the induction of autophagy is inhibited in the ovarian cancer cell line OVCAR-3,when the expression of Ambral gene is inhibited.The results are shown in figures 3D and 3E.After 12 hours of treatment,the expression of Beclinl protein in the OVCAR-3(Ambra1-)cell group was lower than that in the OVCAR-3(Ctrl)cell group of ovarian cancer,and the difference was statistically significant(p<0.05).It was also found that after 24 hours of treatment,the expression of Beclinl protein in the OVCAR-3(Ambral-)cell group was lower than that in the OVCAR-3(Ctrl)cell group of ovarian cancer,and the difference was statistically significant(p<0.01).It is indicated that the expression of autophagy related Beclinl protein is inhibited in ovarian cancer OVCAR-3 cells after Ambral gene expression is inhibited.The results are detailed in figure 3F.Conclusions:1.shRNA-Ambral can significantly inhibit the expression level of target gene Ambral,and achieve the interference effect.2.silencing Ambral gene expression inhibits cisplatin induced autophagic vesicle formation in ovarian cancer cells.3.after silencing the expression of Ambral gene,the ratio of autophagy related protein LC3-II/LC3-I decreased,and the expression of Ambral and Beclin 1 protein decreased.4.Ambral plays a positive role in the induction of autophagy in cisplatin treated ovarian cancer OVCAR-3 cells.Part III The effect of shRNA mediated Ambral knockout on the apoptosis of ovarian cancer OVCAR-3 cells treated with cisplatinObjective:The effects of cisplatin on the growth,cell activity and apoptosis of ovarian cancer OVCAR-3 cells were determined after Ambral knockout,and the effect of Ambral knockout on the sensitivity of cisplatin in OVCAR-3 cells was determined.Methods:1.Colony forming assay was used to detect cell colony formation after treatment with 10μM of cisplatin in two groups of OVCAR-3(Ctrl)and OVCAR-3(Ambra1-).2.MTT test was used to detect the changes in the activity of OVCAR-3(Ctrl)and OVCAR-3(Ambral-)cells treated with cisplatin in 10μM.3.Flow cytometry was used to detect the apoptosis rate of OVCAR-3(Ctrl)and OVCAR-3(Ambral-)cells treated with cisplatin in 10μM.Result:1.Changes in colony formation of OVCAR-3 cells induced by cisplatin in ovarian cancer after the Ambra1 genes are knockout2.As shown in figure A:10μM of cisplatin in ovarian cancer cells can significantly reduce colony formation in the OVCAR-3(Ctrl)cells,differences are statistically significant(p<0.01 in Figure4B,the third and the first columns),the similar decreased colony formation also appeared in OVCAR-3(Ambral-)cells,and the differences are statistically significant(p<0.01,in Figure4A,the fourth and the second columns).More significant is that in OVCAR-3(Ctrl)and OVCAR-3(Ambral-)group,cell colony formation is different,the cell colony decline in OVCAR-3(Ambral-)group is more significant(p<0.05,in Figure 4B,the fourth column and the third column).3.Changes in cell activity after cisplatin treatment of ovarian cancer OVCAR-3 cells after Ambral genesare knockoutThere is no difference in cell activity between OVCAR-3(Ctrl)and OVCAR-3(Ambral-)two groups.As shown in figure 5A,however,when the cells were treated with 10μM cisplatin,the cell viability of OVCAR-3(Ambral-)group was significantly lower than that of OVCAR-3(Ctrl)group,and the difference was statistically significant(in Figure 5B,24 hours after treatment,p<0.05 and 48 hours after treatment,p<0.01).4.Changes in apoptosis after cisplatin treatment of ovarian cancer OVCAR-3 cells after Ambral genes are knockoutAfter treating two groups of cells with cisplatin in 10μM,the apoptotic rate of OVCAR-3(Ambral-)group increased significantly than that of OVCAR-3(Ctrl)group,and the difference was statistically significant(in Figure 5C,24 and 48 hours after treatment,all p<0.01).Conclusions:1.Silencing Ambral gene can improve the sensitivity of OVCAR-3 to cisplatin and increase the rate of apoptosis.2.Ambral may be the key regulator to maintain the balance between autophagy and apoptosis in ovarian cancer cells.Ambral targeting inhibition may be an effective method for ovarian cancer cells to be sensitive to chemotherapeutic drugs.更多还原