【作者】 庞迎新；

【导师】 刘培淑；

【作者基本信息】 山东大学 ， 临床医学（专业学位）， 2014， 博士

【摘要】 研究背景卵巢癌(ovarian cancer, OCa)是女性生殖器官肿瘤中常见的恶性肿瘤,其死亡率在妇科恶性肿瘤高居于首位。卵巢癌发病隐匿,大于2/3的患者出现症状时已经属于晚期。肿瘤细胞减灭术和以铂类化疗药物为主的联合化疗是晚期卵巢癌的主要治疗策略,可暂时缓解病情,然而70%的患者在3年内复发。复发后的卵巢癌耐药性增强,恶性程度增加,严重影响患者的预后及生存质量,使患者5年生存率大大下降。因此,探讨EOC的分子发病机制,寻找新型的和选择性强的抗卵巢癌药物,提高化疗敏感性或逆转耐药,对指导卵巢癌治疗具有重要意义。新型抗肿瘤药物研究的热点主要集中在以下两方面：(1)针对OCa的发生发展机制,探寻新分子作用靶点；(2)从天然产物(植物、海洋生物等)中寻找新的活性成分。MicroRNA(简称1miRNA)是近年来发现的一类长约22nt内源性非编码的小RNA分子,主要通过靶向到mRNA的3’UTR进而调控基因的表达,引起靶mRNA降解或蛋白翻译的抑制。研究发现,miRNA参与了多种生命活动,包括个体发育、细胞凋亡、增殖和代谢等。miRNA的表达具有组织特异性；在多肿瘤组织中往往表达失调,与包括卵巢癌在内的多种肿瘤的发生发展、耐药等病理进程密切相关,对肿瘤的诊断、治疗和判断预后有重要的指导意义。约有半数的miRNA基因位于肿瘤相关的基因组区或脆性位点,通过调控体内相应的癌基因或抑癌基因的表达,进而发挥重要的致癌或抑癌因子的功能。与正常卵巢组织相比,卵巢癌组织有特异的miRNA表达谱。文献显示miR-519d在晚期卵巢癌中表达下调,但下调机制有待进一步阐明。而且,miRNA通过影响细胞凋亡或相关信号通路参与卵巢癌化疗耐药的形成。因此,恢复失衡的miRNA的表达已经成为一个崭新的、有发展前景的巢癌治疗的新方向。天然药物是人类防病治病、药物研发的重要源泉。如卵巢癌一线化疗药物紫杉醇即为从红豆杉属植物中分离纯化得到的一类二萜类化合物。其抗癌机制主要为促进微管聚合、稳定微管,阻断正常的有丝分裂,发挥治疗卵巢癌的作用。大多数卵巢癌患者治疗效果较好,但20%以上的患者对紫杉醇耐药。因此,新型的天然抗肿瘤药物的寻找又成为热点。双联苄类化合物是从苔藓类植物中提取并分离得到的一类大环多酚类化合物,具广泛的生物学活性,如抗炎、抗细菌、抗真菌、抗肿瘤等,是一类发展前景良好的抗癌新药。Dihydroptychantol (DHA)是双联苄化合物的一种,本课题组前期研究发现,其在多种肿瘤细胞株中可显著抑制细胞增殖、促进细胞凋亡；DHA还可诱导其他类型的细胞死亡,如自噬性死亡。然而,双联苄化合物的抗卵巢癌活性未有报道,其具体作用机制有待于进一步研究。第一部分：miR-519d通过靶向调控XIAP抑制卵巢癌细胞增殖及增强顺铂敏感性的研究研究目的：检测miR-519d在卵巢癌细胞系和组织中的表达,探讨其对卵巢癌细胞增殖的影响和与顺铂敏感性的关系。通过预测并验证miR-519d靶基因,揭示miR-519d的作用机制。材料方法：应用TaqMan探针法检测卵巢癌细胞系(OVCAR3、SKOV3及A2780)上皮性卵巢癌组织及正常卵巢组织的miR-519d的表达。转染miR-519d mimics或inhibitors实现miR-519d在卵巢癌细胞的上调或下调,采用MTT法检测对卵巢癌细胞的增殖能力,流式细胞术检测细胞凋亡率,western blot法检测凋亡相关蛋白表达。生物信息学预测miR-519d的靶基因,构建双荧光报告基因系统验证miR-519d与靶基因XIAP的关系。实验结果：1. miR-519d在卵巢癌细胞系及卵巢癌组织中的表达情况应用TaqMan探针实时定量PCR(qRT-PCR)法检测卵巢癌细胞系(OVCAR3、 SKOV3及A2780)及上皮性卵巢癌组织7例和正常卵巢组织2例中的1miR-519d的表达。结果显示：卵巢癌细胞系OVCAR3、SKOV3及A2780中miR-519d的表达水平显著低于正常卵巢组织,差异具有统计学意义(P<0.001)。上皮性卵巢癌7例组织中miR-519d的表达均较正常组织明显降低,差异具有统计学意义(P<0.001)。2.转染miR-519d对卵巢癌细胞增殖的影响在A2780及SKOV3细胞中转染miR-519d mimics及inhibitors48h后,应用TaqMan探针qRT-PCR法检测miR-519d的表达。结果显示：转染miR-519d mimics48h后,A2780及SKOV3细胞内的miR-519表达明显上调,分别为对照组的3985.33±400.05倍和2260.97±541.25倍。而转染miR-519d inhibitors后,A2780及SKOV3细胞的miR-519d表达显著被抑制,分别是对照组的0.32±0.07倍和0.08±0.02倍。在A2780及SKOV3细胞中转染miR-519d mimics48h及72h后,应用四甲基偶氮唑盐(MTT)法检测A2780及SKOV3细胞增殖能力。结果显示：上调A2780细胞内miR-519d水平后,48h及72h的A2780细胞增殖抑制率分别为：(15.50±3.62)%和(29.70±8.31)%,与对照相比差异具有统计学意义(P值分别为0.004及0.03)。上调SKOV3细胞内的miR-519d水平对其增殖能力无明显影响。3.转染miR-519d对卵巢癌细胞的顺铂敏感性的影响3.1.转染miR-519d对顺铂诱导的卵巢癌细胞增殖抑制作用的影响在A2780及SKOV3细胞中转染miR-519d mimics48h后,联合应用不同浓度梯度的顺铂(25,50,100μM)作用24h。应用MTT法检测A2780及SKOV3细胞的增殖能力,结果显示：顺铂25μM作用于A2780细胞24h后,miR-519d转染组与阴性对照组的细胞增殖率分别为(27.01±1.65)%和(38.18±1.48)%(P<0.05)；顺铂501μM作用时,miR-519d转染组与阴性对照组的A2780细胞增殖率分别为(19.40±1.46)%和(30.07±5.76)%(P<0.05)；顺铂100μM作用时,miR-519d转染组与阴性对照组的A2780细胞增殖率分别为(13.38±1.34)%和(20.92±2.23)%,差异具有统计学意义(P<0.05)。转染miR-519d的SKOV3细胞经顺铂作用后,细胞增殖率变化趋势与A2780细胞一致。3.2.转染miR-519d对顺铂诱导的卵巢癌细胞凋亡作用的影响在A2780及SKOV3细胞中转染miR-519d mimics48h后,联合应用不同浓度的顺铂(25,50μM)作用24h,应用流式细胞术检测A2780及SKOV3细胞的凋亡率,结果显示：顺铂25μM作用于A2780细胞24h后,miR-519d转染组与阴性对照组的晚期凋亡率分别为(60.72±3.59)%和(46.82±2.38)%(P<0.05)。50μM顺铂作用时,转染1niR-519d组与阴性对照组的晚期凋亡率分别为(77.65±4.78)%和(67.26±5.35)%(P<0.05)。对于SKOV3细胞,早期凋亡、晚期凋亡及坏死细胞均较对照组增加。应用western blot法检测A2780及SKOV3细胞中凋亡相关蛋白的表达,结果显示：顺铂50μM作用于A2780和SKOV3细胞24h后,miR-519d转染组的酶原形式Caspase-3表达均较对照组降低,而Caspase-3及其底物PARP的剪切形式较对照组增加,表明1miR-519d加速了细胞凋亡进程。4. miR-519d靶基因的预测及其在A2780和SKOV3细胞中的验证TargetScan在线预测XIAP的3’UTR有两个miR-519d的结合位点,分别是1228-1234位点及4925-4931位点。在A2780及SKOV3细胞中转染miR-519d mimics或inhibitors48h后,western blot法及qRT-PCR法分别检测XIAP的蛋白及mRNA表达。结果显示：转染miR-519d mimics48h后,A2780及SKOV3细胞内XIAP的蛋白表达水平分别是对照组的0.39±0.06倍和0.62±0.06倍(P值均小于0.05)；转染miR-519d inhibitors48h后,A2780及SKOV3细胞内XIAP的蛋白表达水平分别是对照组的1.32±0.03倍和1.29±0.02倍(P值均小于0.05); XIAP mRNA的表达与蛋白的表达趋势完全一致。分别构建含有Targetscan预测的两个结合位点的XIAP3’UTR片段的双荧光素酶表达载体,与miR-519d共转染SKOV3细胞48h后,检测相对荧光素酶的表达活性。结果发现：野生型XIAP3’UTR (position1228-1234)双荧光报告基因载体组的荧光素酶相对活性明显低于突变型XIAP3’UTR组(89.28±15.83vs.167.37±21.34),差异有统计学意义(P<0.05)。构建的4925-4931位点的荧光素报告载体的实验结果与1228-1234位点的相一致。5. XIAP siRNA对卵巢癌细胞增殖及顺铂敏感性的影响5.1. XIAP siRNA在A2780及SKOV3细胞中干扰作用的验证XIAP siRNA作用于A2780及SKOV3细胞48h后, western blot法检测XIAP的蛋白表达,结果显示：转染XIAP siRNA的A2780及SKOV3细胞内XIAP蛋白的表达均为阴性。5.2. XIAP siRNA对卵巢癌细胞增殖的影响在A2780及SKOV3细胞中转染XIAP siRNA48h和72h后,MTT法检测A2780及SKOV3细胞增殖能力,结果显示：转染XIAP siRNA48h和72h后的A2780细胞增殖抑制率分别为(18.53±7.75)%和(24.84±3.70)%,与对照相比差异具有统计学意义(P<0.05)；XIAP siRNA对SKOV3细胞的增殖能力无明显影响(P>0.05)。5.3. XIAP siRNA对顺铂诱导的卵巢癌细胞凋亡作用的影响在A2780及SKOV3细胞中转染XIAP siRNA48h后,联合应用不同浓度的顺铂作用24h,应用MTT法检测卵巢癌A2780及SKOV3细胞的增殖能力,结果显示：顺铂50μM作用于A2780细胞24h后,XIAP siRNA干扰组与阴性对照组的A2780细胞增殖率分别为(37.75±1.95)%和(49.57±2.26)%,差异有统计学意义(P<0.05)；顺铂100μM作用时,XIAP siRNA干扰组与阴性对照组的A2780细胞增殖率分别(16.52±5.29)为和(31.02±1.0)%,差异具有统计学意义(P<0.05)。转染XIAP siRNA的SKOV3细胞经顺铂作用后,细胞增殖率变化趋势与A2780细胞一致。在A2780及SKOV3细胞中转染XIAP siRNA48h后,联合应用不同浓度的顺铂作用24h,应用western blot法检测A2780和SKOV3细胞中凋亡相关蛋白的表达,结果显示：顺铂50μM作用于A2780和SKoV3细胞24h后, XIAP siRNA干扰组的Caspase-3及其底物PARP的剪切形式增加。6.卵巢癌细胞及组织中XIAP的表达情况应用TaqMan探针qRT-PCR法检测OVCAR3、SKOV3及A2780细胞内的miR-519d的表达情况,结果以2-ΔCT×103值表示,分别为1.572±0.033,1.474±0.026及0.299±0.007。应用qRT-PCR法及western blot法分别检测三株卵巢癌细胞系XIAP mRNA和蛋白水平的表达,结果显示：XIAP mRNA定量结果以2-ΔcT×103值表示分别为0.003±0.002,47.62±2.33及49.68±21.32,XIAP蛋白水平的表达趋势与mRNA水平完全一致,表明XIAP的表达与miR-519d表达呈负相关。应用western blot法检测7例上皮性卵巢癌组织与2例正常卵巢组织XIAP蛋白的表达,结果显示：卵巢癌组织中XIAP蛋白表达水平与正常卵巢组织相比均显著上调,与miR-519d表达呈负相关。结论：1. miR-519d抑制卵巢癌SKOV3及A2780细胞增殖,增强其对顺铂作用的敏感性。2. miR-519d通过调控XIAP表达发挥抑癌基因功能。第二部分双联苄化合物DHA2通过调控XIAP及AKT/mTOR信-号-通路抑制卵巢癌作用机制的研究研究目的：检测DHA2对卵巢癌细胞凋亡和自噬的影响,探讨其作用机制,动物实验验证DHA2的抑瘤效果。材料方法：DHA2作用于卵巢癌细胞后,MTT法检测卵巢癌细胞的增殖能力,western blot检测细胞中凋亡、自噬相关蛋白及AKT/mTOR通路蛋白的表达变化,PI/Annexin V双染流式细胞仪检测细胞凋亡率,PI染色流式细胞仪检测细胞死亡率,吖啶橙染色流式细胞仪检测AVOs的形成,免疫荧光染色检测细胞中LC3的表达,针对XIAP基因,设计并构建XIAP真核表达质粒,H&E染色及免疫组化染色检测裸鼠移植瘤的凋亡、自噬及增殖相关蛋白的表达变化。实验结果：1.DHA及DHA1-3活性的细胞系筛选及其对卵巢癌细胞的生长抑制作用应用不同浓度的DHA及DHA1-3处理四种卵巢癌细胞系24h后,MTT法检测四种卵巢癌细胞系的增殖。结果显示：DHA及其衍生物DHA1-3对卵巢癌细胞系SKOV3、A2780、3A0和OVCAR3均有不同程度的增殖抑制作用,且呈剂量依赖性。DHA2对SKOV3,3AO和A2780细胞增殖的IC50值分别为13.44±0.84μmol/L,4.04±0.33μmol/L和19.80±1.08μmol/L, DHA2对正常视网膜上皮细胞RPE1细胞则毒性很小,IC50值为471.72±94.02μmol/L。2.DHA2诱导卵巢癌SKOV3细胞死亡的机制研究2.1DHA2对卵巢癌SKOV3细胞形态学的影响不同浓度的DHA2作用于SKOV3细胞24h后,镜下观察细胞形态变化,结果显示：在DHA25μM作用时,细胞体积略增加,细胞质密度增加；在DHA2作用时,细胞胞浆内出现大小不等的空泡,细胞边缘不规则；在DHA215μM时,细胞变圆,皱缩,与邻近细胞脱离。2.2DHA2对卵巢癌SKOV3细胞死亡的影响不同浓度的DHA2作用于SKOV3细胞24h后,应用流式细胞仪检测SKOV3细胞凋亡率,结果显示：DHA25μM,10μM和15μM诱导的SKOV3的细胞死亡率分别为25.01%,56.03%和58.44%。通过测定LDH的泄漏率来评价细胞损伤的程度,结果显示：细胞内的LDH释放呈剂量依赖性增加,DHA215μM处理时,LDH的泄漏率较对照增加4倍左右(904±97vs.238±12,P<0.01)。应用广谱Caspase家族抑制剂z-VAD-fmk(20μM)或坏死性凋亡抑制剂necrostatin-1(30μM)预处理细胞2h后,加入12.5μM DHA2继续作用12h,流式细胞术检测SKOV3细胞死亡率。结果显示：广谱Caspase抑制剂z-VAD-fmk对DHA2诱导的SKOV3细胞死亡无显著逆转作用(18.75±4.28%vs.14.04±2.74%)(P>0.05)。应用坏死抑制剂necrostatin-1对DHA2诱导的SKOV3细胞死亡亦无明显作用(23.18±1.86%vs.24.04±3.79%)(P>0.05)。2.3DHA2对卵巢癌SKOV3细胞内凋亡相关蛋白表达的影响不同浓度的DHA2(5,10,15pM)作用于SKOV3细胞24h后,western blot方法检测SKOV3细胞中凋亡相关蛋白的表达情况。结果显示：DHA2诱导的死亡伴随着抗凋亡蛋白XIAP、Bcl-2的下调,促凋亡蛋白Bax的上调,Bcl-2/Bax比值下调,呈时间和剂量依赖性；Caspase的底物PARP的剪切形式在SKOV3细胞中无明显增加。2.4. XIAP在DHA2诱导的SKOV3细胞死亡的作用构建XIAP过表达载体,在SKOV3细胞中转染XIAP过表达载体24h后,继续应用12.5μMDHA2处理12h,应用流式细胞仪检测SKOV3细胞死亡率,结果显示：过表达XIAP几乎可完全逆转DHA2诱导的SKOV3细胞死亡(31.87±3.53%vs.7.44±4.37%)(P<0.05)。设计XIAP siRNA,转染SKOV3细胞48h后,继续应用12.5μM DHA2处理12h,应用流式细胞仪检测SKOV3细胞死亡率。结果显示：干扰XIAP表达能显著增加卵巢癌细胞对DHA2的敏感性(20.41±6.98%vs.33.79±5.02%)(P<0.05)。3.DHA2对卵巢癌SKOV3细胞自噬的影响3.1.DHA2对卵巢癌SKOV3细胞自噬溶酶体的作用DHA212.5μM处理SKOV3细胞12h和24h,应用AO染色流式细胞术检测处理后SKOV3细胞内的酸性自噬溶酶体空泡(AVOs)的形成。结果显示：随着DHA2作用时间的延长,AO染色的荧光强度明显向右偏移,提示DHA2诱导细胞自噬体的形成。3.2.DHA2对SKOV3细胞内自噬相关蛋白的表达影响DHA212.5μM处理SKOV3细胞不同时间后,应用免疫荧光染色检测LC3蛋白的表达。结果显示：DHA2处理的SKOV3细胞自6h开始于胞浆内逐渐出现红色荧光斑点,随着作用时间的延长,荧光斑点数量明显增加,荧光强度也随之增强。不同浓度的DHA2(5,10,15μM)作用于SKOV3细胞24h,或者]2.5μMDHA2作用于SKOV3细胞不同时间后,应用western blot法检测SKOV3细胞中自噬相关蛋白的表达,结果显示：DHA2在卵巢癌SKOV3细胞中以剂量依赖性及浓度依赖性的方式诱导LC3-II的表达上调,同时促进了LC3-I向LC3-II的转化；自噬降解底物p62表达随着药物作用时间的增加逐渐降低；其他自噬相关蛋白如ATG5、Beclinl的表达变化不明显。qRT-PCR法检测自噬相关基因的mRNA的表达水平,结果显示：经DHA2作用后,自噬相关基因Atg5、Atg7、Atg12、 Beclinl等的mRNA表达无明显变化。3.3.自噬抑制剂对DI-IA2诱导的SKOV3细胞死亡的影响应用自噬抑制剂(E64D/Pepstatin A或氯喹)预处理SKOV3细胞2h后,再应用DHA212.5μM处理SKOV3细胞12h,流式细胞术检测SKOV3细胞死亡率。结果显示：E64D/Pepstatin A联合DHA2作用比DHA2单独作用时细胞死亡率明显增加(40.06±7.84%vs.27.93±2.31%,P<0.05)；应用氯喹阻断自噬后也明显增强DHA2介导的细胞死亡(40.06±7.84%vs.27.93±2.31%,P<0.05)。在SKOV3细胞中转染ATG5siRNA48h后,再应用DHA212.5μM处理12h,流式细胞术检测SKOV3细胞死亡率。结果显示：应用ATG5siRNA能有效阻断LC3-Ⅰ向LC3-Ⅱ的转化,并增强DHA2诱导的细胞死亡。4.DHA2诱导卵巢癌SKOV3细胞自噬的机制研究4.1.DHA2对AKT/mTOR信号通路的影响不同浓度的DHA2(5,10,15μM)作用于SKOV3细胞24h,或者12.5pM DHA2作用于SKOV3细胞不同时间后,western blot方法检测细胞中AKT/mTOR通路中相关蛋白的表达情况。结果显示：15μM DHA2可明显抑制p-AKT.p-mTOR的活性,而对AKT和mTOR总蛋白的表达没有明显影响。在作用时间上,DHA2作用3h即可引起p-AKT的明显下调,随作用时间的延长,p-AKT的活性持续下调。对于p-mTOR, DHA2作用9h也出现显著下调。4.2. XIAP对DHA2诱导的SKOV3细胞自噬的影响XIAP siRNA处理SKOV3细胞48h后,应用12.5μMDHA2继续作用12h,应用western blot法检测SKOV3细胞中相关蛋白表达情况。结果显示：转染XIAP siRNA的SKOV3细胞中,LC3-II/-I值为阴性对照组的1.6倍。DHA2单独作用的LC3-Ⅱ/-Ⅰ值为8.83,而敲除XIAP后使DHA2引起的LC3-Ⅱ/-Ⅰ值上调至12.24。4.3.AKT在DHA2诱导的SKOV3细胞死亡与自噬中的作用在SKOV3细胞中转染AKTl-Myr载体48h后,应用12.5μM DHA2继续作用12h, western blot检测SKOV3细胞中相关蛋白表达,流式细胞术检测SKOV3细胞死亡率。结果显示：转染AKT1-Myr载体48h后,p-AKT的水平明显上调,并伴随着LC3-II的表达下调,而且能部分逆转DHA2引起的细胞死亡(31.47±2.68%vs.12.34±3.05%,P<0.01)。应用PI3K抑制剂LY294002预处理SKOV3细胞2h后,应用12.5μMDHA2继续作用12h, western blot检测SKOV3细胞中相关蛋白表达,流式细胞术检测SKOV3细胞死亡率。结果显示：LY294002可显著降低p-AKT的活性,增加LC3-II的积累,并显著增强DHA2诱导的细胞死亡(17.53±2.62%vs.32.26±2.04%,P<0.01)。5.DHA2对SKOV3卵巢癌裸鼠移植瘤的影响5.1.DI-IA2对SKOV3卵巢癌裸鼠移植瘤的生长抑制作用建立卵巢癌SKOV3细胞荷瘤裸鼠动物模型,建模成功后随机分为对照组、DHA2低浓度组(15mg/kg)和DHA2高浓度组(30mg/kg),隔日腹腔用药,连续两周。结果显示：低剂量和高剂量DHA2均显著地抑制肿瘤的生长,瘤块体积和瘤重的减少较对照组有统计学差异(P<0.05)。根据不同时间点的肿瘤体积绘制肿瘤的生长曲线,结果显示：DHA2给药组能显著地抑制肿瘤生长；随着给药时间的延长,抑瘤效果更加明显。低剂量组和高剂量组的肿瘤体积抑制率分别为48.76%和41.79%,两剂量组间无统计学差异。应用western blot方法检测了各组肿瘤组织中凋亡相关及自噬相关蛋白的表达,结果显示：DHA2给药组的肿瘤组织中XIAP的表达低于对照组,p-AKT、p-mTOR及LC3的表达变化与细胞实验完全一致。采用H&E染色法对肿瘤组织进行形态学特征的观察,结果显示：DHA2抑制了肿瘤细胞的分化,并伴随着局部肿瘤组织的坏死。应用免疫组化检测Ki67的表达情况,结果显示：三组的Ki67比率分别为对照组(55.62±22.92)%,低剂量组(1.31±11.14)%,高剂量组(5.4±7.81)%,差异具有统计学意义(P<0.01vs.对照组)。免疫组化结果显示：DHA2给药组中XIAP、p-AKT和p-mTOR表达较对照组降低,而LC3表达高于对照组。5.2.DHA2对移植瘤模型的骨髓抑制作用及肝肾毒性研究血常规结果显示,各组间白细胞、血红蛋白及血小板的水平无明显差异,提示DHA2对骨髓造血功能无明显的抑制作用。对各组的肝、脾、肾器官进行H&E染色未发现有明显的组织学异常改变。肝肾功能血液学结果显示,高剂量组中的谷草转氨酶(AST)和谷丙转氨酶(ALT)水平较对照组和低剂量组有所升高,但差异无统计学意义(P>0.05)；各组间的血清肌酐(SCr)水平无明显差异,而高剂量组中的尿素氮(BUN)水平比对照组和低剂量组有所升高(P<0.05)。结论：1.DHA2在卵巢癌中具有生长抑制作用。2.AKT在DH_A2诱导的SKOV3细胞死亡中发挥重要调控作用。3.DHA2有可能成为治疗卵巢癌的新型天然药物。更多还原

【Abstract】 Backgroud:Ovarian cancer is the major lethal gynecological malignancy with the characteristics of insidious onset, tendency to metastasis and poor prognosis. However, specific mechanism is unclear. The mainstay of treatment for ovarian cancer is cytoreductive surgery and platinum-based adjuvant chemotherapy. Up to two thirds of patients in ovarian cancer initially suffer from disease at advanced stage, resulting poor therapeutic effects. More than80%of the patients present chemotherapy resistance which finally cause tumor recurrence and metastasis. Thus, the five-year survival rate for ovarian cancer patients is less than30%. So far, searching for newfashioned antitumor drugs and reversing multi-drug resistance for ovarian cancer are always the research hotspots and difficulties. The most hot research area focuses on two components in particular:(1) to explore novel molecular targets directly targeting the mechanism of tumor onset and progression;(2) to find newly active ingredients from natural products (for example, plants and marine organism).MicroRNAs (miRNAs) are a new class of small (-22nucleotides-long) endogenous non-coding RNAs which regulate gene expression at post-transcriptional level and induce translational repression, mRNA cleavage, or destabilization by base-pairing to the3’-untranslated region (3’UTR) of the target mRNAs in animals and plants. Studies have demonstrated that miRNAs play important role in various life activities, including individual development, cellular apoptosis, proliferation and metabolism. miRNAs are closely related to onset and progression of various tumor including ovarian cancer, and development of chemotherapy resistance. miRNAs apparently exhibit a tissue-specific expression, and aberrant versions of miRNAs are involved in various cancers and their diagnosis and prognosis. Nearly half of miRNA genes are positioned at tumor-related genomic regions or fragile sites. Through regulating corresponding oncogenes or tumor suppressors, miRNAs function as both oncogenes and tumor suppressors. Compared to normal ovarian tissues, ovarian cancer tissues have specific expression profiles. A recent study showed that miR-519d was significantly downregulated in advanced ovarian cancer. However, concrete mechanism is still unclear. Moreover, miRNAs play important roles in development of chemotherapy resistance via mediating cellular apoptosis and related signaling pathway. In conclusion, restoration of the deregulated miRNAs may be useful to develop novel strategies of targeted therapies.Natural plants have always been an important part of disease prevention and control, and drug development. For example, paclitaxol, an important clinical first line chemotherapeutic agent, is a kind of diterpenoid compound mainly isolated from plants of Taxus. Its anticancer mechanisms involve in facilitating microtubule assembly, stabilizing microtubule and blocking routine mitosis. However, paclitaxol has some disadvantages, for example, poor solubility, frequent adverse reactions and occasional development of chemotherapy resistance. Therefore, searching for new alternatively effective regimens or chemicals for ovarian cancer treatment is urgently needed. Bisbibenzyls, a novel class of characteristic macrocyclic compontents derived from liverworts, attract more and more attention due to their broad spectrum of biological significance including anti-inflammatory, anti-bacterial, antifungal and antitumor. Thus, bisbibenzyls have bright prospect as new anticancer candidate drugs. Dihydroptychantol (DHA) belongs to bisbibenzyls. In our previous study, DHA significantly inhibited proliferation and induced cell apoptosis in various cancer cell lines. In addition, DAH also triggered other forms of cell death, such as, autophagy. However, its anticancer activity in ovarian cancer has not been investigated and the detail mechanism needs to be further studied.PART I:miR-519d represses ovarian cancer cell proliferation and enhances cisplatin-mediated cytotoxicity in vitro by targeting XIAPObjective:We aimed to detect expression of miR-519d in ovarian cancer cell lines and ovarian cancer tissues, and to analyze the effects of miR-519d on cellular proliferation and ciaplatin sensitivity. We tried to reveal the regulatory mechanism of miR-519d in ovarian cancer after predicting and verifying potent targets of miR-519d.Methods:The expression of miR-519d in ovarian cancer cell lines and ovarian tissues was determined by TaqMan quantitive reverse transcriptase-PCR. We used chemically synthesized oligonucleotides, mimics and inhibitors, for forced up-regulation or down-regulation of miR-519d. Cell proliferation was detected by3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis rate was analyzed by flow cytometry. Apoptosis related proteins were determined by western blot assay. miRNA potential candidates were predicted by TargetScan5.2software. A luciferase reporter assay was built to validate the direct binding interactions between miR-519d and X-linked inhibitor of apoptosis protein (XIAP).Results:1. Expression levels of miR-519d in ovarian cancer cell lines and ovarian cancer tissues Compared with normal ovarian tissues, all ovarian cancer cell lines (OVCAR3, A2780and SKOV3) exhibited significantly lower expression of miR-519d (P<0.001). A2780cells had the highest expression of miR-519d, followed by SKOV3cells. Similarly, miR-519d was also significantly downregulated in seven fresh frozen ovarian cancer tissues compared to normal ovarian samples.2. Effects of miR-519d overexpression on cellular proliferation in ovarian cancer cellsMTT assay showed that upregulation of miR-519d significantly suppressed cellular proliferation in A2780cells, and the inhibition rates of miR-519d overexpression were around15.50±3.62%and29.70±8.31%at48hours and72hours, respectively. After analyzed by student’s t-test, there were significant differences between cells transfected with miR-519d mimics, compared with control cells. P values at48and72h were0.004and0.03, respectively. However, there was no statistical difference in SKOV3cells after transfection with miR-519d (P>0.05).3. Effects of miR-519d overexpression on cisplatin resistance in ovarian cancer cellsOvarian cancer cells were transfected with the mimics of miR-519d for48h, and treated with various concentrations of cisplation for continuous12or24h. Changes in cell viability were detected by MTT assay. Upregulation of miR-519d enhanced cisplatin-induced proliferation suppression in ovarian cancer A2780and SKOV3cells. Similarly, addition of miR-519d significantly facilitated cisplatin-induced apoptosis. Transfection of miR-519d in A2780cells increased conversion of early apoptosis to late apoptosis, and the rate of late apoptotic cells increased significantly. Both early and late apoptosis as well as necrotic rates were increased in miR-519d-treated SKOV3cells, compared with negative control cells. Furthermore, we also showed that overexpression of miR-519d significantly facilitated cisplatin-induced cleavage of Caspase-3and PARP-1, a well-known substrate of active Caspase-3. Our findings suggested miR-519d serves as a tumor suppresive factor in ovarian cancer cells. 4. Prediction and verification of direct targets of miR-519dTargetScan was used to identify direct miR-519d target genes.3’UTR of XIAP is predicted to contain two candidate miR-519d-binding sites. One is postion1228-1234and the other is postion4925-4931. Upregulation of miR-519d remarkably reduced XIAP protein level, whereas downregulation of miR-519d by miR-519d inhibitors enhanced XIAP protein expression in A2780and SKOV3cells. Luciferase reporter assay were next performed to further validate whether XIAP is a direct target of miR-519d. Relative luciferase activities were markedly inhibited for both candidate sites of XIAP3’UTR.5. Effects of knockdown of XIAP on ovarian cancer cell growth and cisplatin-mediated cytotoxicityTo further verify whether miR-519d-mediated effects were mediated by targeting XIAP, siRNA strategy was performed to downregulate XIAP. XIAP siRNA effectively suppressed XIAP protein expression. Changes in cell viability were detected by MTT assay. Knockdown of XIAP significantly suppressed cellular proliferation in A2780cells, and inhibition rates of XIAP downregulation were about18.53±7.75%and24.84±3.70%at48hours and72hours, respectively (P<0.05). However, there was no statistical difference in SKOV3cells after transfection with XIAP siRNA. Knockdown of XIAP also enhanced cisplatin-induced growth inhibition in ovarian cancer A2780and SKOV3cells. In addition, downregulation of XIAP significantly facilitated cleavage of apoptosis-related proteins, Caspase-3and PARP-1.6. Expression levels of XIAP in ovarian cancer cell lines and ovarian cancer tissuesWe analyzed the relative expression of miR-519d in all ovarian cancer cell lines. The expression of miR-519d was presented as2-△Ctx103. Relative abundance of miR-519d in OVCAR3, SKOV3and A2780cell lines was1.572±0.033,1.474±0.026and0.299±0.007, respectively. Both of XIAP mRNA and protein expression levels exhibited an inverse correlation with miR-519d expression levels. Relative expression of XIAP mRNA in OVCAR3, SKOV3and A2780cell lines was0.003±0.002,47.62±2.33and49.68±21.32, respectively. In addition, ovarian cancer tissues had relative higher expression of XIAP protein compared to normal ovarian tissues, which exhibited negative correlation with miR-519d.Conclusion:1. Overexpression of miR-519d suppressed ovarian cancer cell proliferation and sensitized cells to cisplatin-induced cell death.2. miR-519funtion as a tumor suppressor through regulation of XIAP. PART Ⅱ:DHA2, a synthesized derivative of bisbibenzyl, exerts antitumor activity against ovarian cancer through inhibition of XIAP and Akt/mTOR pathwayObjective:We aimed to detect the effects of DHA-2on cell death and autophagy in ovarian cancer cells and to reveal the anticancer mechanism of this process. We further try to confirm the anticancer effects of DHA2in an ovarian xenograft models in vivo.Methods:Cells were treated with chemicals as indicated. Cell viability was determined via3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assay. Western blot analysis was performed to detect expression of apoptosis-and autophagy-related proteins. Cell apoptosis and death rates were analyzed by flow cytometry. Formation of AVOs was determined by acridine orange staining and detected by flow cytometry. LC3puncture dots were dectected by immunofluorescence, and fluorescence images were captured using a confocal microscopy LSM700. XIAP overexpression vector was designed and built to force upregulation of XIAP. H&E staining and immunohistochemical analysis were performed to detect expressions of XIAP, Bcl-2, p-AKT, p-mTOR, LC3B and Ki67in xenograft tumor samples.Results:1. Effects of DHA and its derivatives on cell growth inhibition in ovarian cancer cell linesMTT assay showed that DHA and its derivatives suppressed cellular proliferation st24h in dose-and time-dependent manners. Among the chemical, DHA2showed most effective effects on cell growth inhibition in ovarian cancer cells. The IC50value of DHA2was about13.44±0.84,4.04±0.33,19.80±1.08μmol/L in SKOV3,3AO and A2780cells, respectively. However, DHA2exhibited less growth inhibition in normal retina pigment epithelium RPE1cells with the IC50value of471.72±94.02μmol/L.2. Mechanism of DHA2-induced cell death in ovarian cancer cellsPI/Annexin V staining was performed to detect cell death rates. Data showed that DHA2caused dose-dependent cell death (both apoptosis and necrosis). LDH release was also dose-dependent, especially at the high concentration of DHA2in SKOV3cells. Pretreatment of cells both with a broad spectrum inhibitor, z-VAD-fmk, and a specific inhibitor of necroptosis, Necrostatin, had almost no effect on DHA2-induced cell death. Western blotting analysis indicated that DHA2decreased expression levels of XIAP and Bcl-2, and increased Bax in dose-and time-dependent manners. DHA2induced evident cleavage of PARP in A2780cells, whereas cleaved PARP was almost undetectable in SKOV3cells. Further studies showed that overexpression of XIAP almost reversed cell death induced by DHA2treatment, while knockdown of XIAP by siRNA depletion had the opposite effect. 3. Effects of DHA2on autophagy in ovarian cancer SKOV3cellsDHA2induced activation of autophagy as indicated by evident accumulation of AVOs, conversion of LC3B-Ⅰ to LC3B-Ⅱ as well as increased autophagic flux. Autophagy inhibitors, chloroquine and pepstatin/E64D, significantly enhanced DHA2-induced cell death. In addition, knockdown of ATG5, a crucial component involved in autophagic vesicle nucleation, also facilitated DHA2-mediated cell death.4. Mechanism of DHA2-induced protective autophagy in ovarian cancer SKOV3cellsHigh concentration of DHA2significantly inhibited activities of p-AKT and p-mTOR, but had no effect on total protein levels of AKT and mTOR. DHA2attenuated p-AKT at3h and became more evident with the increase of incubation period. As for p-mTOR, downregulation began at9h of DHA2treatment. Moreover, forced expression of Aktl-myr resulted in increased expression of XIAP and suppressed expression of LC3B-Ⅱ, contributing partially to the reversal of DHA2-mediated cell death. In contrast, pretreatment of cells with LY294002, an inhibitor of PI3K, suppressed the function of Akt and markedly enhanced DHA2-induced cell death.5. Effects of DHA2on tumor growth inhibition in an ovarian xenograft mouse modelHuman SKOV3xenografts were developed in nude mice to detect the effect of DHA2on tumor growth inhibition in vivo. After14d treatments, either initial or final body weight in tumor-bearing mice almost remained unchanged by administration of DHA2compared to placebo group. Monitoring blood parameters displayed that population of white blood cells, red blood cells, hemoglobin and platelet were not affected by DHA2treatment regardless the dosages used. Both of15mg/kg and30mg/kg of DHA2could effectively reduce tumor mass compared to control group. Western blot analysis showed higher expression levels of XIAP, p-AKT and p-mTOR in tumor-bearing mice, and pronouncedly reduced XIAP expressions, and abolished p-AKT and p-mTOR abundance in DHA2-treated groups. DAH2had a mild effect on Bcl-2expressions, consistent with the results in culture cells. Immunohistochemistry revealed that a statistically significant increase in the Ki67positivitywas observed in placebo control tumor cells (55.62±22.92%) compared to DHA2treated tumor cells (11.31±11.14%in15mg/kg treated group and5.4±7.81%in30mg/kg treated group)(P<0.05).Conclusion:1. DHA2suppressed ovarian cancer proliferation and induced cell death.2. AKT palyed key roles in DHA2-induced cell death and autophagy.3. DHA2is a potential candidate for ovarian cancer therapy.更多还原