【作者】 王芳；

【导师】 崔金全；

【作者基本信息】 郑州大学 ， 妇产科学（专业学位）， 2020， 博士

【摘要】 引言卵巢癌作为世界上最具威胁性的女性恶性肿瘤之一,是导致女性死亡的重要疾病。据美国疾病预防工作组(USPSTF)2019年的统计数据表明,每年仅美国地区就有万人以上的患者死于卵巢癌。目前虽然各种化疗药物,例如铂类、紫杉醇等的应用降低了一部分细胞的过度增殖或者迁移情况,抑制卵巢癌的发生发展,改善患者的预后,但是卵巢癌患者总体的五年生存率通常仅为30%左右。虽然盆腔检查、经阴道超声检查和血清CA125在临床诊断中已广泛应用,但很难在早期发现疾病,降低死亡率,因此亟需寻找一种能够在早期发现并且确诊卵巢癌的方法。尽管以铂类和紫杉醇为基础的化疗,最初的临床反应率通常很高,但随后的复发和使用细胞毒性化疗的重复治疗导致对这些治疗的获得性抵抗。因此,针对卵巢癌的发病机制以及卵巢癌的早期诊断和预防机制是当前有关卵巢癌研究的重要方向。找到卵巢癌细胞中影响细胞特性的基因或者分子,使用特异性的抗体或抑制剂,有可能可以有效遏制肿瘤生长和转移,缓解疾病进展。黏蛋白(mucin antigen,MUC)家族是一类高度糖基化的蛋白,其中多为跨膜蛋白。黏蛋白主要负责形成粘膜的保护屏障,此外黏蛋白还具有许多更特殊的功能,如调节溶质运输或为共生和致病菌提供附着位点,以及和白细胞靶向作用相关,同时黏蛋白还与细胞再生、分化、整合、信号传递、粘附和凋亡相关。MUC16(mucin antigen sixteen,MUC16)是一种在卵巢癌、子宫内膜癌等多种肿瘤中高表达的跨膜黏蛋白。已有报道显示MUC16作为抗黏附分子可以抑制NK细胞与肿瘤细胞之间形成免疫突触,进而抑制NK细胞对肿瘤细胞的识别和杀伤。同时,MUC16能够与B细胞以及单核细胞结合而调控其免疫行为。另有研究表明MUC16在卵巢癌中高表达,与卵巢癌的转移和浸润密切相关。本研究首先从TCGA数据和临床样本中证明了MUC16在卵巢上皮性癌中高表达,且与卵巢癌细胞的增殖有关;随后验证了MUC16对卵巢癌细胞的增殖的促进作用。之后本研究对其机制进行了研究,结果表明MUC16可以促进卵巢癌细胞GLUT1的表达,促进卵巢癌细胞的糖摄取能力,加快糖酵解的进程。最后通过免疫缺陷小鼠荷瘤实验,观察和测量小鼠体重和肿瘤大小的变化差异,验证MUC16对卵巢癌细胞的促增殖作用。本研究首次从糖代谢的角度证明了在卵巢上皮性癌中,MUC16可以通过促进GLUT1的表达来促进肿瘤细胞的糖摄取,从而促进肿瘤细胞的糖代谢,为肿瘤细胞的生长提供更多的能量物质,最终通过促进肿瘤细胞的增殖来促进疾病的进展。证明了MUC16不仅可以作为临床诊断的指标,同时可以作为卵巢癌治疗的靶点,为卵巢癌的靶向治疗提供理论依据。1 TCGA数据及临床标本分析MUC16与卵巢癌相关性目的分析MUC16在卵巢癌患者临床标本及TCGA数据库中的表达情况。将MUC16表达与MKI67表达进行相关性分析,探讨MUC16的表达在卵巢上皮性癌发生发展中的作用。方法(1)TCGA数据库分析MUC16在卵巢癌组织和正常卵巢组织中的表达差异以及和生存的相关性;(2)TCGA数据库分析卵巢癌组织中MUC16与MKI67表达的相关性;(3)利用RT-PCR和免疫组化分别在在基因水平和蛋白水平检测临床样本中MUC16在卵巢上皮性癌组织和正常卵巢组织中的表达差异及与增殖基因MKI67的相关性;(4)分析MUC16的表达对卵巢上皮性癌患者预后的影响。结果(1)TCGA数据库分析显示,MUC16在卵巢癌组织中的表达明显高于正常卵巢组织,MUC16高表达的患者总生存期明显低于MUC16低表达患者。(2)TCGA数据库分析显示,卵巢癌组织中MUC16的表达与肿瘤细胞增殖基因MKI67的表达明显正相关。(3)RT-PCR和免疫组化结果表明,在临床样本中,MUC16在卵巢上皮性癌组织中的表达水平明显高于正常卵巢组织。同时MUC16的表达与MKI67的表达明显正相关。(4)卵巢上皮性癌患者生存分析显示,MUC16高表达的患者总生存期及无瘤生存期明显低于MUC16低表达患者。小结MUC16在卵巢上皮性癌中高表达,是一种促癌基因。MUC16高表达的卵巢上皮性癌患者总生存期和无瘤生存期显著低于MUC16低表达的卵巢上皮性癌患者,MUC16表达与卵巢上皮性癌患者的不良预后相关。MUC16在卵巢上皮性癌的表达与增殖基因MKI67正相关,MUC16与卵巢癌细胞的增殖相关。2 MUC16在卵巢癌细胞系中的表达情况及对细胞系增殖的影响目的检测MUC16在卵巢癌细胞系中的表达情况,构建敲低和过表达MUC16的细胞系,在细胞系水平检测MUC16的表达与卵巢癌细胞增殖的关系。方法(1)利用流式细胞术检测MUC16在卵巢癌细胞系OVCAR3、HO8910及SKOV3中的表达情况;(2)利用RNA干扰技术敲低高表达MUC16细胞系中MUC16的表达;(3)利用慢病毒技术对低表达MUC16的细胞系进行MUC16过表达;(4)利用CCK8、平板克隆的方法检测MUC16的表达对卵巢癌细胞增殖的影响,利用流式细胞术检测MUC16的表达对KI67表达的影响。结果(1)MUC16在OVCAR3、HO8910两株卵巢癌细胞系中高表达,而在SKOV-3细胞系中低表达。(2)RNA干扰技术成功构建MUC16敲低表达的OVCAR3、HO8910细胞系,慢病毒技术成功构建MUC16过表达的SKOV-3细胞系。CCK8及平板克隆结果显示高表达MUC16的两株细胞系OVCAR3、HO8910在MUC16敲低后增殖明显降低,而低表达MUC16的细胞系SKOV-3在MUC16表达上调后其增殖能力明显变强。(3)高表达MUC16的两株细胞系OVCAR3、HO8910在MUC16敲低后KI67的表达明显降低,而低表达MUC16的细胞系SKOV-3在MUC16过表达后KI67的表达明显升高。小结MUC16在OVCAR3、HO8910两株卵巢癌细胞系中高表达,而在SKOV-3细胞系中低表达。MUC16能够促进卵巢癌细胞中KI67的表达,促进卵巢癌细胞的增殖。3 MUC16对卵巢癌细胞增殖影响的机制研究目的通过TCGA数据库分析、临床标本检测及卵巢癌细胞系实验,探究MUC16表达对卵巢癌细胞增殖影响的机制,检测MUC16与GLUT1、KI67之间的调控关系。方法(1)TCGA数据库分析GLUT1在卵巢癌组织和正常卵巢组织中的表达差异以及GLUT1与MUC16表达的相关性;(2)利用RT-PCR技术和免疫组化分别在在基因水平和蛋白水平检测临床样本中GLUT1在卵巢上皮性癌组织和正常卵巢组织中的表达差异及与MUC16的相关性;(3)Western Blot技术检测MUC16与GLUT1之间的相关性;(4)小分子抑制剂的方法检测MUC16与GLUT1的调控关系;(5)2-NBDG摄取实验检测MUC16对卵巢癌细胞糖摄取能力的影响。结果(1)TCGA数据库分析显示,GLUT1在卵巢癌组织中的表达明显高于正常卵巢组织,卵巢癌组织中GLUT1的表达与MUC16的表达明显正相关。(2)RT-PCR和免疫组化结果表明,在临床样本中,GLUT1在卵巢上皮性癌组织中的表达水平明显高于正常卵巢组织。同时GLUT1的表达与MUC16的表达明显正相关。(3)Western Blot结果显示MUC16能够调控GLUT1的表达。(4)小分子抑制剂实验的结果显示MUC16通过调控GLUT1的表达,从而促进卵巢癌细胞的增殖。(5)当卵巢癌细胞系OVCAR3、HO8910的MUC16表达被敲低后,卵巢癌细胞的糖摄取能力明显减弱,同时当卵巢癌细胞系SKOV-3的MUC16表达被升高后,卵巢癌细胞的糖摄取能力明显增强。小结GLUT1在卵巢上皮性癌中高表达,MUC16与GLUT1和MKI67之间有明显的正相关的关系。MUC16通过调控GLUT1的表达,促进卵巢癌细胞的糖摄取和糖酵解能力,影响卵巢癌细胞的增殖。4小鼠模型验证MUC16在卵巢癌增殖中的作用目的以小鼠荷瘤模型为研究对象,通过检测不同组别荷瘤小鼠的体重、肿瘤体积和重量等差异,以及肿瘤中相关基因的表达情况,探究MUC16表达对卵巢癌细胞增殖及成瘤能力的影响。方法(1)以卵巢癌细胞系OVCAR3为研究对象,构建MUC16敲低的OVCAR3细胞系(OVCAR3-MUC16-SH),以及作为对照的MUC16正常表达的细胞系(OVCAR3-MUC16-SH-NC);(2)通过皮下注射的方法给免疫缺陷小鼠(NOD-SCID)注射卵巢癌细胞系,小鼠荷瘤后分别检测不同组别小鼠体重、肿瘤体积和重量等差异;(3)处死小鼠后将小鼠肿瘤组织取出,利用RT-PCR技术检测不同组别小鼠肿瘤中MKI67和GLUT1基因水平表达差异;(4)处死小鼠后将小鼠肿瘤组织取出,利用免疫组化技术检测小鼠肿瘤中KI67蛋白的表达差异。结果(1)MUC16敲低的OVCAR3荷瘤小鼠的肿瘤体积和重量明显小于对照组小鼠。(2)MUC16敲低的OVCAR3荷瘤小鼠的体重明显大于对照组小鼠;说明MUC16的表达对荷瘤小鼠的体重有明显影响。(3)MUC16敲低的OVCAR3荷瘤小鼠肿瘤MKI67及GLUT1基因表达明显下降。(4)MUC16敲低的OVCAR3荷瘤小鼠肿瘤KI67蛋白表达明显下降。小结MUC16对荷瘤小鼠的肿瘤体积、重量及小鼠体重均有明显影响。MUC16可以促进荷瘤小鼠肿瘤KI67及GLUT1的表达。MUC16的表达能够促进卵巢癌细胞在小鼠体内的成瘤及增殖能力。结论MUC16是一种在卵巢上皮性癌中高表达的促癌基因,并且跟肿瘤细胞增殖和卵巢上皮性癌患者的不良预后相关。MUC16在OVCAR3、HO8910两株卵巢癌细胞系中高表达,而在SKOV-3细胞系中低表达。MUC16能够促进卵巢癌细胞系中KI67的表达,促进卵巢癌细胞的增殖。在卵巢上皮性癌中MUC16与GLUT1和MKI67之间有明显的正相关的关系。MUC16通过调控GLUT1的表达,促进卵巢癌细胞的糖酵解,从而调控KI67的表达,影响卵巢癌细胞的增殖。MUC16对荷瘤小鼠的肿瘤体积、重量及小鼠体重均有明显影响。MUC16可以促进荷瘤小鼠肿瘤KI67及GLUT1的表达,促进卵巢癌细胞在小鼠体内的成瘤及增殖能力。更多还原

【Abstract】 IntroductionOvarian cancer(OC)is one of the most life-threatening gynaecological malignancies for women globally.According to the statistics by The US Preventive Service Task Force(USPSTF),there are thousands deaths from ovarian cancer each year in the United States alone.Although the use of various chemotherapeutic agents,like paclitaxel,platinum et al,remarkably decreased cells proliferation,migration and invasion and improve the prognosis of ovarian cancer,the 5-year survival rate of advanced stage ovarian cancer patients is presently only approximately 30%.Current diagnostic methods mainly include pelvic examination,transvaginal ultrasound,and measurement of the serum biomarker CA125,but difficult to detect early.Identifying a method to early detection of ovarian carcinomas.Despite the high response to platinum-and taxane-based chemotherapy,ut the postoperative recurrence and chemotherapy resistance always result in the treatment failure.Thus,it is necessary to explore the specific molecular pathogenesis and find early diagnosis and treatment strategies for ovarian cancer.To find regulators contribute to the growth,transformation,migration and invasion of ovarian cancer cells,using a specific inhibitor or a neutralizing antibody to improve comfort as the disease progresses.The transmembrane mucin family is a category of glycosylated transmembrane protein.They are mostly responsible for formation of the protective barrier.Moreover,these proteins have a number of peculiar properties,such as regulation of solute transport or provision of attachment sites for commensal and pathogenic microbes,as well as for leukocyte targeting,they are associated with cellular regeneration,differentiation,integration,signalling,adhesion and apoptosis.MUC16(mucin antigen sixteen,MUC16)is a membrane protein expressed on a variety of cancers including those of the ovary and endometrium.It has been reported that MUC16 as an anti-adhesion molecule can inhibit the formation of immune synapses between NK cells and tumor cells,thereby inhibiting the recognition and killing of tumor cells by NK cells,and promoting tumor progression.At the same time,MUC16 can bind to B cells and monocytes to regulate its immune behavior.Other studies have shown that MUC16 is highly expressed in ovarian cancer and is closely related to the metastasis and invasion of ovarian cancer.In this study,clinical data and TCGA database confirmed the effect of high MUC16 expression on ovarian epithelial cancer.Subsequently,the promotion effect of MUC16 on the proliferation was verified.Later,this study investigated its mechanism,and the results showed that MUC16 can promote the expression of GLUT1 in ovarian cancer cells,promote the glucose uptake capacity of ovarian cancer cells,and accelerate the process of glycolysis.Finally,through tumor-bearing experiments in immunodeficient mice,the differences in body weight and tumor size of the mice were observed and measured to verify the proliferative effect of MUC16 on ovarian cancer cells.This study demonstrated that MUC16 could promote the glucose uptake of tumor cells by promoting the expression of GLUT1 in epithelial ovarian cancer.firstly,thereby promoting the glucose metabolism of tumor cells and providing more energy for the growth of tumor cells.Substances ultimately promote disease progression by promoting tumor cell proliferation.It is proved that MUC16 can not only be used as an index for clinical diagnosis,but also be a target for ovarian cancer treatment,and provide a theoretical basis for targeted treatment of ovarian cancer.1 TCGA data and clinical specimens analyze the correlation between MUC16 and ovarian cancerObjectiveThe expression of MUC16 in ovarian cancer patients and database was analyzed.The correlation between MUC16 expression and disease progression was analyzed,and the role of MUC16 in the progression of ovarian cancer was initially explored.Methods(1)Analyze the differential expression of MUC16 in ovarian cancer tissues and normal ovarian tissues and its correlation with survival using the TCGA database.(2)Analysis the correlation between MUC16 expression and tumor cells proliferation in ovarian cancer tissues using the TCGA database.(3)RT-PCR was used to detected the expression of MUC16 in epithelial ovarian cancer tissues and normal ovarian tissues and its correlation with MKI67.(4)The immunohistochemical method was used to detect the expression difference of MUC16 in epithelial ovarian cancer tissues and normal ovarian tissues and its correlation with KI67.(5)Survival analysis was used to evaluate the influence of MUC16 on patient outcomes.Result(1)The results of TCGA database analysis showed that the expression of MUC16 in cancer tissues was significantly higher than that normal ovarian tissues,and the high expression of MUC16 was significantly negatively correlated with the prognosis of patients.(2)The results of TCGA database analysis showed that MUC16 expression in ovarian cancer tissues was positively correlated with tumor cell proliferation gene MKI67.(3)RT-PCR and immunohistochemistry results showed that the expression level of MUC16 in cancer tissues was significantly higher than that normal ovarian tissues in clinical samples.At the same time,the expression level of MUC16 is significantly correlated with the proliferative gene MKI67 in cancer tissues.(4)The clinical data analysis of ovarian cancer patients showed that the expression of MUC16 in cancer tissues was negatively correlated with the survival and disease-free survival of patients.SummaryMUC16,highly expressed in epithelial ovarian cancer,is a tumor-promoting gene.The expression of MUC16 is associated with poor prognosis in patients with epithelial ovarian cancer.The expression of MUC16 in epithelial ovarian cancer is positively correlated with the proliferation gene MKI67.2 Expression of MUC16 in ovarian cancer cell lines and its effect on cell line proliferationObjectiveThe expression of MUC16 in ovarian cancer cell lines was detected,and the relationship between MUC16 expression and tumor cell proliferation was verified at the cell line level.The results of the first part were verified and the experimental basis for the later mechanism study was provided.Methods(1)Detection of MUC16 expression in different cell lines was performed by flow cytometry.(2)RNA interference was used to knock down the expression of MUC16 in cell lines.(3)Lentiviral technology was used for over-expression of MUC16 in cell lines.(4)The effect of MUC16 expression on cell line proliferation was detected by CCK8,flow cytometry and plate cloning.Results(1)Flow cytometry results showed that MUC16 is highly expressed in various ovarian cancer cells(2)RNA interference technology can significantly inhibit the expression of MUC16 in OVCAR3 and HO8910,and the lentiviral technology significantly increases the expression of MUC16 in SKOV-3.CCK8 and plate cloning results showed that the proliferation of OVCAR3 and HO8910 was reduced because the knockdown of MUC16,while the proliferation of SKOV-3 was significantly enhanced because the overexpression of MUC16.(3)The expression of KI67 in OVCAR3 and HO8910 was significantly reduced after MUC16 knockdown,while the expression of KI67 in SKOV-3 was significantly enhanced after MUC16 overexpression.SummaryMUC16 is highly expressed in two ovarian cancer cell lines,OVCAR3 and HO8910,and lowly expressed in SKOV-3 cell line.MUC16 can promote the expression of KI67 in ovarian cancer cells and promote the proliferation of ovarian cancer cells.3 Mechanism of MUC16 on tumor cell proliferationObjectiveEffects of MUC16 on the growth of human ovarian cancer cells were investigated by TCGA database analysis,clinical specimen detection and ovarian cancer cell line experiments,and the regulatory relationship between MUC16 and GLUT1 and KI67 was detected.Methods(1)TCGA data analysis was used to find the correlation between MUC16 and GLUT1 as well as MKI67.(2)The correlation between MUC16 and GLUT1 was verified using Western Blot technology.(3)The regulation relationship between MUC16 and GLUT1 was further verified by small molecule inhibitors.Results(1)TCGA database analysis showed that the expression of GLUT1 in ovarian cancer tissues was significantly higher than that in normal ovarian tissues,and the expression of GLUT1 in ovarian cancer tissues was significantly positive correlated with the expression of MUC16.(2)Western Blot results showed that MUC16 can regulate the expression of GLUT1.(3)The results of small molecule inhibitor experiments show that MUC16 promote the proliferation of tumor cells by regulating GLUT1.SummaryGLUT1 is highly expressed in epithelial ovarian cancer,and there is a significant positive correlation between MUC16 and GLUT1,MKI67.MUC16 promotes the glucose uptake and glycolytic ability of ovarian cancer cells by regulating the expression of GLUT1,and affects the proliferation of ovarian cancer cells.4 Mouse model validates the role of MUC16 in the proliferation of ovarian cancerObjectiveThe mouse tumor-bearing model was used to explore the effect of MUC16 expression on the proliferation and tumorigenic ability of ovarian cancer cells in vivo.The results of the first three parts were further explained and fully verified by in vivo results,which finally proved that MUC16 was the promotion of tumors during the development of ovarian cancer.Methods(1)OVCAR3 with MUC16 knocked down(OVCAR3-MUC16-SH)and OVCAR3 with normal expression of MUC16 as control(OVCAR3-MUC16-SH-NC)were constructed.(2)The ovarian cancer cell line was injected into the immunodeficient mice(NOD-SCID)by subcutaneous injection.The tumor volume and weight were detected after tumor bearing.(3)After the mice were sacrificed,the tumor tissues were taken out,the mRNA of the tumor tissues was extracted,and the related gene expression was detected.(4)After the mice were sacrificed,the tumor tissues were taken out,the tumor tissues in different groups were paraffin-embedded,and the expression of related proteins was detected by immunohistochemistry.Results(1)There is a significant difference in the tumor volume and weight in two groups.(2)There is a significant difference in the mouse weight in two groups.(3)The expression of MUC16 and MKI67 in mouse tumors was significantly different in two groups.(4)The immunohistochemical results of mouse tumors from two groups showed significant differences in the expression of KI67.SummaryMUC16 has significant effects on tumor volume,weight and body weight of tumor-bearing mice.MUC16 promote the expression of KI67 and GLUT1 in tumor-bearing mice.MUC16 promote tumorigenesis and proliferation in mice.ConclusionMUC16 is a tumor-promoting gene that is highly expressed in epithelial ovarian cancer,and is associated with tumor cell proliferation and poor prognosis in patients with epithelial ovarian cancer.MUC16 is highly expressed in two ovarian cancer cell lines,OVCAR3 and HO8910,and lowly expressed in SKOV-3 cell lines.MUC16 can promote the expression of KI67 in ovarian cancer cell lines so that promote the proliferation of ovarian cancer cells.There is a positive correlation between MUC16 and GLUT1 as well as MKI67 in epithelial ovarian cancer.MUC16 promotes glycolysis of ovarian cancer cells by regulating the expression of GLUT1,thereby regulating the expression of KI67 and affecting the proliferation of ovarian cancer cells.MUC16 has significant effects on tumor volume,weight and body weight of tumor-bearing mice.MUC16 can promote the expression of KI67 and GLUT1 in tumor-bearing mice,so that promote tumorigenesis and proliferation in mice.更多还原