【作者】 王玉；

【导师】 张淑兰；

【作者基本信息】 中国医科大学 ， 妇产科学， 2018， 博士

【摘要】 目的:子宫内膜癌是严重威胁女性健康的生殖道恶性肿瘤之一,近年来发病率逐年增加。手术治疗是子宫内膜癌主要的治疗手段,术后需要辅以化疗或其他辅助治疗,预后欠佳,缺乏有效的预防措施。目前公认长期无孕激素拮抗的雌激素作用是子宫内膜癌发生的主要原因,而高血压、肥胖、糖尿病被认为是子宫内膜癌发病的高危因素,通常被称为“子宫内膜癌三联征”。随着我国肥胖女性数量增加,子宫内膜癌也呈现发病率高及年轻化的趋势,很多未生育女性因子宫内膜癌或癌前病变切除子宫失去生育能力。流行病学数据表明,肥胖至少与40%的子宫内膜癌发生有关,肥胖人群子宫内膜癌发病率是普通人群的2-3倍。原因在于超重或肥胖女性的内源性雌激素水平升高,子宫内膜长期暴露于雌激素的作用下,会导致子宫内膜增生及潜在的不典型增生。糖尿病会增加子宫内膜癌的发病风险也已经得到了充分证实。糖尿病妇女患子宫内膜癌的风险比非糖尿病妇女高。有学者认为可以将子宫内膜癌看作是一组代谢异常的征候群,针对发生子宫内膜癌的高危因素改变个人生活习惯、节制饮食、加强锻炼,控制肥胖、糖尿病、高血压等代谢性疾病可以帮助降低子宫内膜癌的发病风险。由于子宫内膜癌发病率高,又缺乏有效的治疗措施,因此寻找有效的预防手段对于降低子宫内膜癌的发病率和保护女性健康及生育功能具有重要意义。利用安全有效的药物进行化学预防是非常有前景的研究方向,尤其对于子宫内膜早期病变以及有保留生育功能要求的女性患者。癌症化学预防(Chemorevention)这个名词在1976年由Michael Sporn创造,现在被美国国立癌症研究所(NCI)以及其它多个机构和个人所公认的定义为:利用天然、合成或生物物质来阻止、减缓或者逆转癌症发生发展过程,从而达到降低癌症的发生率和死亡率的方法、策略。小檗碱(Berberine,BBR)是一种拥有多个作用靶点的药物,在体应用BBR能够促进高能量介导的分解代谢。无论动物试验还是临床研究,BBR在糖代谢、脂代谢、糖尿病治疗、内皮作用及心血管系统的作用均得到了令人满意的结果。综上,肥胖、糖尿病是子宫内膜癌的高危因素,控制肥胖、糖尿病等代谢性疾病可以帮助减少子宫内膜癌的发生;而BBR对肥胖、糖尿病等糖脂代谢紊乱性疾病具有很好的治疗效果,同时由于BBR应用历史悠久,具有很好的安全性,因此可以尝试利用BBR进行子宫内膜癌的化学预防与治疗。本课题拟通过研究BBR对子宫内膜癌细胞增殖的抑制作用,并深入研究其作用机制,探讨将BBR应用于治疗子宫内膜癌的可能性。方法:1、研究对象:子宫内膜癌RL95-2、HEC-1-A及及AN3 CA细胞系;采集共15个EC组织和匹配的病灶周围正常子宫内膜组织,所有标本均采自因EC于中国医科大学附属盛京医院手术治疗的患者。手术切除后立即在液氮中冷冻,并在-80℃保存直至使用。5-6周龄裸鼠。2、研究方法:(1)第一部分:首先,体外培养人子宫内膜癌RL95-2细胞系及HEC-1-A细胞系,分别用不同浓度的BBR作用于两种细胞系,MTS法检测BBR对子宫内膜癌细胞增殖的抑制作用,采用流式细胞术方法检测BBR作用对子宫内膜癌细胞周期的影响。然后采用Western blotting方法检测BBR诱导细胞凋亡过程中凋亡相关蛋白caspase-3、caspase-9的变化,然后用JC-1染色法研究BBR作用前后细胞内线粒体膜电位的变化。采用葡萄糖氧化酶法和乳酸氧化酶法分别检测BBR作用前后子宫内膜癌细胞内葡萄糖消耗量及乳酸生成量的变化。Western blotting方法检测BBR作用前后子宫内膜癌细胞内p-AMPK,AMPK,p-mTOR,mTOR蛋白表达变化情况,然后向细胞内加入AMPK特异性抑制剂compound C,再次检测BBR作用前后子宫内膜癌细胞内p-AMPK,AMPK,p-mTOR,mTOR蛋白表达变化。(2)第二部分:培养人类EC细胞AN3 CA和HEC-1-A细胞,并采集共15个EC组织和匹配的正常组织。用脂质体?2000分别转染miR-101,及含有杂乱miRNA的miR-101抑制剂为阴性对照(NC)。逆转录-定量聚合酶链反应(RT-qPCR)检测mRNA的表达的差异。细胞活力测定和集落形成试验,使用细胞计数试剂盒8(CCK8)评估细胞存活率。BBR处理细胞后分析细胞的生存能力。克隆形成实验评价细胞集落形成能力的变化。采用24孔trans-well小室进行细胞侵袭和迁移试验检测EC细胞侵袭和迁移变化。Western blotting分析COX-2蛋白表达变化。酶联免疫吸附试验(ELISA)测定培养基中释放的PGE2水平。报告质粒构建,pGL3-Basic-pri-miR-101-2启动子及对照质粒pRL-SV40转染入EC细胞,BBR处理细胞后,进行双荧光素酶报告基因分析,测量萤火虫和海肾荧光素酶的活性。肿瘤异种移植,HEC-1-A细胞皮下植入5-6周龄裸鼠右侧腹壁,当肿瘤体积约为150-200毫米~3,选择性的分别给小鼠口服0.5%MC、不同剂量BBR,用卡尺每3天测量各组肿瘤体积。EC肺转移小鼠模型,通过向裸鼠尾静脉注射HEC-1-A cells细胞建立肺转移小鼠模型。处理4周后,计数宏观肺转移数。结果:MTS结果显示BBR作用于两种细胞后,两种细胞增殖速率均明显降低,且这种作用呈现时间及剂量依赖性(10%FBS)。流式细胞术结果显示,BBR作用于两种细胞后,细胞周期受到阻滞,处于G1/G0期的细胞所占的比例明显增加,而处于S、G2/M期的细胞所占的比例则明显降低。western blotting结果显示,BBR作用后,Rl95-2细胞内cleaved-caspase-3和cleaved-caspase-9的表达量显著增加,而在HEC-1-A细胞内,cleaved-caspase-3蛋白表达无明显差异。JC-1染色结果显示,BBR能使两种细胞线粒体膜电位均减弱。葡萄糖氧化酶法及乳酸氧化酶法结果显示,BBR作用后,两种细胞内均表现葡萄糖消耗量及乳酸生成量明显增加。western blotting结果显示,BBR作用于RL95-2细胞后,细胞内p-AMPK逐渐增加,而p-mTOR逐渐减少;而预先加入AMPK特异性抑制剂后,BBR激活AMPK的作用被减弱,但p-mTOR蛋白表达与单纯BBR作用时没有明显差异。BBR能以剂量和时间依赖性显著抑制AN3 CA和HEC-1-A EC细胞的增殖。同样,细胞集落形成实验也表明BBR处理后显著抑制AN3 CA和HEC-1-A EC细胞的集落形成能力。动物实验显示,口服黄连素50毫克/千克和100毫克/千克显著诱导肿瘤模型的肿瘤生长的抑制作用。改良的Boyden小室法检测细胞迁移及侵袭能力变化,结果表明,BBR显著抑制AN3 CA和HEC-1-A EC细胞的迁移能力,BBR治疗能够引起AN3 CA和HEC-1-A EC细胞的浸润明显减少。小鼠肺转移模型观察BBR对EC细胞转移的影响。结果发现给予BBR后,导致转移结节数明显减少,且呈现剂量依赖性。BBR作用于AN3 CA和HEC-1-A EC细胞后,以浓度依赖性的方式显著的抑制COX-2的表达。ELISA方法检测BBR治疗后前列腺素E2蛋白水平的变化,结果表明BBR同样以剂量依赖性的方式显著抑制PGE2水平。我们在HEC-1A细胞中存在或缺失COX-2过表达的情况下分别用BBR处理细胞,结果发现COX-2过表达使细胞存活率增加2.3倍,而在50μMBBR作用后COX-2的作用被抑制。HEC-1A细胞过表达COX-2时显示细胞迁移和侵袭能力显著上调。而BBR治疗后削弱了COX-2对于细胞活力及浸润能力的影响。与相匹配的正常组织相比,EC肿瘤组织中miR-101的表达显著下调,而COX-2蛋白水平明显上调。荧光定量PCR和Western blotting分析也证实了通过miR-101介导的对COX-2的调节作用。用的BBR处理细胞后发现,BBR刺激miR-101的表达,且呈现剂量依赖性。BBR诱导荧光素酶活性增加,而AP-1结合位点敲除后削弱了BBR诱导荧光素酶活性的作用。此外,miR-101抑制剂和BBR共同作用于HEC-1A细胞时,BBR诱导COX-2表达增加的作用被抑制。结论:BBR能够抑制子宫内膜癌细胞的增殖,促使细胞周期阻滞,对表达和不表达雌激素受体的子宫内膜癌细胞均有显著的抑制作用;BBR抑制子宫内膜癌细胞线粒体氧化功能,促进细胞无氧代谢;BBR通过对线粒体的抑制发挥对子宫内膜癌细胞的抑制作用;BBR能够以不依赖于AMPK通路的方式抑制RL95-2细胞的代谢核心分子mTOR。BBR抑制体外和体内EC细胞的生长、细胞迁移和侵袭,对肿瘤转移的调控起着至关重要的作用;BBR通过COX-2/PGE2轴介导抑制EC细胞存活、迁移和侵袭;miR-101参与了BBR对COX-2的调控。更多还原

【Abstract】 Objective:Endometrial cancer is a serious threat to women’s health as reproductive tract malignant tumors,the incidence increased year by year in recent years.Surgery is the main treatment,by now it’s lack of effective preventive measures.Exposed to estrogen for a long time without progesterone antagonist is thought to be the main cause of endometrial carcinoma,and high blood pressure,obesity,diabetes is regarded as the risk factors of endometrial cancer,called"trilogy of endometrial carcinoma".Along with the increase of obese women in our country,the endometrial cancer incidence is also increase,and shows younger trend,many childless women with endometrial cancer or precancerous lesion lose their uterus and fertility.Therefore,the chemical prevent of endometrial cancer is very important.Epidemiological data indicate that obesity is at least related to 40%endometrial carcinoma,the incidence of endometrial cancer is 2-3 times in obesity higher than that in the general population for increased level of endogenous estrogen.Exposure to estrogen for long-term can lead to endometrial hyperplasia and potential atypical hyperplasia.Diabetes will increase the incidence of endometrial cancer has been fully confirmed.The risk of suffering endometrial cancer in Diabetic women is higher than women without diabetes.Some scholars think that endometrial carcinoma can be regarded as a set of metabolic abnormalities syndrome,and changes in lifestyle,diet,strengthening exercise,control of metabolic diseases such as obesity,diabetes,high blood pressure helps to reduce the occurrence of endometrial carcinoma.Due to the high incidence of endometrial carcinoma,and lack of effective treatment measures,so looking for effective preventive measures to reduce the incidence of endometrial cancer is important.Using safe and effective drugs as chemical prevention are very promising research direction,especially for early endometrial lesions and those female patients who want to reserve the reproductive function.Cancer chemoprevention was coined in 1976 by Michael Sporn,now the national cancer institute(NCI)and several other institutions and individuals recognized is defined as the use of natural,synthetic or biological substances to prevent,slow or reverse the process of cancer development,strategies to reduce cancer incidence and mortality rates.Berberine is a drug with multiple targets,the application of berberine in the body can lead to the change of gene expression,thus promotes high-energy mediated catabolism.Both animal experiments and clinical studies of berberine on glucose metabolism,lipid metabolism,diabetes treatment,the role of regulating endothelial function and cardiovascular system are obtained the satisfactory results.In conclusion,obesity and diabetes are high risk factors of endometrial cancer,controlling obesity,diabetes and other metabolic diseases could help reduce the occurrence of endometrial cancer.Berberine has very good treatment effect on glucolipid metabolic disorder disease such as obesity and diabetes,so we can try to use berberine for chemical prevention and treatment of endometrial cancer.This topic proposed through the study of berberine inhibiting endometrial cancer cell proliferation,and further study of its mechanism of action,to discuss the possibility of berberine application in the treatment of endometrial cancer.Methods:1.Study population:RL95-2,HEC-1-A and AN3 CA cell lines of endometrial carcinoma.A total of 15 EC tissues and matched normal endometrial tissues around the lesion were collected.All specimens were taken from patients undergoing EC surgery in Shengjing Hospital affiliated to China Medical University.Immediately after operation,it was frozen in liquid nitrogen and stored at-80 C until it was used.5-6week old nude mice.2.Study methods:(1)The first part:People endometrial carcinoma RL95-2 and HEC-1-A cell lines were incubated in vitro,with different concentrations of berberine in the two cell lines.To detect endometrial cancer cell proliferation inhibition by MTS method,and the effects of berberine on endometrial cancer cell cycle by cycleflow cytometry test.Then detect the change of apoptosis related proteins caspase-3 and caspase-9 induced by berberine by western blotting,and JC-1 staining method is used to study the mitochondrial membrane potential changes in the cell before and after berberine effect.Glucose oxidase method and lactate oxidase method is used to detect the change in glucose consumption and lactic acid production in endometrial cancer cells after berberine effect.Western blotting is used to test the expression of p-AMPK,AMPK,p-mTOR and mTOR before and after berberine effect,and then to add AMPK specific inhibitors into the cell,again test the protein expression changes of p-AMPK,AMPK,p-mTOR and mTOR in the cell.(2)The second part:A total of 15 EC tissues and matched normal tissues were from Shengjing Hospital,Human EC cells(AN3 CA and HEC-1-A)were cultured.Transfection of miR-101 mimic or miR-101 inhibitor with scrambled miRNA as a negative control was performed with Lipofectamine?2000.RT-qPCR method was used to calculate differences in mRNA expression.Cell viability was assessed with Cell Counting Kit 8(CCK8,Beyotime,Shanghai,China).The viability of the cells was analyzed after incubation for 72h.Invasion and migration activity of EC cells were analyzed by using 24-well trans-well chambers.Protein isolation and western blot analysis was used to detect the expression of protein COX-2,and PGE2 levels released in the medium were determined by ELISA.Luciferase Reporter Assay,Following transfection of pGL3-Basic-pri-miR-101-2 promoter and a control vector pRL-SV40into EC cells,cells were treated with BBR.Dual-Luciferase Reporter Assay were performed to measure the firefly and Renilla luciferase activities.Tumor xenograft,HEC-1-A cells were subcutaneously implanted into the right flank of five six-week old nude mice,When the tumor volume was around 150-200 mm~3,the mice were randomly grouped into three groups with 6 mice per group.The selected mice were orally administrated with 0.5%MC(vehicle control),BBR(15 mg/kg,p.o.qd)or BBR(50 mg/kg,p.o.qd),respectively.Tumor volume of each group was calculated by caliper every 3 days.EC lung metastases mouse model were established by tail vein injections of HEC-1-A cells into nude mice.After 4 weeks treatment,the lungs were excised,fixed and paraffin embedded for counting of macroscopic lung metastases.Results:The first part:The MTS results show that the proliferation rate of two kinds of cell were significantly lower after treated by berberine,and the rendering is time and dose dependent.Flow cytometry results showed that the G1/G0 cell proportion increased significantly,while in S and G2/M stage cells proportion is significantly reduced after the effect of berberine on two kinds of cells.According to the results of western blotting,after the effect of berberine,the expression of cleaved-caspase-3 and cleaved-caspase-9 in RL95-2 cell is increased significantly,but in the HEC-1-A cell,there is no obvious difference was found between the above two kinds of protein expression.JC-1 staining results showed that the mitochondrial membrane potential were weakened in two kinds of cells after the effect of berberine.According to the results of glucose oxidase method and lactate oxidase method,glucose consumption and lactic acid production were significantly increased after the effect of berberine in two kinds of cells.According to the results of western blotting,during the effect of berberine on RL95-2 cell,the expression of p-AMPK increase gradually,and p-mTOR reduce gradually;And after adding specific inhibitors of AMPK in advance,the role of berberine activates AMPK is abate,but the expression of p-mTOR was no significant difference than that of pure berberine effect.The second part:Our findings revealed that BBR significantly inhibited cell proliferation in AN3 CA and HEC-1-A EC cells in both dose-and time-dependent manner.Similarly,colony formation assays also showed that BBR treatment dramatically suppressed the ability of colony formation in AN3 CA and HEC-1-A EC cells,In athymic mice model,oral administration of BBR at 50 mg/kg and 100 mg/kg significantly induced a tumor growth inhibition in tumor models.The effect of BBR on cell migration was assessed with a modified Boyden chamber assay.The results showed that BBR significantly repressed the migratory ability of AN3 CA and HEC-1-A EC cells.and BBR treatment caused a remarkable reduction of the invasion in AN3 CA and HEC-1-A EC cells.Next,we established a tail vein injection induced lung metastasis mouse models to evaluate the effect of BBR on EC cell metastasis.Administration of BBR resulted in a dramatic decrease in the number of metastatic nodules in a dose-dependent manner.We determined the protein levels of COX-2 in AN3 CA and HEC-1-A EC cells in response to treatment with BBR,BBR significantly inhibited the protein level of COX-2 in a concentration-dependent manner.We also assessed the protein level of PGE2 upon treatment with BBR with ELISA assay.The results showed that BBR dose dependently and remarkably inhibited PGE2 level.To further clarify whether COX-2 contributed to the action of BBR on tumor growth and metastasis in EC cells,HEC-1A cells were treated with BBR in the presence or absence of overexpression of COX-2.We demonstrated that overexpression COX-2 induced around 2.3-fold increase of cell survival while addition of BBR at 50μM abolished the effect of COX-2.BBR treatment wakened the effect of COX-2 on cell motility and invasion.miR-101 expression was significantly downregulated in EC tumor tissues relative to matched normal tissues,whereas COX-2 protein level was markedly upregulated.The regulation of COX-2 by miR-101 was also confirmed with Q-PCR and western blot assays.BBR dose dependently stimulated miR-101 expression.We found that BBR induced increase of luciferase activity while depletion of AP-1 binding sites diminished BBR induced luciferase activity.Furthermore,cotreatment of miR-101inhibitor and BBR abolished BBR-induced COX-2 increase in HEC-1-A cells.Conclusions:.Berberine can inhibit proliferation of human endometrial cancer RL95-2and HEC-1-A cell,and induce cell cycle arrest in two kinds of cells.Berberine exert its inhibitory effect in RL95-2 and HEC-1-A cell by inhibiting the mitochondrial function.After the Mitochondrial function are suppressed,both intracellular glucose consumption and lactic acid generation are increased.Berberine inhibit RL95-2 cell by inducing apoptosis through the mitochondrial pathway,but the inhibition of HEC-1-A cell is not achieved by apoptosis.The mechanism of Berberine inducing apoptosis in RL95-2 cells through mitochondrial way is to inhibit the core metabolic molecular mTOR,and the inhibition do not rely on AMPK pathway.BBR inhibits cell migration,invasion and metastasis in EC.BBR inhibited cell survival,migration and invasion mediated by COX-2/PGE-2 axis in EC cells.We conclude that miR-101 is involved in the regulation of COX-2 by BBR.更多还原