研究背景： 卵巢癌是严重威胁女性健康的一种生殖系统恶性肿瘤，发病隐匿，易早期转移，生存率低。研发新的治疗药物，了解其抗卵巢癌的分子机制，是改善卵巢癌患者预后的重要途径。前期研究发现，从红参中提取的中药活性单体——人参皂苷20(S)-Rg3能够抑制卵巢癌细胞的增殖和侵袭，其抗癌作用的主要机制之一是拮抗卵巢癌细胞的瓦伯格效应，但相关分子机制尚需阐明。 瓦伯格效应是肿瘤细胞葡萄糖代谢的一个特征性途径，是指肿瘤细胞在有氧情况下利用糖酵解而非氧化磷酸化途径进行葡萄糖代谢，由己糖激酶-2(hexokinase-2， HK2)、磷酸果糖激酶-1(phosphofructokinase-1，PFK-1)、丙酮酸激酶M2(pyruvate kinase isozyme M2，PKM2)和乳酸脱氢酶A(1actate dehydrogenase A，LDHA)等关键酶催化。瓦伯格效应不仅为肿瘤细胞的快速生长提供能量，还可提供其合成代谢的底物，形成利于肿瘤细胞增殖和侵袭的微环境，表明瓦伯格效应具有作为肿瘤治疗靶点的研究价值。前期发现20(S)-Rg3通过影响肿瘤微环境因素、或在转录、转录后、翻译和翻译后修饰等多个水平拮抗卵巢癌细胞的瓦伯格效应，并确定microRNA是其中一类重要的调控分子。通过microRNA测序和实验验证，已确定了两个20(S)-Rg3上调并拮抗瓦伯格效应的microRNA，但20(S)-Rg3是否调控更多的microRNA，形成拮抗卵巢癌细胞瓦伯格效应的miR网络而发挥其抑制卵巢癌细胞生长和侵袭的作用，值得深入研究。 研究目的： 确定人参皂苷20(S)-Rg3抑制卵巢癌细胞瓦伯格效应的新的microRNA调控途径，进一步奠定20(S)-Rg3作为卵巢癌候选药物的理论基础。 研究方法： 1.分析课题组前期通过高通量microRNA测序筛选出20(S)-Rg3处理卵巢癌细胞SKOV3后差异表达的microRNA，进行反转录实时定量PCR(qRT-PCR)验证后选择miR-603作为研究对象。 2.选择人卵巢癌细胞株SKOV3和A2780作为细胞模型，对SKOV3和A2780细胞分别进行如下处理：(1)转染miR-603的mimic过表达miR-603，或在用20(S)-Rg3处理细胞的同时转染miR-603的inhibitor抑制miR-603的活性；(2)用甲基化抑制剂5-Aza-CdR处理细胞；(3)利用siRNA敲低DNMT3A或在20(S)-Rg3处理细胞的同时过表达DNMT3A；(4)在过表达DNMT3A的同时转染miR-603的mimic或在用siRNA敲低DNMT3A的同时转染miR-603的inhibitor。随后采用qRT-PCR检测miR-603的水平，Western blotting检测DNMT3A和HK2的蛋白水平，甲基化特异性PCR(MSP)检测miR-603前体基因启动子区甲基化水平，试剂盒检测细胞葡萄糖摄取量和乳酸生成量，细胞增殖与毒性检测试剂盒(CCK8)检测细胞活力，平板克隆形成实验检测细胞的增殖能力，Transwell小室检测细胞迁移和侵袭能力的变化。 3.利用生物信息学软件预测miR-603与HK2的结合位点，通过双荧光素酶报告基因系统验证miR-603对HK2的直接靶向抑制作用。 4.构建SKOV3细胞的裸鼠皮下移植瘤模型，瘤内注射特殊化学修饰的miR-603激动剂(agomir-603)或阴性对照(agomir-NC)，每隔3d测定裸鼠体重及皮下移植瘤体积，用microPET／CT显像检测裸鼠移植瘤的葡萄糖摄取的变化，用免疫组织化学方法检测皮下移植瘤组织中HK2的表达水平。 5.采用qRT-PCR检测miR-603在21例卵巢癌患者卵巢癌及癌旁组织中的表达水平。 研究结果： 1.20(S)-Rg3能够上调miR-603的表达水平 microRNA测序发现20(S)-Rg3处理后miR-603在SKOV3细胞中的表达上调， qRT-PCR证实20(S)-Rg3可使SKOV3和A2780细胞中的miR-603的表达增加2倍以上。 2.20(S)-Rg3通过上调miR-603抑制卵巢癌细胞的瓦伯格效应 过表达miR-603使HK2表达下降，抑制SKOV3和A2780细胞的葡萄糖摄取和乳酸生成，并抑制细胞的增殖、迁移和侵袭。在20(S)-Rg3处理细胞的同时抑制miR-603可逆转20(S)-Rg3对HK2表达水平、葡萄糖摄取和乳酸生成、细胞增殖和迁移侵袭能力的抑制活性；双荧光素酶报告基因系统证实HK2是miR-603的直接靶点，提示20(S)-Rg3通过上调miR-603拮抗卵巢癌细胞瓦伯格效应。 3.DNMT3A介导的miR-603前体基因启动子区甲基化可以抑制miR-603的表达 生物信息学分析显示miR-603前体基因启动子区存在CpG岛，提示miR-603的表达可能受到DNA甲基化的负调控。用DNA甲基转移酶抑制剂5-Aza-cdR处理SKOV3和A2780细胞后，miR-603前体基因启动子区甲基化水平显著降低，miR-603表达水平显著升高，表明DNA甲基化抑制了miR-603表达。20(S)-Rg3显著降低DNMT3A的蛋白水平，进而下调miR-603前体基因启动子区的甲基化水平，最终导致miR-603表达升高。过表达DNMT3A可以逆转20(S)-Rg3导致的miR-603前体启动子区甲基化水平的降低和miR-603表达水平的上调；过表达miR-603可以拮抗DNMT3A过表达促进的瓦伯格效应和细胞迁移侵袭能力的增强；过表达DNMT3A可逆转20(S)-Rg3对HK2表达水平、葡萄糖摄取和乳酸生成以及迁移侵袭能力的抑制。敲低DNMT3A可以降低miR-603前体基因启动子区甲基化水平，并上调miR-603，抑制SKOV3和A2780细胞HK2的表达、葡萄糖摄取和乳酸生成以及细胞增殖、迁移和侵袭；在敲低DNMT3A的同时抑制miR-603的表达可逆转DNMT3A水平下降对HK2表达水平、葡萄糖摄取和乳酸生成以及增殖、迁移侵袭能力的抑制。 4.miR-603可以抑制卵巢癌细胞裸鼠移植瘤的生长 在用SKOV3细胞构建的裸鼠皮下移植瘤模型中，miR-603的agomir治疗组的皮下移植瘤生长受抑，肿瘤体积明显小于对照组，移植瘤组织中HK2的表达水平也明显降低。microPET／CT显像检测发现miR-603的agomir治疗组移植瘤的葡萄糖摄取率明显低于对照组。 5.miR-603在患者卵巢癌组织中表达低于癌旁组织 qRT-PCR检测发现，21例患者卵巢癌组织中有18例组织的miR-603表达水平显著低于癌旁组织，两组差异具有统计学意义。 结论： 1.miR-603通过靶向抑制HK2而拮抗卵巢癌细胞瓦伯格效应，在人卵巢癌中发挥抑癌作用； 2.DNMT3A介导的DNA甲基化负调控miR-603的表达； 3.人参皂苷20(S)-Rg3通过DNMT3A／miR-603／HK2途径抑制卵巢癌细胞的瓦伯格效应、增殖和侵袭； 4.miR-603可能成为治疗卵巢癌的一个候选分子； 5.人参皂苷20(S)-Rg3是治疗卵巢癌的一种候选药物。 关键词：卵巢癌；瓦伯格效应；人参皂苷；microRNA 论文类型：应用基础 *本研究得到国家自然科学基金基金(编号：81702576和30973429)资助。

 Backgrounds: Ovarian cancer is a malignant tumor of the reproductive system that seriously threatens women's health. It is occult, easy to metastasize, and has a low survival rate. The development of new therapeutic drugs and to understand its molecular mechanism of anti-ovarian cancer are important ways to improve the prognosis of patients with ovarian cancer. Previous studies have found that ginsenoside 20(S)-Rg3, an active monomer of traditional Chinese medicine extracted from red ginseng, can inhibit the proliferation and invasion of ovarian cancer cells. One of the main mechanisms of its anticancer effect is to antagonize the Warburg effect of ovarian cancer cells, but the molecular mechanisms involved need to be clarified. The Warburg effect is a characteristic mode of glucose metabolism in tumor cells. It refers to the glucose metabolism of tumor cells under aerobic conditions by glycolysis rather than oxidative phosphorylation pathway and is catalyzed by hexokinase-2, phosphofructokinase-1, pyruvate kinase M2 and lactate dehydrogenase. The Warburg effect not only provides energy for the rapid growth of tumor cells, but also provides its anabolic substrate, forming a microenvironment that is conducive to the proliferation and invasion of tumor cells, indicating that the Warburg effect has research value as a tumor therapy target. It was previously discovered that 20(S)-Rg3 antagonized the Warburg effect of ovarian cancer cells by affecting tumor microenvironment factors, or at various levels such as transcriptional, post-transcriptional, translational and post-translational modifications, and microRNAs are one of the important regulatory molecules. Through microRNA sequencing and experimental verification, two microRNAs up-regulated by 20(S)-Rg3 and antagonizing the Warburg effect have been identified, but whether 20(S)-Rg3 regulates more microRNAs and forms an miR network of anti-Warburg effect to play its role in inhibiting the growth and invasion of ovarian cancer cells is worthy of further study. Objectives: To determine the new microRNA regulatory pathway of ginsenoside 20(S)-Rg3 to inhibit the Warburg effect of ovarian cancer cells, and to lay the theoretical basis for 20(S)-Rg3 as a drug candidate for ovarian cancer. Methods: 1.High-throughput miR sequencing was used to screen differentially expressed microRNAs after treatment with 20(S)-Rg3 in SKOV3 cells. After qRT-PCR validation, miR-603 was selected as the research object. 2.Ovarian cancer cell lines SKOV3 and A2780 were selected as cell models, and SKOV3 and A2780 cells were treated as follows: (1) mimic-603 was transfected to overexpress miR-603, or inhibitor-603 was transfected into 20(S)-Rg3-treated cells; (2) the SKOV3 and A2780 cells were treated with DNA methylation inhibitor 5-Aza-CdR; (3) DNMT3A was knock-down with siRNA or overexpressed in 20(S)-Rg3-treated SKOV3 and A2780 cells; (4) mimic-603 was transfected while overexpressing DNMT3A or inhibitor-603 transfected while knocking down DNMT3A with siRNA. Then the level of miR-603 was detected by qRT-PCR; the protein level of DNMT3A and HK2 was detected by Western blotting; the methylation level of promoter region of miR-603 precursor gene was detected by MSP; the glucose consumption and lactate production were measured by special kits; the viability of cells was detected by CCK8; the ability of proliferation was detected by plate clone assay; cell migration and invasion were detected by Transwell chamber. 3.Bioinformatics software was used to predict the binding sites of miR-603 and HK2, and the direct inhibition of HK2 by miR-603 was verified by the dual luciferase reporter assay. 4.SKOV3 cells were subcutaneously injected into nude mice to establish xenograft models. Intratumoral injection with agomir-603 and its negative control was conducted. The body weight and subcutaneous tumor volume of nude mice were monitored every 3 days. The changes of glucose uptake in nude mice were detected by microPET/CT imaging. Tumor tissues were collected to evaluate the HK2 protein expression by immunohistochemical methods. 5.qRT-PCR was used to detect the expression of miR-603 in ovarian cancer and adjacent tissues in 21 patients with ovarian cancer. Results: 1.20(S)-Rg3 up-regulated miR-603 levels microRNA sequencing revealed that miR-603 was up-regulated in SKOV3 cells after 20(S)-Rg3 treatment, and qRT-PCR confirmed that 20(S)-Rg3 increased the expression of miR-603 in SKOV3 and A2780 cells by more than 2-fold. 2.20(S)-Rg3 inhibited Warburg effect by up-regulating miR-603 Overexpression of miR-603 could down-regulate the expression of HK2, inhibited glucose consumption, lactate production, cell proliferation, cell migration and invasion in SKOV3 and A2780 cells. Downregulation of miR-603 could reverse the inhibitory effects of 20(S)-Rg3 on HK2 expression, glucose consumption, lactate production, cell proliferation, cell migration and invasion. Dual luciferase reporter assay confirmed that HK2 was a direct target of miR-603, suggesting that 20(S)-Rg3 antagonized the Warburg effect of ovarian cancer cells by up-regulating miR-603. 3.DNMT3A-mediated methylation of the promoter region of miR-603 precursor gene inhibited expression of miR-603 Bioinformatics analysis revealed the presence of CpG islands in the promoter region of miR-603 precursor gene, suggesting that DNA methylation may negatively regulate miR-603. After treatment of SKOV3 and A2780 cells with 5-Aza-cdR, the methylation level of the promoter region of miR-603 precursor gene significantly decreased and miR-603 level increased, which indicated that DNA methylation inhibited the expression of miR-603. 20(S)-Rg3 significantly inhibited the protein expression of DNMT3A and down-regulated the methylation level of the promoter region of miR-603 precursor gene and eventually led to the increased expression of miR-603. Overexpression of DNMT3A reversed the decrease in methylation level of the promoter region of miR-603 precursor gene and the upregulation of miR-603 expression levels caused by 20(S)-Rg3. Overexpression of miR-603 could antagonize the Warburg effect, cell migration and invasion promoted by overexpression of DNMT3A. Overexpression of DNMT3A reversed the inhibitory effect of 20(S)-Rg3 on HK2 expression, glucose uptake and lactate production, as well as cell migration and invasion. Knockdown of DNMT3A reduced the methylation level of the promoter region of miR-603 precursor gene and increased miR-603 expression, thus inhibited HK2 expression, glucose uptake and lactate production, cell proliferation, cell migration and invasion in SKOV3 and A2780 cells. Inhibiting the expression of miR-603 could reverse the decrease of HK2 expression level, glucose consumption, lactate production, cell proliferation, cell migration and invasion caused by knock-down of DNMT3A. 4.miR-603 could inhibit the growth of ovarian cancer xenografts in nude mice In the subcutaneous xenograft model of nude mice constructed with SKOV3 cells, the growth of subcutaneous xenografts in the miR-603 agomir treatment group was inhibited, the tumor volume was significantly smaller than that of the negative control group, and the expression level of HK2 was also significantly decreased in the tumor tissues of miR-603 agomir treatment group. microPET/CT imaging showed that the glucose uptake rate of subcutaneous tumors in miR-603 agomir treatment group was significantly lower than that in the negative control group. 5.miR-603 is expressed lower in ovarian cancer tissues than the adjacent qRT-PCR showed that the expression of miR-603 in 18 out of 21 patients with ovarian cancer was significantly lower in cancer tissues than that in adjacent tissues. The difference between the two groups was statistically significant. Conclusions: 1.miR-603 exerts a tumor suppressor effect in ovarian cancer by antagonizing Warburg effect and directly targeting HK2; 2.DNMT3A-mediated DNA methylation negatively regulates miR-603; 3.Ginsenoside 20(S)-Rg3 inhibits the Warburg effect, proliferation and invasion of ovarian cancer cells through the DNMT3A/miR-603/HK2 pathway; 4.miR-603 may be a candidate for the treatment of ovarian cancer; 5.Ginsenoside 20(S)-Rg3 is a candidate drug for the treatment of ovarian cancer. KEY WORDS: Ovarian cancer; Warburg effect; Ginsenoside; microRNA TYPE OF DISSERTATION: Application Fundamentals *The study was funded by the National Natural Science Foundation of China (No. 81702576 and 30973429).