【作者】 白莎莎；

【导师】 庞义存；

【作者基本信息】 河北医科大学 ， 妇产科学， 2012， 硕士

【摘要】 目的:绒癌（choriocarcinoma CC）是一种高度恶性的肿瘤，早期即可发生血行转移，导致全身播散。虽然目前化疗效果比较好，但随着耐药病例的出现，使绒癌的治疗出现了新的困难。因此，如何提高这部分患者的治愈率及生存率是临床医生所面临的一个难题。microRNA（miRNA）是一类广泛存在于真核生物中，长度约为22个核苷酸的非编码单链小分子RNA，能通过与靶mRNA特异性的碱基配对引起靶mRNA的降解或抑制其翻译，从而在转录后水平对基因的表达进行调控。它们与人类的生长发育、干细胞分化与自我更新以及肿瘤、心脏疾病、艾滋病等多种疾病关系密切，尤其在肿瘤的发生发展过程中，扮演着重要角色。miR-34最先发现于线虫，并在一些非脊椎动物中保持同源性，是一类保守的、非编码miRNA。人类miR-34包括miR-34a、miR-34b和miR-34c等，基因结构的预测结果表明：成熟mir-34a序列位于其主基因原初转录本外显子1下游约30kb的外显子2内；成熟mir-34b序列位于该基因原始转录本的内含子1内；mir-34c序列位于同一主基因原初转录本的外显子2内。根据miRBase序列数据库及ensembl数据库资料显示：has-mir-34a定位于1p36.23，其茎环结构序列位于1号染色体第9、134、314位至第9、134、423位负链上，共计110个核甘酸序列；has-mir-34b和has-mir-34c共同位于11q23.1，前者茎环结构序列位于11号染色体第110、888、873位至第110、888、956位正链，共计84个核苷酸序列；后者茎环结构序列位于该染色体第110、889、374位至第110、889、450位正链，共计77个核苷酸序列。miR34在多种肿瘤中都呈现非正常表达。miR-34通过被p53激活，抑制E2F3、Bcl-2、c-myc、CDK4、CDK6、Cyclin D1以及CyclinE2的表达，使肿瘤细胞停滞在G1期，抑制肿瘤细胞的生长，诱导肿瘤细胞凋亡，并通过E2F3、SIRT1与p53形成正反馈环路，不断增强其自身和p53的作用。最近研究证实，miR34能够在多种肿瘤细胞中表达：人类结肠癌的发展过程中miR34a的表达下调，在胰腺癌肿瘤细胞和儿童神经母细胞瘤（neuroblastoma）中miR34a的表达经常缺失；同时人为提高miR34a的表达能够抑制细胞增殖并激活细胞凋亡途径。这些研究表明miR34a表达的下调或缺失与肿瘤的发生和细胞增殖之间存在着联系，miR34a的表达对抑制细胞增殖，激活细胞凋亡途径有重要意义。ValeryTarasovl等研究证实，miR34a是P53基因的直接靶标之一，染色体免疫沉淀反应和荧光素酶分析证实P53基因活化miR34基因转录，miR34家族的miRNA可以和P53的其它靶标，如P21和BAX一起，在可促使恶性肿瘤发生的应激条件下促进细胞生长停滞和死亡的发生。P53基因做为一种细胞增殖负调节因子,调控细胞的生长和分化,维持基因组DNA的稳定,其突变或缺失所致的P53失功能可能在肿瘤发生、发展中起着重要的作用。研究发现P53基因与滋养细胞肿瘤的生物学行为有密切关系。P53基因的表达与葡萄胎滋养细胞的增生程度有关，并且P53的细胞凋亡调控水平失衡对于葡萄胎由良性转变为恶性起了重要作用。同时研究发现，在MCF-7细胞系中加入能够激活p53基因的依托泊苷（etoposide）能够诱发大小约为3.7kb的mir-34a原初转录本（pri-mir-34a）的表达。因此，本实验通过应用依托泊苷作用于人绒毛膜癌细胞株JEG-3，以期上调microRNA34a （miR34a）在人绒毛膜癌细胞株JEG-3中的表达，同时观察不同药物作用时间内miR34a表达情况，并检测不同时间JEG-3绒癌细胞增殖和凋亡的情况，探讨miR34a在绒癌发生发展过程中的作用，为绒癌的诊断和基因治疗提供理论依据。方法:1.用含15%胎牛血清的RPMI-1640培养JEG-3细胞，37℃、5%CO₂细胞培养箱中培养。2.利用临床应用化疗药依托泊苷体外上调人绒毛膜癌JEG-3细胞中miR34a表达，将培养细胞分为：对照组（常规培养，未进行任何处理）、加药6小时、12小时、24小时和48小时组。3.倒置显微镜下观察细胞形态学变化。4.一步法Trizol提取细胞中总RNA，实时定量PCR方法检测各组细胞miR34a表达情况，实验重复三次。5.四甲基偶氮唑蓝（MTT）法检测各组细胞增殖能力，实验设4个平行复孔。6.流式细胞仪检测各组细胞凋亡率，实验重复三次。7.采用SPSS13.0统计软件对所有数据进行统计学分析。数据以均数±标准差（x±S）表示，两组间均数比较用t检验，多组间均数比较正态、方差齐采用单因素方差分析，非正态或方差不齐采用秩检验，p>0.05为无显著性差异，p<0.05为具有显著性差异，所有实验均重复3次。结果:1.绒毛膜癌JEG-3细胞用含有15%（体积分数）胎牛血清的1640培养基，在37℃、5%（体积分数）CO₂培养箱中培养。其生长状态良好，细胞贴壁生长、椭圆形或圆形、排列紧密、大小均匀、边界清楚、细胞膜完整，细胞核界限清晰，胞浆中可见黑色分泌颗粒。2.用药6-24小时，部分贴壁细胞死亡，漂浮于培养液中，剩余细胞形态尚好，用药48小时，大部分贴壁细胞死亡，漂浮于培养液中，剩余细胞形态渐不规则，细胞大小不等，有的细胞可有长的伪足，胞浆中黑色颗粒增多。3.绒毛膜癌JEG-3细胞中有miR34a表达。将对照组miR34a表达水平作为1，使用依托泊苷作用6小时、12小时、24小时miR34a表达水平分别为1.378±0.511，1.667±0.982，1.742±0.405，较对照组分别上调37.8%，66.7%，74.2%，但与对照组比较差异无明显统计学意义（P＞0.05）；48小时miR34a表达水平为5.423±3.611，较对照组上调442.3%，与对照组比较差异有统计学意义（P<0.05）。4.MTT法检测各组细胞增殖能力：6小时下降16.97%,12小时下降42.56%，24小时下降49.00%，48小时下降70.57%，与对照组比较差异均有统计学意义（P<0.05）。5.流式细胞术检测细胞凋亡率：6小时增加4.92倍，12小时增加7.46倍，24小时增加8.76倍，48小时增加9.23倍，与对照组比较差异均有统计学意义（P<0.05）。结论:人绒毛膜癌细胞株JEG-3中有microRNA34a的表达；依托泊苷可诱导microRNA34a高表达，随作用时间的延长上调明显；microRNA34a可能有降低细胞的增殖能力的作用，但microRNA34a表达量与细胞增值率两者变化不同步，考虑microRNA34a只是导致细胞增殖能力下降的原因之一；microRNA34a可能有激活细胞凋亡途径的作用，但microRNA34a表达量与细胞凋亡率两者变化不同步，考虑microRNA34a只是影响细胞凋亡的原因之一；microRNA34a可能对绒癌的发生发展起抑制作用。更多还原

【Abstract】 Objective:Choriocarcinoma （CC） is a high malignant tumor,at earlyperiod,it can hematogenous metastasis, spreading all over the body. Althoughthe effect of chemotherapy, but with the emergence of drug-resistant cases, thetreatment of choriocarcinoma have new difficulties. Therefore, how toimprove the cure rate and survival rate of these patients is a problem faced byclinicians. microRNA （miRNA） is a kind of non-coding single-stranded RNAmolecules，which is widespread in eukaryotes with about22nucleotidelength and through base pairing with the specificity target mRNA to causedegradation or inhibition of its target mRNA translation, then to regulate geneexpression at the posttranscriptional level. They are closely related to humangrowth and development, stem cell differentiation, self-renewal and tumor,heart disease, AIDS and other diseases, in particular, play an important role inthe tumor development process. mir-34is a conservative, non-coding miRNAwhich was first discovered in C. elegans, and keep homology in someinvertebrates. Human miR-34, including miR-34a, miR-34b and miR-34c,gene structure prediction showed that the mature mir-34a sequence is in theoriginal transcription exon2of its major gene, the position is located³0kb inthe downstream of this original transcription exon1. At the same time, themature mir-34b and mir-34c sequence in the original transcript of the samemajor gene intron1and exon2. According to the miRBase SequenceDatabase and ensemble database information, has-mir-34a located in the1p36.23,which sequence of the stem-loop structure is located on the1,9,134,314to9,134,423minus-strand of chromosome1,for a total of110nucleotide sequence; has-mir-34b, has-mir-34c are all located in11q23.1, thestem-loop structure sequences were located on chromosome11in the first110,888,873to110,888,956bits chain and the first110,889,374to 110,889,450positive-strand, respectively, a total of84and77nucleotidesequence. miR34have shown abnormal expression in a variety of tumors.miR34through p53activation, inhibition of the expression of E2F3, Bcl-2,c-myc, CDK4, CDK6,Cyclin D1, and Cyclin E2, to arrest the tumor cells inG1, inhibiting tumor cell growth and inducing tumor cell apoptosis,meanwhile，E2F3, of SIRT1and p53form a positive feedback loop, andconstantly enhance the role of itself and p53. Recent studies have confirmedthat miR34can be expressed in a variety of tumor cells: miR34a expression inhuman colon cancer development process down regulation, it’s often missingin the in pancreatic cancer cells and neuroblastoma cell tumor; meanwhile,increase miR34a expression artificially can inhibit cell proliferation andactivate cell apoptotic pathway. These studies suggest that there is a linkbetween the miR34a expression down regulation or missing and tumorigenesisand cell proliferation, miR34a expression have important significance ininhibiting cell proliferation and activation of the apoptotic pathway, ValeryTarasovl et al. research confirms that miR34a is one of P53gene directlytarget, chromosome immunoprecipitation reaction and luciferase enzymeanalysis confirmed the P53gene activate miR34gene transcription, miR34family can promote cell growth arrest and death occurred with P53othertarget gene, such as P21and BAX under stress which can contribute tomalignancy. As a cell proliferation negative regulator, P53gene regulate cellgrowth and differentiation, to maintain the stability of the genomic DNA, theloss of P53function caused by P53mutation or deletion may plays animportant role in the tumor occurring and development. The study found thatP53gene related closely with the biological behavior of trophoblastic tumor.P53gene expression was related to the degree of mole trophoblastichyperplasia, and P53regulation of apoptosis level imbalance played animportant role to mole changing from benign to malignant. Also found thatetoposide can activate the p53gene in the MCF-7cell lines can induceoriginal transcription of mir-34a （pri-mir-34a） expression of3.7kb size.Therefore, this experiment through the use of etoposide administered in human choriocarcinoma cell line in JEG-3, in order to increase m icroRNA34a（miR34a） expression, and observing the expression of m iR34a withindifferent drug action time, detecting JEG-3choriocarcinoma cell proliferationand apoptosis at different time. And exploring the role of miR34a inchoriocarcinoma occurring and developing to provide a basis theory for thediagnosis and gene therapy of choriocarcinoma.Method:1. Culturing JEG-3cells with RPMI-1640which containing15%fetal bovine serum at37°C,5%CO₂cell incubator culture.2. Useclinical application chemotherapy drug etoposide raised the miR34aexpression of human choriocarcinoma JEG-3cells in vitro, the cultured cellsare divided into: control group （conventional training, did not carry out anyprocessing）,6hours,12hours,24hours and48hours of dosing group.3.Under the inverted microscope to observe the cell morphological changes4.one step way Rrizol extracted total RNA, real-time PCR to detect miR34aexpression of different groups, the experiment was repeated three times.5.Four methyl thiazolyl tetrazolium （MTT） method detect cell proliferationability, the experimental set up four parallel hole6. flow cytometry detect cellapoptosis rate, the experiment was repeated three times.7All data werestatistically analyzed using SPSS13.0statistical software. Data presented asmean±standard differential （x±S）, between the two groups, the number witht-test, interclass use one-way ANOVA or rank test, p>0.05for no significantdifference, p<0.05as has significantly difference, all the experimental resultswere repeated three times.Results:1choriocarcinoma JEG-3cell cultured in1640mediumcontaining15%（volume fraction） fetal bovine serum,,at37°C,5%（volumefraction） CO₂incubator. The cell is in good growth state, adherent growth,oval or round, tightly packed, uniform size, clear boundary, cell membraneintegrity, cell nucleus boundary is clear, visible secretory granules in thecytoplasm.2at6-24hour after administered：part of the adherent cells weredead, floating in the culture medium, the remaining cell morphology is stillgood；at48hour after administered：most of the adherent cells were dead, floating in the culture medium, the remaining cells form becoming irregular.the cells vary in size, some cells may have long pseudopodia, black particleincreased in the cytoplasm.3miR34a expressed in choriocarcinoma JEG-3cells. Control group miR34a expression level as1, the etoposide role6hour,12hour,24hour,48hour miR34a expression level is1.378±0.511,1.667±0.982,1.742±0.405, compared with the control group increases37.8%,66.7%,74.2%, but the difference was not have statistically significant （P﹥0.05）;48hours miR34a expression level was5.423±3.611, compared withthe control group increase of442.3%, and the difference was statisticallysignificant （P <0.05）.4.MTT method to detect the proliferation ability:6hoursdecreased by16.97%,12hours decreased by42.56%,24hours decreased by49.00%, decreased by70.57%in48hours, the differences were statisticallysignificant compared with the control group （P <0.05）.5. flow cytometry todetect apoptosis rate:6hours increased4.92times,12hours increased7.46times,24hours increased8.76times,9.23times in48hours, compared withthe control group differences were statistically significant （P <0.05）.Conclusion: MiR34a expressed in chorionic carcinoma JEG-3cells; miR34a expression can be up-regulated through chemotherapy drug etoposide，；microRNA34a maybe inhibits cell proliferation, but microRNA34a expressionand cell proliferation rate change are not in-phase，microRNA34a maybe onlyone of the inhibiting cell proliferation rate factor；microRNA34a maybeinducing cell apoptosis, but microRNA34a expression and cell apoptosis ratechange are not in-phase，microRNA34a maybe only one of the inducing cellapoptosis rate factor； microRNA34a may inhibits the occurring anddeveloping of choriocarcinoma.更多还原