【作者】 李成林；

【导师】 王雅棣；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2014， 博士

【摘要】 我国食管癌发病和死亡人数约占全球的二分之一。放射治疗是目前食管癌主要的治疗手段之一，但是5年生存率仅有10%～30%。研究表明，肿瘤乏氧细胞的产生和存在使肿瘤对放疗的抗拒性增加，是影响肿瘤放射治疗疗效的主要原因之一。乏氧诱导因子-lα（hypoxia inducible factor-1，HIF-lα）是介导肿瘤细胞的乏氧适应性变化的关键调控因子。近年来的研究表明，miR-210作为HIF-1α新发现的下游靶基因，参与了多种细胞生物学功能的调控，如：能量代谢、细胞周期、DNA损伤修复等。已有研究证实，miR-210过表达可引起肿瘤细胞G1/S期和/或G2/M期阻滞，干扰有丝分裂进程，抑制肿瘤生长。其中G1/S期阻滞与miR-210特异性抑制E2F转录因子3（E2Ftranscription factor3， E2F3）和成纤维细胞生长因子受体样因子1（Fibroblast Growth Factor Receptor-like1，FGFRL1）的表达有关。然而目前关于miR-210诱导G2/M期阻滞所涉及的靶基因还鲜有报道，具体调控机制也尚不明了。有研究称原癌基因（c-MYC）的竞争性抑制剂Max结合蛋白（Max’s next tango，MNT）也是miR-210的靶基因，在乏氧条件下，miR-210可以通过抑制它的表达加速细胞周期进程。因此，miR-210调控细胞周期的机制及其对细胞增殖的具体影响都还有待进一步揭示。循环miRNA具有单链小分子特性，多以RNA/蛋白复合体的形式存在，稳定性比其他核酸分子高，RNA酶不易降解，适合作为分子标志物。此前有3项研究分别报道了循环miR-210在胰腺、乳腺、肾脏肿瘤中高表达。尽管上述研究的样本量较小，但结果的一致性表明循环miR-210的差异性表达或可为肿瘤诊断提供帮助。目前还未见食管癌患者循环miR-210表达情况的报道，而且此前的研究多关注于手术对肿瘤患者循环miRNA表达的影响，对于根治性放疗前后循环miRNA表达的变化还鲜有报道。本研究首先观察并检测了人食管鳞癌Eca109细胞在乏氧条件下miR-210的表达情况，通过转染miR-210mimics观察miR-210过表达对Eca109细胞增殖、周期、凋亡的影响，并探讨其可能的作用机制；其次，应用生物信息技术筛选出miR-210调控G2/M期转换的可能靶基因Plk1，通过构建荧光素酶报告质粒pmiR-RB-REPORTTM-Plk1，检验Plk1基因mRNA的3’UTR区域是否包含miR-210的作用结合位点，并检测过表达miR-210对Plk1蛋白表达水平的影响，验证Plk1是否是miR-210的直接靶基因；最后，通过检测食管癌患者及健康志愿者血浆miR-210的表达水平，并对比食管癌患者根治性放疗前后血浆miR-210的表达变化，了解血浆miR-210对食管癌的诊断及放疗疗效评估价值。第一部分miR-210在乏氧食管Eca109细胞中的表达及其对细胞生物学行为的影响目的：检测miR-210在乏氧食管Eca109细胞中的表达水平，并探讨其对细胞增殖、周期、凋亡的影响。方法：RT-PCR检测不同乏氧时相食管鳞癌Eca109细胞中miR-210的表达。采用CCK-8增殖实验、EdU增殖实验、流式细胞仪检测miR-210过表达对Eca109细胞增殖、周期、凋亡的影响结果：经RT-PCR检测， Eca109细胞乏氧培养12h后miR-210的表达就有明显增高，24h后达峰值，较常氧组差异有统计学意义（P<0.001）。CCK-8及EdU细胞增殖实验显示，miR-210mimics转染24、48h后Eca109/miR-210组增殖细胞活力比例均较Eca109/Control组和Eca109/ncRNA组显著下降，差异有统计学意义（P<0.001）。流式细胞分析显示，转染24h后，同Eca109/Control和Eca109/ncRNA相比，Eca109/miR-210组出G2/M期细胞比例明显增高，差异有统计学意义（P<0.05）；转染48h后，Eca109/miR-210组G2/M期增多更为明显（P<0.01）。然而，Eca109/miR-210、Eca109/Control和Eca109/ncRNA三组转染24、48h后细胞凋亡比例未见明显变化。结论：miR-210在乏氧食管Eca109细胞中高表达，miR-210过表达抑制细胞增殖，其机制可能与G2/M期阻滞相关。第二部分miR-210靶向调控Plk1基因表达的研究目的：筛选miR-210调控G2/M期转换的可能靶基因，并验证miR-210对预测靶基因的调控关系。方法：应用生物信息学手段对miR-210的可能靶基因进行预测，经酶切及基因测序鉴定构建含有预测靶基因3’-UTR报告载体pmiR-RB-REPORTTM，应用荧光素酶报告系统检测细胞内荧光活性的变化。WesternBlot检测miR-210对预测靶基因蛋白表达的影响。结果：通过生物信息学预测，筛选Plk1作为miR-210调控G2/M期转换的潜在靶基因。经过酶切及基因测序鉴定，成功构建含有Plk1基因3’-UTR荧光素酶报告载体pmiR-RB-REPORTTM-Plk1。与共转染pmiR-RB-Report_PLK1+mimicsNC相比，HEK293T/17细胞共转染pmiR-RB-Report_PLK1+mimics210后荧光素酶活性显著下（P<0.01）。转染miR-210mimics48h后，对Eca109细胞进行Plk1蛋白的检测，结果提示与Eca109/Control组及Eca109/NC组比较，Eca109/miR-210组Plk1蛋白表达水平显著下降（P<0.01）。结论：miR-210与靶基因Plk1mRNA3’-UTR能够有效结合，miR-210过表达抑制Plk1基因蛋白表达。Plk1是miR-210的直接靶基因。第三部分放疗对食管鳞癌患者血浆miR-210、miR-21表达水平的影响目的：检测食管癌患者血浆miR-210、miR-21表达水平，并评估根治性放疗对血浆miR-210、miR-21表达水平的影响。方法：采集食管癌初治患者根治性放疗前后及健康志愿者血液标本，应用TaqMan探针实时荧光定量RT-PCR法检测血浆miR-210和miR-21的表达水平。结果：22例初治食管鳞癌患者及15例健康志愿者入组，共采集合格血液样本59份。RT-PCR显示，食管癌组血浆miR-210、miR-21的表达较健康对照组明显升高（P<0.01）。血浆miR-210、miR-21表达水平与食管癌病变位置和分化程度无关。根治性放疗后，食管癌组血浆miR-210、miR-21的表达均显著升高，其中miR-210升高的更为显著（P<0.01，P<0.05）。结论：miR-210、miR-21在食管癌患者血浆中高表达，根治性放疗影响二者的表达水平。更多还原

【Abstract】 Esophageal carcinoma is one of the most common malignant diseases inChina, with high incidence and high mortality characteristics.90%of esopha-geal tumors are esophageal squamous cell carcinoma (ESCC) in in Asia-pacific region. Currently radiotherapy is the mainstay in the treatment ofesophageal cancer, but the local failure has remained a major concern, withpersistent or recurrent disease being reported in around60-80%of patients,and an overall5-year survival rate of10%.Intratumoral hypoxia is a hallmark of most solid tumors and results fromincreased oxygen consumption and/or insufficient blood supply. Evidenceobtained from radiochemical and radiobiological studies has revealed theseproblems to be caused, at least in part, by a tumor-specific microenvironment,hypoxia. Many of the hypoxia-induced cellular responses are mediatedthrough the hypoxia-inducible factors (HIFs), which regulate genes involvedin angiogenesis, survival, cell metabolism, invasion and other functions.Recently, a number of miRNAs induced during hypoxia have been identified.One of these miRNAs, miR-210, is strongly induced by HIF-lα and haspleiotropic effects. Several recent studies also demonstrated that miR-210inhibited tumor cell proliferation by inducing G0/G1and/or G2/M phase cellcycle arrest. In contrast to other solid tumors, miR-210is frequently under-expressed in ovarian cancers, which potentially leads to increased expressionof E2F transcription factor3(E2F3) which participates in the regulation of thecell cycle. Likewise, miR-210is down-regulated in esophageal squamous cellcarcinoma. MiR-210inhibits cancer cell proliferation and induces G0/G1phase cell cycle arrest by derepressing fibroblast growth factor receptor-like1(FGFRL1) that in turn accelerates cell cycle progression. However, onecannot generally state that miR-210induction in hypoxia negatively regulates cell cycle progression and proliferation. In hepatic cancer cells, miR-210activates the myc pathway, via downregulation of the c-Myc antagonist MNT,and loss of MYC abolished miR-210-mediated override of hypoxia-inducedcell cycle arrest. Therefore, the net impact of miR-210on cell cycle regulationseems to be context dependent.In previous studies, the miR-210up/down-regulated expression isreported in other malignancy tissues, such as cancers of the head and neck,pancreas, breast, and ovarian. Recently, studies on the role of circulating miR-210in cancer and its potential utility as prognostic markers have emerged.Lately, the correlation of circulating miR-210levels with breast cancermortality is a striking one. Eun-Jung Jung and colleagues reported plasmamiR-210levels correlate with sensitivity to trastuzumab, tumor presence, andlymph node metastases in breast cancer patients. Another two studies showeda statistically significant four-fold increase of circulating miR-210in expres-sion in pancreatic and clear cell renal cancer patients compared with normalcontrols. Although the results of these three studies were obtained from smallcohorts, the presence of elevated miR-210in the plasma of cancer patientssuggests that this may be an important feature of these diseases, which mayfurther advance our understanding of the underlying pathogenesis of cancers.In present study, we investigated the functional role of miR-210in thegrowth of carcinomas and the mechanism by which it acts. Next, we analyzedthe serum miR-210expression level in ESCC patients for the first time.Furthermore, our work also revealed the potential role of serum miR-210levelas response markers during radiotherapy for ESCC patients.Part1The expression of miR-210in hypoxic esophageal carcinomaEca109cells and its effects on biological behiviorObjective: To detect the expression of miR-210in hypoxic esophagealcarcinoma Eca109cells and explore its effects on proliferation, cell cycle andapoptosis.Methods: After being treated with hypoxia, miR-210at different hypoxicculture phases were detected by RT-PCR. Furthermore, and the proliferation, cycle distribution and apoptosis of Eca109cells were detected with CCK-8,EdU and flow cytometry after transfection.Results: Under hypoxia, the level of miR-210increased obviously.Transfection of miR-210significantly decreased the proliferation of cancercells in CCK-8test and reduced the uptake of EdU, respectively. Transfectionof miR-210resulted in a significant increase in the proportion of cells in G2/Mphase. No significant apoptotic changes were found.Conclusion: miR-210was inducted by hypoxia in ESCC cells; miR-210may inhibit proliferation of mainly by inducing cell cycle arrest in G2/Mphase.Part2miR-210targeted the expression ofPlk1geneObjective: To explore the candidate target of miR-210that reugulatesG2/M arrst.Methods: The candidate target of miR-210that reugulate G2/M arrst waspredicted by bioinformation.The dual luciferase vector which contains3’-UTRof the target was constructed, and was regared as the binding sites withmiR-210. The expression of the target gene in Eca109cells was investigatedafter transfection by Western blot.Results: Plk1was predicted as the specific target gene of miR-34a bybioinformation method.3’-UTR of Plk1which was contained in the dualluciferase recombinant vector was successfully constructed and vertified byenzyme digestion and gene sequence methods. The results of luciferaseWhenmiR-210oligos were transfected into293T/17cells with the reporter constructpmiR-RB-Report_Plk1, luciferase activity was repressed more than50%compared with transfection of scramble oligos. The level of Plk1proteinexpression in transfected cells with miR-210oligos was significantly decree-ased compared with that of cells with stable integration of the scramblesequence after48hours.Conclusion:3’-UTR of Plk1was the binding sites with miR-210andmiR-210inhabited its protein expression, which suggested Plk1was the targetgenes ofmiR-210. Part3The effect of radiotherapy on plasma miR-210and miR-21inesophageal cancer patientsObjective: To analyze the miR-210and miR-21expression level inesophageal cancer patients and reveale the potential role of their level asresponse markers during radiotherapy.Methods: Plasma miR-210and miR-21expression level of ESCCpatients during radiotherapy and healthy controls were measured by usingreal-time RT-PCR.Results: A totle of55plasma samples from22ESCC patients before＆after radiotherapy and15healthy controls were measured. Plasmaconcentration of miR-210and miR-21in ESCC patients were significantlyhigher than that in healthy controls (P=0.0024;P=0.0004). No significantassociation was found between the levels of the two miRNAs and sex, age,tumor location and differentiation. Furthermore, a significant elevation in theserum miR-210and miR-21levels were observed in the postradiotherapysamples versus the preradiotherapy samples (P=0.0025;P=0.0316).Conclusion: The results suggested that a statistically significant increaseof plasma miR-210miR-21expression in esophageal cancer patientscompared with normal controls.Their potential utility as prognostic andresponse markers during radiotherapy markers had emerged.更多还原