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学科主题: 肿瘤学
题名:
MELK在胃癌中作为不良预后的指标和潜在的治疗靶点
作者: 李沈
答辩日期: 2016-05-25
导师: 季加孚
专业: 肿瘤学
授予单位: 北京大学
授予地点: 北京大学临床肿瘤学院
学位: 博士
关键词: 胃癌 ; MELK ; 预后 ; 转移 ; PDX模型
其他题名: Maternal embryonic leucine zipper kinase serves as a poor prognosis marker and therapeutic target in gastric cancer
分类号: R735.2
摘要:

背景与目的:

在全世界范围内,胃癌是第四常见的恶性肿瘤,在肿瘤相关性死亡中位居第二位。尽管胃癌的诊断和治疗水平在不断的提高,但是胃癌的5年生存率依旧很低。原因在于胃癌在早期阶段没有症状或者症状不特异,当被诊断为胃癌时,患者经常处于进展期,或者转移到了其他脏器。然而,远处转移是引起胃癌患者死亡的只要原因之一。因此我们需要对胃癌进展的分子机制进行进一步研究,以便发现更有效的药物治疗靶点。母系胚胎亮氨酸拉链激酶(MELK)是丝氨酸、苏氨酸蛋白激酶snf1/AMPK家族中的一员。MELK参与各种细胞活动的过程,包括细胞周期、细胞增殖、凋亡、RNA的剪切和胚胎的发育。而且,MELK参与多种蛋白的相互作用,并通过这种相互作用作用于肿瘤形成的多个阶段,已有研究显示其在多种肿瘤中发挥重要作用,但在胃癌中的表达和作用机制尚不清楚,因此本课题将对MELK在胃癌中的功能机制进行深入研究。

材料与方法:

本研究我们通过免疫组化的方法检测178例胃癌组织中MELK基因的表达情况,分析其与临床病理参数和预后的关系。同时我们通过Western Blot检测了MELK在胃癌细胞系中和配对胃癌组织中的表达情况。然后我们选择MELK高表达的胃癌细胞系进行体外功能实验。通过敲低MELK的表达,或者利用小分子药抑制其激酶活性来观察其对细胞增殖、细胞周期、细胞凋亡、侵袭和迁移等恶性表型的影响。根据体外实验的结果,选择移植肿瘤动物模型进行体内功能研究。通过相关实验观察,了解其过表达对肿瘤生物学行为的影响。最后,利用胃癌PDX模型来验证MELK小分子抑制剂对胃癌的治疗疗效。

结果:

MELK在胃癌细胞系及胃癌组织中均呈现高表达。免疫组化结果显示,MELK高表达与肿瘤浸润深度(P=0.004)、Lauren分型(P=0.004)、淋巴结转移(P=0.010)和肿瘤远处转移(P=0.024)密切相关,MELK表达阳性患者预后明显比阴性表达者差(P=0.008),并且多因素回归显示其为独立预后因子(P= 0.017)。敲低MELK的表达或者用小分子化合物抑制MELK激酶的活性明显抑制了胃癌细胞的增殖,经进一步分析发现这可能与MELK通路的阻断导致了胃癌细胞发生G2/M期的阻滞和细胞凋亡有关。MELK的敲低能降低胃癌细胞的侵袭转移能力,并且能在小鼠移植模型中能减少转移克隆的形成,更有意义的是我们第一次发现MELK参与到EMT过程当中。且在胃癌患者来源的小鼠移植瘤(PDX)治疗模型中,我们发现MELK小分子抑制剂OTSSP167具有明显的抗瘤效果。

结论:

MELK在胃癌中普遍高表达,是胃癌的独立预后因子,抑制胃癌细胞凋亡、促进G2/M期的转化,并通过EMT途径促进胃癌细胞的侵袭和转移。

MELK小分子抑制OTSSP167在胃癌PDX模型中有明显的抑瘤效果, MELK是胃癌治疗的一个潜在的治疗靶点。

英文摘要:

Background and Objectives:

Gastric cancer is the fourth most common malignant tumor and the second leading cause of cancer-related deaths. Despite improvements in detection and management, the 5-year survival rate for GC remains low. Because patients in the early stages of GC are either asymptomatic or report only nonspecific symptoms, by the time of diagnosis the tumor has often progressed to an advanced stage or has even metastasized to distant organs. Metastasis is the most common cause of death in patients with GC. Further study of the molecular mechanisms of GC development and progression may help identify new molecular targets for more effective therapies. Maternal embryonic leucine zipper kinase (MELK) is a member of the snf1/AMPK family of protein serinethreonine kinases. Data collected previously using an Affimetrix HG-133 array showed a 3.84-fold increase in MELK expression in 79 GC tissues as compared to 24 non-cancerous tissues, making it one of the most upregulated genes in GC (P<0.05). MELK participates in diverse processes, including cell cycle, cell proliferation [10], apoptosis, RNA processing, and embryonic development. Furthermore, MELK is involved in multiple protein interactions that affect many stages of tumorigenesis. MELK plays important functions in several cancer types but its expression in gastric cancer remains unknown. This study was also designed to investigate MELK expression in gastric cancer.

Materials and methods:

MELK expression in primary GCs (n=178) was examined by IHC. GC cells with depleted endogenous MELK expression was established using lentivirus that encoded small hairpin RNA (shRNA) against MELK, and proliferation assay, migration, invasion and flow cytometry-based assay were down. GC cell-derived xenograft models were used to assess the influence of MELK depletion on tumor growth and metastasis. Anti tumor efficacy of MELK inhibitor OTSSP167 was evaluated in the GC patient-derived xenograft (GC-PDX) mouse models.

Results:

MELK was frequently overexpressed in primary GCs, and higher MELK levels correlated with worse clinical outcomes. Reducing MELK expression or inhibiting kinase activity resulted in growth inhibition, G2/M arrest, apoptosis and suppression of invasive capability of GC cells in vitro and in vivo. MELK knockdown led to alteration of epithelial mesenchymal transition (EMT)-associated proteins. Furthermore, targeting treatment with OTSSP167 in GC patient-derived xenograft (PDX) models had anticancer effects.

Conclusion:

MELK promotes cell growth and invasiveness by inhibiting apoptosis and promoting G2/M transition and EMT in GC. These results suggest that MELK may be a promising target for GC treatment.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/125693
Appears in Collections:北京大学临床肿瘤学院_学位论文

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作者单位: 北京大学临床肿瘤学院

Recommended Citation:
李沈. MELK在胃癌中作为不良预后的指标和潜在的治疗靶点[D]. 北京大学临床肿瘤学院. 北京大学. 2016.
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