【作者】 帖君；

【导师】 樊代明； 聂勇战； 潘阳林；

【作者基本信息】 第四军医大学 ， 内科学， 2010， 博士

【摘要】 【背景】随着诊断和治疗技术的进步,早期胃癌患者5年的生存率已明显提高,但伴有淋巴结转移和远处转移的进展期胃癌患者的预后仍然没有改善。由于其确切的机制仍不清楚,目前仍然缺乏早期诊断和有效治疗的方法。因此,胃癌转移已成为影响胃癌患者疗效和获得长期生存的关键问题。继续深入开展胃癌转移的基础研究,阐明胃癌转移的分子机制,仍然是解决胃癌转移的预测、早诊,提高胃癌患者疗效、改善胃癌患者预后的突破口。近年来研究发现的一种内源性、单链、非编码的小RNA分子,即microRNA(miRNA),通过负性调控靶基因的表达,在肿瘤转移中起着非常重要的作用。由于miRNA“一对多”的作用方式,即它可同时调控多个蛋白质编码基因以及多条与肿瘤生长、凋亡、转移相关的分子通路,所以靶向miRNA的干预比蛋白质编码基因能更有效地控制、逆转涉及多基因改变的肿瘤转移表型。然而,miRNA是否参与了胃癌转移的发生?有哪些miRNA参与了胃癌转移?它们起着什么样的作用?通过什么样的途径起作用?靶向miRNA的干预是否能有效逆转胃癌细胞的转移表型?这些问题都不清楚,miRNA在胃癌转移中的作用及作用机制还需进一步的研究和探索。【目的】筛选、鉴定胃癌转移相关的miRNA,研究miRNA分子在胃癌转移过程中的作用及其作用机制,进一步从新的角度理解胃癌转移的发生,为胃癌转移的预防、早期诊断、分子分型以及选择合适的靶点进行干预和治疗提供新的理论依据。【方法】1.利用体外反复的Tanswell侵袭实验分别筛选、建立高转移潜能(MKN28-M,SGC7901-M)和低转移潜能(MKN28-NM,SGC7901-NM)的胃癌细胞亚系,然后对各细胞亚系的生物学特性进行鉴定。用MTT、裸鼠成瘤检测各细胞亚系的生长、增殖能力;用流式细胞仪检测细胞周期和细胞凋亡;用Tanswell迁移和侵袭实验、划痕实验、裸鼠尾静脉注射内脏转移实验检测细胞的侵袭和转移能力。2.采用高通量的miRNA芯片检测高转移潜能( MKN28-M ,SGC7901-M)和低转移潜能(MKN28-NM,SGC7901-NM)胃癌细胞亚系的miRNA表达谱,筛选不同转移潜能胃癌细胞间差异表达的miRNA的分子,用qRT-PCR分别在细胞、组织标本中对芯片的结果进行验证。3.结合芯片结果及文献报导,对miR-218进行深入的功能研究。收集40例胃癌、癌旁及部分转移灶的冰冻和石蜡包埋组织标本,用qRT-PCR检测胃癌及癌旁组织中miR-218的表达水平,分析miR-218的表达水平与胃癌临床病理参数的关系。4.构建miR-218的表达载体,分别将miR-218的表达载体及寡核苷酸抑制剂转染胃癌细胞,上调和下调miR-218的表达,用MTT、裸鼠成瘤、Tanswell迁移和侵袭实验、裸鼠尾脉注射内脏转移实验分别在体外和体内观察miR-218对胃癌细胞生长、增殖和转移的影响。5.用生物信息学软件(PicTar、MiRand、TargetScan)预测miR-218的靶基因,对预测的靶基因用DAVID数据库(http://david.abcc.ncifcrf.gov/ )进行功能的聚类分析,筛选出与细胞迁移、侵袭能力相关的基因,再分析其与miR-218的匹配程度,挑出与细胞的迁移、侵袭能力关系最密切,与miR-218结合最紧密的靶基因进行研究。通过生物信息学预测,Robo1可能是miR-218的靶基因之一。6.我们用qRT–PCR检测GES, MKN28-NM,SGC7901-NM,MKN28-M和SGC7901-M细胞中miR-218和Robo1 mRNA的表达水平并分析其表达相关性。用miR-218的表达载体和抑制剂分别上调和下调miR-218在胃癌细胞中表达,用Western blot检测miR-218表达变化后对Robo1的影响。用免疫组化检测Robo1在癌旁、胃癌、胃癌转移灶中的表达水平,并分析miR-218与Robo1在表达上的相关性。7.生物信息学分析miR-218在Robo1 mRNA 3′-UTR上的结合位点,构建含miR-218结合位点的Robo1的3′-UTR的荧光报告基因表达载体,与miR-218共转染,观察miR-218对荧光报告基因的表达调控作用。分别构建miR-218结合位点突变的Robo1的表达载体及靶向Robo1的siRNA载体,用突变的Robo1表达载体和miR-218的表达载体共转染MKN28-M细胞,使Robo1和miR-218同时过表达,观察Robo1对miR-218抑制胃癌细胞转移的拮抗作用;用Robo1的siRNA载体转染MKN28-M细胞,观察是否会出现转染miR-218相似的表型变化。8.用qRT-PCR检测胃癌组织中miR-218、miR-218-1、miR-218-2、Slit2、Slit3的表达水平,并分别分析miR-218、miR-218-1、Slit2,miR-218、miR-218-2、Slit3之间的表达相关性,揭示miR-218表达下调的原因以及miR-218同时与Slit/Robo1这一对配体、受体分子相互作用的独特的受体信号调控模式。【结果】1.体外及体内的实验结果表明, MKN28-M , SGC7901-M与MKN28-NM,SGC7901-NM相比,其侵袭、转移能力明显增强,而生长、增殖能力和细胞周期没有明显差别。2. miRNA芯片共筛选出45个在高转移潜能( MKN28-M ,SGC7901-M)和低转移潜能(MKN28-NM,SGC7901-NM)胃癌细胞亚系间差异表达的分子(>1.5倍),表达上调的11个,表达下调的34个。其中miR-218表达下调较为显著,且既往文献报导其在胃癌组织中表达下降,提示miR-218可能是一个既参与胃癌发生,又影响胃癌转移的关键分子。在细胞及组织中验证的结果表明,miR-218在高转移潜能的胃癌细胞及大部分(7/10)胃癌转移组织中表达明显下调,与芯片结果一致。3. miR-218的表达水平与胃癌临床病理参数的分析结果表明,其表达降低的程度与胃癌的临床分期、淋巴结转移以及胃癌患者的预后密切相关。4.体外及体内的功能研究表明miR-218可以抑制胃癌的侵袭和转移,对胃癌细胞的生长增殖能力没有明显的影响。5.生物信息学预测提示,Robo1可能是miR-218的靶基因之一。在细胞和临床组织标本中,Robo1在胃癌尤其是高转移潜能的胃癌中,表达增强,与miR-218的表达负相关。6.用miR-218的表达载体上调miR-218的表达时,Robo1表达下调;而用miR-218的抑制剂抑制其功能时,Robo1表达上调。萤光报告基因实验也证实,miR-218可以抑制含有其结合位点的萤光报告基因的活性,而对突变体及对照质粒没有影响。用miR-218结合位点突变的Robo1的表达载体使miR-218与Robo1同时过表达时,Robo1可以对抗miR-218对胃癌细胞迁移、侵袭的抑制作用,而用siRNA下调Robo1的表达,可达到与miR-218过表达相似的效果。从而证实了miR-218通过负性调控Robo1参与胃癌转移。7. Slit的受体Robo1是miR-218的靶基因,受miR-218的负调控。同时,miR-218的两个编码基因miR-218-1、miR-218-2分别位于Slit2、Slit3基因的内含子中。qRT-PCR检测的结果表明,在胃癌组织中,miR-218-1,miR-218-2与Slit2, Slit3的表达明显正相关,提示在胃癌中,miR-218-1、miR-218-2分别与其宿主基因Slit2、Slit3共表达;而miR-218的表达与miR-218-2表达明显正相关,与miR-218-1的表达没有明显的相关性,表明在胃癌中miR-218的表达降低主要是由于miR-218-2的表达下调引起,而与miR-218-1无关;与此一致的是,在胃癌中,Slit3的表达明显下调,而Slit2的表达没有明显变化,从而在Slit,miR-218和Robo1之间形成一个负性调控的反馈环,miR-218以一个负性调控分子的角色维持着这一信号通路的动态平衡,这一信号通路活性的改变参与胃癌的侵袭和转移。【结论】1.成功建立了胃癌转移的细胞模型,为胃癌转移的相关研究提供了良好的平台。2.在高转移潜能的胃癌细胞和低转移潜能的胃癌细胞间有众多差异表达的miRNA分子,提示miRNA在胃癌转移中起着重要的作用。3. miR-218在胃癌中表达降低,尤其是高转移潜能的胃癌细胞中。恢复miR-218的表达,可通过负性调控Robo1抑制胃癌的侵袭和转移。4.在胃癌中miR-218的表达降低主要是由于miR-218-2/Slit3的表达下调引起,而与miR-218-1/Slit2无关。miR-218表达缺失,失去对Robo1的翻译抑制,使Robo1表达上调,通过与Slit2相互作用,促进了胃癌的侵袭和转移。更多还原

【Abstract】 【Background】The five-year survival rate for early gastric cancer (GC) has been improved significantly with the advances in diagnosis and therapy approaches. However, the prognosis of advanced GC with extensive invasion and metastasis remains poor. Owing to the mechanisms of GC metastasis are not fully understood, there is a lack of early diagnosis and effective treatment of GC metastasis. Further understanding the molecular mechanisms of metastasis and improving the prognosis of patients with GC are the most important and urgent issues in the field of oncology research.Recently, microRNAs (miRNAs), a set of naturally occurring short non-coding RNA molecules, have been identified to play important roles in tumor metastasis by negatively regulating target genes expression. Compared with protein-coding genes, miRNAs is more effective in inhibition of tumor metastasis because each miRNA can regulate more than one target genes involved in tumor growth, invasion and metastasis. Thus, we wondered whether there was a specific miRNA signature for GC metastasis, what kind of roles the miRNAs play in the process of GC metastasis, what is their mechanism of action and whether therapeutic intervention on miRNA expression could effectively reverse the metastatic phenotype of GC cells. It is clear that all these issues still need to be clarified.【Aims】To screen for the metastasis-related miRNAs in GC and to clarify their roles and the underlying mechanisms in metastasis process, with the aim of better elucidating the mechanisms of GC metastasis and providing a new theoretical basis for early diagnosis and treatment of GC metastasis.【Methods】1. We generated cell sublines with high-invasive (MKN28-M and SGC7901-M) or low-invasive potential (MKN28-NM and SGC7901-NM) from established human GC cell lines MKN28 and SGC7901 by using the repeated Transwell approach in vitro. Then MTT assay and tumour formation in nude mice were used for evaluating cell-growth and survival; Flow Cytometry were used for analysis of Cell Cycle; Transwell migration and invasion assays, would-healing assay and tail vein injection metastasis assay were employed for investigating the metastatic properties of each cell subline.2. The differential expression of miRNA between the high-invasive (MKN28-M and SGC7901-M) and low-invasive potential (MKN28-NM and SGC7901-NM) cell sublines were detected by miRNA microarray. According to miRNA bioinformatical analysis, We focused on identifying miR-218 because it might be involved in both tumorigenesis and metastasis. QRT-PCR was carried out to verify the differential expression of candidate miRNAs.3. The expression levels of miR-218 in 40 GC tissues and corresponding non-tumor mucosa were detected by qRT–PCR. Correlations between the miR-218 expression level and clinicopathologic characteristics of GC were analyzed.4. miR-218 was overexpressed in MKN28-M with a miR-218 expression vector (pGenesil-1-miR-218) or silenced in MKN28-NM cells with antisense oligonucleotide respectively. The effect of miR-218 expression on cell growth in vitro and tumorigenicity in vivo were determined by MTT assay and nude mice.Transwell migration and invasion assays and tail vein injection metastasis assay were performed to observe the effect of miR-218 expression on metastasis of GC.5. To ascertain how the low expression of miR-218 contributes to the migration and metastasis of GC, we searched for its potential regulatory targets by computative predicting tools, including miRanda, Pictar, and TargetScan. Although hundreds of different targets were predicted, those involved in migration or invasion may be critical for the pathological functions of miR-218. We then performed a functional classification of the predicted targets using the DIVID program (http://david.abcc.ncifcrf.gov/ ). Of these genes, Robo1 is regarded as a proto-oncogene and harbors migration-promoting activity, suggesting that Robo1 could be a possible target for miR-218.6. To determine whether Robo1 is one of miR-218 target genes, we analyzed the expression of miR-218 and Robo1 mRNA in GES, non-invasive (MKN28-NM and SGC7901-NM), and invasive (MKN28-M and SGC7901-M) GC cells by qRT–PCR. Then we observed the Robo1 mRNA and protein levels when miR-218 was upregulated by transfecting pGenesil-1-miR-218 in MKN28-M cells or was knocked down by transfecting antisense oligonucleotide in MKN28-NM cells. The inverse relationship between miR-218 and Robo1 expression was further confirmed by immunohistochemistry in 40 cases of GC and matched adjacent normal tissues which also were used in clinicopathological studies, as well as 29 matched metastases.7. We investigated the binding site of miR-218 in the Robo1 mRNA 3′-UTR by bioinformatics, and constructed a luciferase reporter (Luc-Robo1) in which the fragment containing the nucleotide base sequences 971-978 of the Robo1 3′-UTR complementary to miR-218 were inserted into the pMIR-REPORT miRNA expression reporter vector. Correspondingly, we also generated both the mutant reporter (Luc-Robo1-mu), in which the first 6 nucleotides in the miR-218 seed-region complementary sites were deleted, and the control reporter, which contained a non-related fragment of cDNA (Luc-Ctrl). miR-218 plasmids were co-transfected with Luc-Robo1 or Luc-Robo1-mu or Luc-Ctrl into MKN28-M cells for assessing the impact of miR-218 on the expression of Robo1. To test whether Robo1 is functionally regulated by miR-218, we generated a Robo1 expression construct containing only a fragment of the predicted miR-218 binding site and Robo1 mutant expression vector entirely lacking the 3′-UTR. We also made the Robo1 siRNA. MKN28-M-miR-218 cells, which stably expressed miR-218 ectopically, were transiently transfected with the Robo1 construct or the mutant construct (with no miR-218 binding site), and MKN28-M cells were transfected with Robo1 siRNA or a negative control siRNA.8. qRT-PCR was employed to examine the expression of the miR-218-1 precursor, the miR-218-2 precursor, mature miR-218, Slit2 mRNA, and Slit3 mRNA in the GC tissues used in the survival analysis. Statistical analysis of the correlation coefficient of the qRT-PCR results uncovered the reason for the downregulation of miR-218 in GC.【Results】1. The metastatic properties of each cell subline were characterized in vitro and in vivo. The results shown that the migration and invasion abilities of MKN28-M was approximately 4-fold greater than that of MKN28-NM cells. In the in vivo studies, tumor cell metastasis was observed in nude mice. Almost no metastatic GC cells were detected in the lungs or livers of nude mice at 10 weeks after injection of MKN28-NM cells, whereas most of the mice injected with MKN28-M cells displayed obvious lung or liver metastases. Similar results were observed for SGC7901-M and SGC7901-NM cells. No significant differences in cell proliferation or cell-cycle distribution were observed among these cell sublines.2. The microarray results revealed that the expression of 124 miRNAs significantly differed between the highly invasive variant MKN28-M and the non-invasive cell subline MKN28-NM. Of these, 83 were upregulated and 41 were downregulated. Compared with SGC7901-NM, 62 miRNAs were differentially expressed in the SGC7901-M cell subline, including 47 downregulated and 15 upregulated miRNAs. In total, 11 miRNAs were found to be upregulated and 34 miRNAs were downregulated in both MKN28-M and SGC7901-M cells. Of the 45 differentially regulated miRNAs, miR-218 was one of those that displayed significantly differential expression. miR-218 has been reported to be downregulated in cervical cancer, GC, lung cancer and prostate cancer, indicating possible involvement in both oncogenic transformation and tumor metastasis. However, miRNA-218 was only one of the many potential miRNAs of interest in cancers. In this study, miR-218 has been investigated in much greater detail. To validate the microarray results, we assessed miR-218 expression in the GC cell sublines previously mentioned and in the immortal gastric epithelial cell line GES by qRT–PCR. It was found that miR-218 expression was significantly decreased in MKN28-M and SGC7901-M cells and was lower in all four GC cell sublines compared to immortalized human gastric epithelial GES cells. Furthermore, we compared miR-218 expression between the primary GC tumor and the metastatic lymph nodes in 10 patients with stage III/ⅣGC using qRT–PCR. The results showed that mature miR-218 levels were significantly decreased in 7 out of 10 metastatic lymph nodes.3. The data of miR-218 expression in GC tissues verified that the miR-218 expression level in GC (-13.81±0.15, mean±SE) tissues was significantly lower than that in non-neoplastic mucosa (-11.62±0.15, mean±SE) (P < 0.0001, t = 10.62, paired t-test). Analysis of correlation between the miR-218 expression level and clinicopathologic characteristics of GC showed that there were statistically significant associations between the miR-218 expression level and clinical stage and between the miR-218 expression level and GC metastasis. The median expression of miR-218 was -14.25±0.17 in the 22 cases with advanced stage (stage III andⅣ) disease, whereas the median expression was -13.27±0.20 (P = 0.0010, Mann-Whitney test) in the 18 cases with early-stage (stages I and II) disease. In the 29 cases of GC with lymph node metastasis, the median expression of miR-218 was -14.09±0.16, which was significantly lower than the median expression (-13.07±0.24 ) in the 11 non-metastatic GC cases (P = 0.0036). The expression of miR-218 in GC patients did not correlate with age, gender, tumor size, or cell differentiation. Moreover, we examined whether the level of miR-218 expression was associated with survival in patients with GC. Patients were subsequently divided into low expression (n = 20) and high expression groups (n = 20) based on miR-218 levels greater or less than the mean (-13.81). Kaplan–Meier survival analyses revealed that patients whose primary tumors displayed low expression of miR-218 had a shorter median survival time. The three-year survival rate of patients with low miR-218 expression was 30%, which was significantly lower than the survival rate in patients with high miR-218 expression (65%; P = 0.0012, log-rank test).4. Functional studies of miR-218 revealed that ectopic expression of miR-218 resulted in an approximately three-fold reduction in migration and invasiveness. Consistent with the above data, there was a three- to four-fold increase in cell migration and invasiveness when we silenced miR-218 with an antisense oligonucleotide inhibitor in the MKN28-NM cells. To test if inhibition of tumor invasion by miR-218 is caused by impairing the invasive ability of tumor cells, we excluded the effect of miR-218 on the proliferation and cell cycle distribution of GC cells. Over-expression of miR-218 did not affect the proliferation and the cell cycle distribution of MKN28-M cells in vitro. To further investigate the inhibition of in vivo tumor metastasis by miR-218, we implanted MKN28-M-miR-218 cells that were stably expressing miR-218 or control cells into nude mice through the lateral tail vein. Lung and liver metastasis of GC was apparent in mice injected with MKN28-M-miR-control cells. In contrast, few metastatic tumors were detected in mice injected with MKN28-M-miR-218 cells. Furthermore, we simultaneously observed the growth of the primary tumors and the incidence of distant metastasis in the nude mice injected subcutaneously with MKN28-M-miR-218 cells or control cells. The results showed lung or liver metastasis was apparent in 3 out of 10 mice injected with MKN28-M-miR-control cells; in stark contrast, no metastasis were found in mice injected with MKN28-M-miR-218 cells.5. Bioinformatics predicted that Robo1 may be a target for miR-218. To further test the hypothesis, we analyzed the expression of miR-218 and Robo1 in GC cell lines. The results showed a negative correlation between the levels of miR-218 and Robo1 mRNA in these cells. Furthermore, we observed that Robo1 mRNA and protein levels were decreased when miR-218 was expressed by pGenesil-1-miR-218 in MKN28-M cells. The reverse was observed for Robo1 expression when miR-218 was knocked down in MKN28-NM cells. The inverse relationship between miR-218 and Robo1 expression was further confirmed by immunohistochemistry in 40 cases of GC, in matched adjacent normal tissues that were also used in clinicopathological studies, and in 29 matched metastases. The results show that Robo1 was upregulated in GC, especially in metastatic GC, in which miR-218 has a relatively low expression.6. Luciferase reporter assay showed showed that the luciferase activity in the Luc-Robo1-transfected cells was significantly decreased compared to the luciferase activity in the mutant and negative control cells (P < 0.05), suggesting that miR-218 reduced the luciferase activity of Luc-Robo1 but had no effect on Luc-Robo1-mu. Furthermore, MKN28-M-miR-218 cells transfected with the Robo1 mutant construct showed a 3.8-fold increase in invasion ability compared to cells transfected with the Robo1 construct. These results indicate that introduction of mutant Robo1 cDNA that lacked the miR-218 binding site into the miR-218-overexpressing cells reversed the effect of miR-218-mediated suppression of cell invasion. Knockdown of Robo1 by siRNA in MKN28-M cells inhibited cell invasion, which fell to levels similar to those observed after transfection with the miR-218-expressing vector.7. miR-218 is an intronic miRNA. Two genes code for mature miR-218, miR-218-1 and miR-218-2, which are located within intron 15 of Slit2 and intron 14 of Slit3, respectively. The intronic location of the two miR-218 genes prompted us to ask whether miR-218-1 and miR-218-2 are transcribed together with their host gene mRNAs. To test this hypothesis, we used qRT-PCR to examine the expression of the miR-218-1 precursor, the miR-218-2 precursor, mature miR-218, Slit2 mRNA, and Slit3 mRNA in the GC tissues used in the survival analysis. Statistical analysis of the correlation coefficient of the qRT-PCR results revealed a significant positive correlation between the levels of Slit2 mRNA and miR-218-1 and between the levels of Slit3 mRNA and miR-218-2. These results indicate that the miR-218 coding genes, miR-218-1 and miR-218-2, are transcribed together with their host genes, Slit2 and Slit3, respectively. A significant positive correlation between the levels of miR-218 and miR-218-2 was seen in GC; however, no such correlation was seen between the levels of miR-218 and miR-218-1. These results indicate that downregulation of miR-218 in GC is promoted by a decrease in miR-218-2, but not in miR-218-1. Consistent with this conclusion, Slit3 expression was significantly reduced in GC (-22.43±0.21, mean±SE) compared to normal gastric tissue (-20.79±0.23, mean±SE), (P < 0.0001, t = 7.67, paired t-test) (Figure 6F), whereas Slit2 expression was not significantly different (P = 0.0772, paired t-test).【Conclusions】1. We successfully established a GC metastatic model, which would provide a research platform for study of GC metastasis.2. Many miRNAs were differentially expressed between the highly invasive variants and the low-invasive cell sublines of GC, suggesting that miRNAs play important roles in GC metastasis。 3. miR-218 was downregulated in GC, especially in metastatic GC. Ectopic expression of miR-218 inhibited tumor cell invasion and metastasis by targeting the Robo1 receptor.4. miR-218 is part of a regulatory circuit involving the Slit-Robo1 pathway. miR-218 coding genes, miR-218-1 and miR-218-2, are transcribed together with their host genes, Slit2 and Slit3, respectively. In metastatic tumor cells, miR-218 was suppressed along with Slit3, one of its host genes. Meanwhile, Robo1, one of several Slit receptors, is upregulated in response to the decrease in miR-218, which in turn induced a reactive upregulation of the Slit-Robo1 pathway through an interaction with Slit2, thus facilitating tumor cell migration and invasion.更多还原