摘要
研究背景和目的
转移是导致结直肠癌患者治疗失败及死亡的重要原因。临床上有一半以上的结直肠癌患者在行根治性手术前已出现了微转移,它是结直肠癌术后转移和复发的直接原因。因此,阐明结直肠癌转移的分子机制,是结直肠癌研究的重要内容。
分子标签是以一定数量的基因作为一个整体,来判断或定义某些生物学特性。与单个的分子模式不同,分子标签以大量对单个基因的功能研究为基础,更加注重基因之间的共同协调作用,从整体和系统水平上对不同的生物学特性进行描述。基因表达谱分析是获得分子标签的重要手段。
我们通过有限克隆稀释法建立结直肠癌单细胞源性子代细胞亚系,进一步利用整体可视化结直肠癌原位及转移动物模型,筛选出具有不同转移潜能的结直肠癌细胞亚系,应用全基因组表达谱芯片技术分析不同转移潜能的细胞亚系全基因组表达谱变化,在此基础上应用生物信息学及文献挖掘的方法,提取结直肠癌转移相关特征性分子标签,并在具有完整随访资料的临床标本中对分子标签的可靠性及临床价值进行了初步的验证。研究从整体水平上揭示了结直肠癌转移的分子特征,为结直肠癌转移早期预警、诊断以及预后判断和靶向治疗的分子体系的建立奠定了基础。
方法
1、按照有限克隆稀释法建立了来源于同一亲本结直肠癌细胞株的单细胞源性子代细胞亚系,通过体外功能实验及整体可视化结直肠癌原位及转移动物模型,比较不同细胞亚系的结直肠癌细胞生物功能方面的差异,获得不同转移潜能的结直肠癌细胞亚系。
2、应用基因芯片技术分析不同转移潜能的细胞亚系全基因组表达谱变化;交集分析获得共同改变的差异基因;应用生物信息学方法分析转移相关的基因表达模式并提取转移特征性分子标签。
3、应用生物信息学方法分析结直肠癌转移相关分子标签所含基因的生物学功能及参与的信号转导通路;结合文献轮廓挖掘确定分子标签分子与肿瘤转移的关系。
4、应用具有完整随访资料的临床标本分析分子标签相关蛋白的表达情况,并与患者的临床特征及预后进行相关性分析,初步验证分子标签的可靠性并初步确定分子标签的临床价值。
5、统计学方法:应用SPSS13.0统计学软件,采用方差分析、相关分析、生存分析等统计学方法。
结果
1、将结直肠癌SW480/EGFP细胞株采用有限克隆稀释法,在体外成功建立了59个单细胞源性子代细胞亚系(SCPs)。
2、对59个SCPs进行了裸鼠皮下成瘤实验,共筛选出29个具有成瘤能力的SCPs;对部分SCPs与其亲本细胞株SW480/EGFP的皮下瘤生长能力进行了比较,其皮下瘤生长能力存在显著差别(F=33.446,P=0.000)。确定部分SCPs成瘤能力不同,具有不同于其亲本母系细胞的生长特性。
3、将29个具有成瘤能力的SCPs及其亲本细胞株SW480/EGFP进行了裸鼠盲肠原位移植试验,建立结直肠癌原位移植及转移整体可视化动物模型,筛选转移能力明显差异的细胞亚系;在此基础上对3个具有高转移潜能亚系、3个具有低转移潜能亚系及母系细胞株SW480/EGFP的转移潜能进行了比较。统计学分析结果表明,其转移能力差别具有显著性(F=4.155,P=0.000)。为了进一步明确亚系之间转移潜能的差异,对其中2个具有高转移潜能及2个具有低转移潜能的细胞亚系重复进行了结直肠癌原位移植及转移整体动物试验。统计学分析结果表明:细胞亚系间转移能力差异具有显著性(F=6.172,P=0.029),并且经过SNK多重比较,转移能力最强的是SCP51,转移能力最弱的是SCP58。综合分析试验结果,最后确定SCP51为具有高转移能力的细胞亚系,SCP58为具有低转移能力的细胞亚系。
4、应用Affymetrix公司制备的HG-U133 plus 2.0原位合成寡核苷酸芯片对具有高转移能力的细胞亚系SCP51、具有低转移能力的细胞亚系SCP58及其亲本母系SW480/EGFP细胞株进行了全基因组表达谱芯片检测,每组均进行3次的生物学重复。应用芯片显著性分析(Significance Analysis ofMicroarray,SAM)软件并结合交集分析,筛选出结直肠癌转移相关差异显著性基因143个,作为结直肠癌转移相关基因表达模式,其中包括85个上调基因及58个下调基因。系统聚类分析,143个基因成功地将三组检测样本分为三类。
5、利用DAVID分析系统以及博奥生物有限公司生物信息学综合分析平台对结直肠癌转移相关基因表达模式所包含基因的生物学功能及参与的信号转导通路进行了初步分析,并对其进行文献挖掘,分析这些基因的生物学功能及临床价值,筛选出生物学功能可能与结直肠癌转移相关的基因29个,初步确定为结直肠癌转移相关分子标签。
6、随机选取结直肠癌转移相关分子标签中5个与实体瘤转移密切相关、但在结直肠癌转移研究中未见报道的基因LYN、SDCBP、MAP4K4、MID1、DKK1进行初步的功能鉴定。
7、应用Real-Time PCR检测SCP51、SCP58及其亲本母系细胞株SW480/EGFP中LYN、SDCBP、MAP4K4、MID1及DKK1基因在mRNA水平的表达情况,检测结果与芯片结果一致。应用RT-PCR检测了LYN、SDCBP、MAP4K4、MID1、DKK1在结直肠癌肿瘤组织与正常粘膜组织中mRNA的表达水平,LYN、SDCBP及MAP4K4.三个在高转移细胞株中表达上调的基因,其在肿瘤组中的表达明显高于正常粘膜组,MID1及DKK1两个在高转移细胞株中表达下调的基因,其在肿瘤组中的表达明显低于正常粘膜组。
8、应用具有完整随访资料的临床结直肠癌标本,对LYN、SDCBP、MAP4K4、MID1以及DKK1在蛋白质水平的表达进行验证,检测这五种基因在患者肿瘤组织中的表达情况,并与患者的临床特性进行相关性分析。免疫组化结果表明:LYN、MAP4K4、MID1与患者的临床转移情况密切相关(P＜0.05),LYN、SDCBP、MAP4K4、MID1与患者的临床预后密切相关(P＜0.05)。
9、将LYN、SDCBP、MAP4K4、MID1及DKK1五个基因作为一个整体标签,与患者的临床特性进行相关性分析。结果表明:作为一个整体标签,这五种基因的表达与患者临床转移及预后均显著相关(P＜0.05)。研究提示,由多种基因构成的整体分子标签可作为新的结直肠癌转移与预后的分子标记物。
结论
1、结直肠癌细胞株SW480/EGFP具有明显的异质性。应用有限克隆稀释法建立了来源于同一亲本结直肠癌细胞株SW480/EGFP的单细胞源性子代细胞亚系(SCPs);
2、来源于同一亲本结直肠癌细胞株的单细胞源性子代细胞亚系存在转移潜能的差异;
3、来源于同一亲本结直肠癌细胞株不同转移潜能的细胞亚系具有不同的分子特征;
4、LYN、SDCBP、MAP4K4、MID1及DKK1可以作为新的结直肠癌转移与预后的分子标记物。它们共同组成的整体分子标签可作为新的结直肠癌转移与预后的分子标记物。
本研究的创新之处
1、将结直肠癌SW480/EGFP细胞株采用有限克隆稀释法,在体外成功建立了59个单细胞源性子代细胞亚系(SCPs);筛选出29个具有成瘤能力的SCPs;获得2个具有高转移潜能及2个具有低转移潜能的细胞亚系。
2、应用结直肠癌细胞单细胞源性子代细胞亚系的筛选策略及基因表达谱技术和生物信息学分析获得原创性、与结直肠癌转移密切相关的分子,为进一步确定结直肠癌转移相关分子标签奠定基础。
3、应用基于临床病理研究的策略确定与结直肠癌转移密切相关分子的功能;获得有明确临床价值的结直肠癌转移相关基因。
4、确定LYN、SDCBP、MAP4K4、MID1及DKK1可以作为新的结直肠癌转移与预后的分子标记物。它们共同组成的整体分子标签可作为新的结直肠癌转移与预后的分子标记物。
BACKGROUND & OBJECTIVE
Metastasis is the main cause leading to deaths of patients with colorectal cancer. Micrometastasis,which is present in more than half of patients with colorectal cancer before radical surgery,resulted in distant metastasis and recurrence in colorectal cancer after radical surgery.However,there is no reliable method to detect potential micrometastases in patients with colorectal cancer.Therefore,to clarify the molecular mechanism of metastasis in colorectal cancer is the main task in the field of colorectal cancer research.
Molecular signature is a whole number of some genes,to determine or define certain biological characteristics.Different with individual molecules,molecular signature pay more attention to the coordination of different genes,it is the overall description of different biological characteristics and it is based on function research of a large number of single gene.An important means of gaining molecular signature is through Analysis of gene expression profiles.
In this study,59 single-cell-derived progeny(SCPs) cell subclones of colorectal cancer were established in vitro and selected in in surgical orthotopic implantation mouse model of colorectal cancer.Several SCPs subclones with different metastatic potential were obtained in our study.We identified genes associated with tumor metastasis by comparing the difference between subclones with different metastatic potential and its parent cell line using genome-wide expression profile chip of Affymetrix.Molecular signature of metastasis in colorectal cancer was extracted from metastasis associated genes based on bioinformatic methods and literature mining methods.The clinical significance of molecular signature and the comprising genes was tested in clinical samples of colorectal cancer.
METHODS
1、Single cell-derived progenies(SCPs) of colorectal cancer were isolated from SW480/EGFP cells.Tumor formation of SCPs was detected by monitoring the tumor growth under subcutaneous tissue of nude mice.In vitro methods and orthotopic implantation mouse model of colorectal cancer were used to screen for SCPs with different metastasis potential
2、Gene expression profiles of SCPs with different metastasis potential and its parent SW480/EGFP were determined using Affymetrix human Genome U133 Plus 2.0 Array containing 47000 transcripts.Hybridization,washing,staining, scanning,and data collection were performed according to the standard Affymetrix protocol.Gene sets shared in two independent comparision between SCPs and its parent SW480/EGFP were selected in our next study.Molecular signature of metastasis in colorectal cancer was extracted from metastasis associated genes based on bioinformatic methods and literature mining methods.
3、Biological functions,pathways and the possible role in tumor metastasis of genes in our molecular signature of metastasis were analyzed.
4、Formalin-fixed and paraffin-embedded tissues of CRC were used in our clinicopathological investigation.Immunohistochemistry was performed using a Dako Envision System(Dako,Carpentaria,CA,USA) following the manufacturer's recommended protocol to study protein expression of five genes in our metastasis signature.Antibodies against LYN、SDCBP、MAP4K4、MID1、DKK1 were used in this study.The clinical significance of molecular signature and the comprising genes was tested in clinical samples of colorectal cancer.
5、All statistical analyses were carried out using the SPSS software program (version 12.0;SPSS Inc.,Chicago,IL).The chi-square test was used to assess differences in metastases and peritoneal dissemination of SCPs and their parental SW480/EGFP.The Mann-Whitney U test was used to analyze the relationship between expression of LYN,SDCBP,MAP4K4,MID1,DKK1 and clinicopathological characteristics of the patients.Survival curves for the patients with different expressions of LYN,SDCBP,MAP4K4,MID1,DKK1 were plotted using the Kaplan-Meier method and compared using the log-rank test.The significance of various survival-related variables was assessed using the Cox proportional hazards model in a multivariate analysis.A p-value of≤0.05 will be considered statistically significant.
RESULTS
1、Fifty-nine single cell-derived progenies(SCPs) from SW480/EGFP was isolated and established by limited dilution method.
2、Tumor formation under subcutaneous tissue of nude mice was found in 29 SCPs.Heterogeneous ability of tumor formation was found in different SCPs from SW480/EGFP(F=33.446,P=0.000).
3、Surgical orthotopic implantation(SOI) nude mice model of colorectal cancer was performed to compare metastatic potentials of 29 SCPs and their parent SW480/EGFP.Different metastatic potentials were found in 29 SCPs in SOI nude mice model of colorectal cancer(F=4.155,P=0.000).Among the SCPs studied,3 SCPs with high metastatic potential isolated as SCP12、SCP21 and SCP51,3 SCPs with low metastatic potential isolated as SCP11、SCP28 and SCP58,were found in SOI nude mice model of colorectal cancer.In order to rule out the randomness of tests,two SCPs with high metastatic potential and 2 SCPs with low metastatic potential were tested repeatedly to confirm their metastatic potential in SOI nude mice model of colorectal cancer.Statistically significant differences of metastasis in different SCPs was observed(F=6.172, P=0.029).A SNK multiple comparison showed that SCP51 had the highest metastatic potential and SCP58 had the lowest metastatic potential among the above SCPs.SCP51 and SCP58 were used in our next study.
4、Gene expression profiles of SCP51、SCP58 and its parent SW480/EGFP were detected by HG-U133 plus 2.0 oligonucleotide array of Affymetrix Inc.SAM analysis was performed to identify the most discriminating genes.One hundred and forty-three genes including 85 up-regulated genes and 58 down-regulated genes were extracted as gene expression pattern of metastasis in colorectal cancer;Hierarchical cluster analysis showed that the cell subclone and its parent cell line can be successfully classified as three categories according to gene expression level of 143 genes.
5、Molecular signature of metastasis in colorectal cancer comprising of 29 genes was extracted using platform of Significance Analysis of Microarrays constructed by Capitalbio company.
6、Biological significance of genes in the molecular signature of metastasis in colorectal cancer was analyzed by literature mining method.Five genes including LYN、SDCBP、MAP4K4、MID1 and DKK1 were found to be associated with tumor metastasis.However,no study about the roles of five genes was found.
7、We found that mRNA expressions of LYN,SDCBP,MAP4K4,MID1,DKK1 in SCP51,SCP58 and SW480/EGFP cells detected by real-Time PCR were the same as the results detected by previous gene chip.We found that mRNA expression levels of LYN,SDCBP,MAP4K4 were higher in the primary colorectal tumor tissue samples than those in their normal counterparts.We also observed that mRNA expression levels of DKK1 and MID1 were lower in the primary colorectal tumor tissue samples than those in their normal counterparts.
8、Immunohistochemistry results showed that LYN,MAP4K4 and MID1 were closely correlated with metastasis of patients(P＜0.05) and LYN,SDCBP, MAP4K4,MID1 were closely associated with the prognosis of patients(P＜0.05).
9、To determine the possibility of using LYN,SDCBP,MAP4K4,MID1 and DKK1 as a molecular signature in colorectal cancer,the sum of 5 independent scores of LYN,SDCBP,MAP4K4,MID1 and DKK1 in clinical samples of colorectal cancer was used as the final score of the molecular signature in colorectal cancer.The results showed the molecular signature comprising of LYN,SDCBP,MAP4K4,MID1 and DKK1 was significantly correlated with metastasis and prognosis of patients(P＜0.05) as a whole.Our results suggested that the molecular signature comprising of LYN,SDCBP,MAP4K4,MID1 and DKK1 can be potential new molecular markers of metastasis and prognosis in colorectal cancer.
CONCLUSIONS
1、SW480/EGFP exhibiting heterogeneity in metastatic potential is suitable for in vivo selection and SCPs selection.
2、Different SCPs from SW480/EGFP exhibit heterogeneity in metastatic potential and tumorgenicity detected in vitro assay,in vivo tumor growth and metastasis assays.
3、One hundred and forty-three genes including 85 up-regulated genes and 58 down-regulated genes were extracted as gene expression pattern of metastasis in colorectal cancer,Molecular signature of metastasis in colorectal cancer comprising of 29 genes was determined using bioinformatic methods and text mining.
4、LYN,MAP4K4 and MID1 were closely correlated with metastasis of colorectal cancer and LYN,SDCBP,MAP4K4,MID1 were closely associated with the prognosis of colorectal cancer.Molecular signature comprising of LYN,SDCBP, MAP4K4,MID1 and DKK1 was significantly correlated with metastasis and prognosis of patients and can be potential new molecular markers of metastasis and prognosis in colorectal cancer.
INNOVATIONS
1、Fifty-nine single cell-derived progenies(SCPs) from SW480/EGFP is isolated and established.
2、SCPs with high metastatic potential isolated as SCP12、SCP21 and SCP51,SCPs with low metastatic potential isolated as SCP11、SCP28 and SCP58,are established and confirmed in SOI nude mice model of colorectal cancer.
3、Gene expression pattern and molecular signature of metastasis in colorectal cancer have been determined using bioinformatic methods and text mining.
4、LYN,SDCBP,MAP4K4,MID1 are the candidates of prognostic indexes of colorectal cancer.Molecular signature comprising of LYN,SDCBP,MAP4K4, MID1 and DKK1 can be potential new molecular markers of metastasis and prognosis in colorectal cancer.
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