Wnt拮抗剂SFRP4在胰腺癌中的异常表达及其机制
摘要
目的
Wnt家族广泛地参与了生物胚胎的一些基本发育过程，以及成体细胞增殖、分化和凋亡过程。SFRPs(Secreted Frizzled-ralated Proteins)为分泌型蛋白质，全称为分泌型Frizzled相关蛋白，该家族成员SFRP_(1-5)一样，大约由300多个氨基酸组成，其中含有一个氨基端附近的CRD区域，该区域内含有10个高度保守的半胱氨酸，与Frizzled的胞外CRD区域有30-40％的同源性。SFRP4是Wnt信号通路的负调控因子，在一些肿瘤组织中它的表达明显减弱或消失，例如在乳腺、结直肠、前列腺、卵巢。SFRP4基因的染色体区域在一些肿瘤中经常缺失，这被认为是削弱了抑癌基因。作为SFRPs家族的一个成员，一些研究提示在肿瘤的发生中SFRP4的表达消失起了重要的作用。
本实验的目的在于探讨SFRP4在胰腺癌中的甲基化及表达情况，同时对SFRP4异常表达与患者临床病理特征之间相关性进行分析。
材料和方法
1、材料
(1)人类胰腺癌细胞系CFPAC-1，PC-3和PANC-1购自上海昆肯生物有限公司。胰腺癌及相应的癌旁组织各36例，均来自于中国医科大学附属盛京医院2005-2006年手术切除标本。标本获取后即冷冻于液氮之中，并保存在—80℃条件下备用。采用HE染色确定肿瘤标本主要由肿瘤组织构成，癌旁组织没有肿瘤细胞浸润。
(2)主要试剂：蛋白酶K(TaKaRa)，RNAiso试剂(Trizol，Takara)，SFRP4多克隆抗体(SANTA CRUZ)，RNA PCR试剂盒(AMV)3.0(TaKaRa)，GENMED基因甲基化检测试剂盒(杰美)，DAB显色试剂盒，山羊抗小鼠IgG
(3)主要仪器：PCR仪(ABI-9700，美国)，低温超速离心机(5810R，德国)，凝胶自动成像分析系统(PERSONALFX，美国)，紫外分光光度计(UVMINI-1204，日本)，深冻冰箱(—80℃，MDF-U32V，日本)，各种显微镜，匀浆器等
2、方法
采用免疫组织化学、RT-PCR及甲基化特异性PCR等方法对SFRP4在胰腺癌细胞CFPAC-1，PC-3和PANC-1、36例原发性胰腺癌及相应癌旁组织中的表达情况及甲基化状态进行分析。采用Fisher's e xact检验对SFRP4异常表达与原发性胰腺癌患者临床病理特征之间的相关性进行分析。
结果
(1)胰腺癌细胞CFPAC-1和PANC-1存在SFRP4基因甲基化，而PC-3未检出SFRP4基因甲基化。PC-3存在SFRP4 mRNA表达，而CFPAC-1和PANC-1未检出表达。采用去甲基化试剂5-aza-2'-deoxycytidine对CFPAC-1和PANC-1进行处理后，SFRP4 mRNA重新出现表达。
(2)在36例原发性胰腺癌中，17例存在SFRP4基因甲基化现象；在其相应的癌旁组织中，5例检出SFRP4甲基化，两者差异显著(P＜0.01)。15例原发性胰腺癌存在SFRP4表达消失；在其相应的癌旁组织中，3例表达消失，两者差异显著(P＜0.01)。
(3)SFRP4表达消失与患者年龄、性别、肿瘤大小及分化程度均无相关性，而与SFRP4基因甲基化状态、肿瘤分期、淋巴结转移情况具有相关性。
结论
(1)SFRP4在部分胰腺癌中表达消失，且其机制主要由SFRP4基因甲基化所致。
(2)SFRP4表达消失与肿瘤侵袭转移能力增加有关。
(3)结果提示，SFRP4表达消失与部分胰腺癌发病有关，且SFRP4表达消失可能与患者不良预后相关。
Objective
The canonical Wnt signaling pathway takes important roles in embryonic development,cell proliferation and cell differentiation. Defects in this pathway have been implicated in the pathogenesis of various types of tumors in human beings. Secreted frizzled-ralated proteins(SFRPs) are a family of Wnt antagonists that habours a cyteine-rich domain(CRD) homologous to the frizzled receptors. SFRPs are able to bind Wnt proteins in the extracellular compartment,thereby inhibiting ligand-receptor interaction and signal transduction.
SFRP4 is a putative inhibitor of Wnt signaling. Its expression is frequently lost or strongly reduced in some tumors.,including breast,colorectal,prostate and ovarian cancers. The SFRP4 gene is located in a chromosomal region that is frequently deleted in some tumors and is thought to harbour a tumor suppressor gene. To be a member of SFRPs family, some studies indicated that silencing of SFRP4 expression played an important role in the carcinogenesis of esophagus and colorectal tumors.
In this study, we aim to investigate the expressions of SFRP4 in pancreatic tumors and matched normal pancreatic tissues,analyze the mechanism of gene silencing. Meanwhile, analyze the relationship between the aberrant expression and clinical pathologic characteristics.
Materials and Methods
Matched tumor/normal samples of pancreatic cancer specimens(n=36) analyzed in the study were obtained from patients at the departments of general surgeny at second affliated hospital,China medical university between 2005 and 2006, and three human pancreatic cancer cell lines purchased from shanghai kunken biochemistry company. We detected the protein expression of SFRP4 with immunohistochemiccal method,detected the level of mRNA with RT-PCR method, and the status of methelation with the methelation-specific PCR method. The clinical pathologic characteristics of all patients were summarized to find the rationship between the loss of expression of the both genes and their clinical pathologic characteristics.
Results
In three human pancreatic cell lines, two of them the SFRP4 mRNA expression silence were discovered. After the process of demethelation in the two cell lines,the SFRP4 mRNA recover again. The loss of SFFRP4 expression was detected in 15 pancreatic cancer samples. In matched normal tissues, the loss of expression occurred in 3 samples. The difference between cancer and match normal tissue is significant. Methylation of SFRP4 promoter was found in 17 pancreatic cancer samples and in 5 matched normal tissues. The different between cancer and matched normal tissue is significant. The loss of SFRP4 expression is correlated with status of methylation , TNM stages and lymph node metastasis of tumors.
Conclusion
1. The loss of SFRP4 expression may take place in pancreatic cancer tissue mostly, the machenism is due to the methelation of promoter.
2. The loss of SFRP4 expression may be correlated with the greater invasive capacity of the cancer and associated with unfavorable prognosis.
3. The loss of SFRP4 expression may be linked to the occurrence of partial pancreatic cancer.
Wnt家族广泛地参与了生物胚胎的一些基本发育过程，以及成体细胞增殖、分化和凋亡过程。SFRPs(Secreted Frizzled-ralated Proteins)为分泌型蛋白质，全称为分泌型Frizzled相关蛋白，该家族成员SFRP_(1-5)一样，大约由300多个氨基酸组成，其中含有一个氨基端附近的CRD区域，该区域内含有10个高度保守的半胱氨酸，与Frizzled的胞外CRD区域有30-40％的同源性。SFRP4是Wnt信号通路的负调控因子，在一些肿瘤组织中它的表达明显减弱或消失，例如在乳腺、结直肠、前列腺、卵巢。SFRP4基因的染色体区域在一些肿瘤中经常缺失，这被认为是削弱了抑癌基因。作为SFRPs家族的一个成员，一些研究提示在肿瘤的发生中SFRP4的表达消失起了重要的作用。
本实验的目的在于探讨SFRP4在胰腺癌中的甲基化及表达情况，同时对SFRP4异常表达与患者临床病理特征之间相关性进行分析。
材料和方法
1、材料
(1)人类胰腺癌细胞系CFPAC-1，PC-3和PANC-1购自上海昆肯生物有限公司。胰腺癌及相应的癌旁组织各36例，均来自于中国医科大学附属盛京医院2005-2006年手术切除标本。标本获取后即冷冻于液氮之中，并保存在—80℃条件下备用。采用HE染色确定肿瘤标本主要由肿瘤组织构成，癌旁组织没有肿瘤细胞浸润。
(2)主要试剂：蛋白酶K(TaKaRa)，RNAiso试剂(Trizol，Takara)，SFRP4多克隆抗体(SANTA CRUZ)，RNA PCR试剂盒(AMV)3.0(TaKaRa)，GENMED基因甲基化检测试剂盒(杰美)，DAB显色试剂盒，山羊抗小鼠IgG
(3)主要仪器：PCR仪(ABI-9700，美国)，低温超速离心机(5810R，德国)，凝胶自动成像分析系统(PERSONALFX，美国)，紫外分光光度计(UVMINI-1204，日本)，深冻冰箱(—80℃，MDF-U32V，日本)，各种显微镜，匀浆器等
2、方法
采用免疫组织化学、RT-PCR及甲基化特异性PCR等方法对SFRP4在胰腺癌细胞CFPAC-1，PC-3和PANC-1、36例原发性胰腺癌及相应癌旁组织中的表达情况及甲基化状态进行分析。采用Fisher's e xact检验对SFRP4异常表达与原发性胰腺癌患者临床病理特征之间的相关性进行分析。
结果
(1)胰腺癌细胞CFPAC-1和PANC-1存在SFRP4基因甲基化，而PC-3未检出SFRP4基因甲基化。PC-3存在SFRP4 mRNA表达，而CFPAC-1和PANC-1未检出表达。采用去甲基化试剂5-aza-2'-deoxycytidine对CFPAC-1和PANC-1进行处理后，SFRP4 mRNA重新出现表达。
(2)在36例原发性胰腺癌中，17例存在SFRP4基因甲基化现象；在其相应的癌旁组织中，5例检出SFRP4甲基化，两者差异显著(P＜0.01)。15例原发性胰腺癌存在SFRP4表达消失；在其相应的癌旁组织中，3例表达消失，两者差异显著(P＜0.01)。
(3)SFRP4表达消失与患者年龄、性别、肿瘤大小及分化程度均无相关性，而与SFRP4基因甲基化状态、肿瘤分期、淋巴结转移情况具有相关性。
结论
(1)SFRP4在部分胰腺癌中表达消失，且其机制主要由SFRP4基因甲基化所致。
(2)SFRP4表达消失与肿瘤侵袭转移能力增加有关。
(3)结果提示，SFRP4表达消失与部分胰腺癌发病有关，且SFRP4表达消失可能与患者不良预后相关。
Objective
The canonical Wnt signaling pathway takes important roles in embryonic development,cell proliferation and cell differentiation. Defects in this pathway have been implicated in the pathogenesis of various types of tumors in human beings. Secreted frizzled-ralated proteins(SFRPs) are a family of Wnt antagonists that habours a cyteine-rich domain(CRD) homologous to the frizzled receptors. SFRPs are able to bind Wnt proteins in the extracellular compartment,thereby inhibiting ligand-receptor interaction and signal transduction.
SFRP4 is a putative inhibitor of Wnt signaling. Its expression is frequently lost or strongly reduced in some tumors.,including breast,colorectal,prostate and ovarian cancers. The SFRP4 gene is located in a chromosomal region that is frequently deleted in some tumors and is thought to harbour a tumor suppressor gene. To be a member of SFRPs family, some studies indicated that silencing of SFRP4 expression played an important role in the carcinogenesis of esophagus and colorectal tumors.
In this study, we aim to investigate the expressions of SFRP4 in pancreatic tumors and matched normal pancreatic tissues,analyze the mechanism of gene silencing. Meanwhile, analyze the relationship between the aberrant expression and clinical pathologic characteristics.
Materials and Methods
Matched tumor/normal samples of pancreatic cancer specimens(n=36) analyzed in the study were obtained from patients at the departments of general surgeny at second affliated hospital,China medical university between 2005 and 2006, and three human pancreatic cancer cell lines purchased from shanghai kunken biochemistry company. We detected the protein expression of SFRP4 with immunohistochemiccal method,detected the level of mRNA with RT-PCR method, and the status of methelation with the methelation-specific PCR method. The clinical pathologic characteristics of all patients were summarized to find the rationship between the loss of expression of the both genes and their clinical pathologic characteristics.
Results
In three human pancreatic cell lines, two of them the SFRP4 mRNA expression silence were discovered. After the process of demethelation in the two cell lines,the SFRP4 mRNA recover again. The loss of SFFRP4 expression was detected in 15 pancreatic cancer samples. In matched normal tissues, the loss of expression occurred in 3 samples. The difference between cancer and match normal tissue is significant. Methylation of SFRP4 promoter was found in 17 pancreatic cancer samples and in 5 matched normal tissues. The different between cancer and matched normal tissue is significant. The loss of SFRP4 expression is correlated with status of methylation , TNM stages and lymph node metastasis of tumors.
Conclusion
1. The loss of SFRP4 expression may take place in pancreatic cancer tissue mostly, the machenism is due to the methelation of promoter.
2. The loss of SFRP4 expression may be correlated with the greater invasive capacity of the cancer and associated with unfavorable prognosis.
3. The loss of SFRP4 expression may be linked to the occurrence of partial pancreatic cancer.
引文
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3． Katoh M.WNT2 and human gastrointestinal cancer.Int J Mol Med.2003;12(5) :811-6．
4． Verras M,Brown J,Li X,Nusse R,Sun Z.Wnt3a growth factor induces androgen receptor-mediated transcription and enhances cell growth in human prostate cancer cells.Cancer Res.2004;64(24) :8860-6．
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6． Katoh Y,Katoh M.Comparative genomics on DKK2 and DKK4 orthologs.Int J Mol Med.2005;16(3) :477-81．
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18． Jung Y,Bang S,Choi K,et al.TC1(C8orf4) enhances the Wnt/beta-catenin pathway by relieving antagonistic activity of Chibby.Cancer Res.2006;66(2) :723-8．
19． Blavier L,Lazaryev A,Dorey F,et al.Matrix metalloproteinases play an active role in Wnt1-induced mammary tumorigenesis.Cancer Res.2006;66(5) :2691-9．
20． Katoh M.WNT2 and human gastrointestinal cancer.Int J Mol Med.2003;12(5) :811-6．
21． Verras M,Brown J,Li X,Nusse R,Sun Z.Wnt3a growth factor induces androgen receptor-mediated transcription and enhances cell growth in human prostate cancer cells.Cancer Res.2004;64(24) :8860-6．
22． Byun T,Karimi M,Marsh JL,et al.Expression of secreted Wnt antagonists in gastrointestinal tissues:potential role in stem cell homeostasis.J Clin Pathol.2005;58(5) :515-9．
23． Katoh Y,Katoh M.Comparative genomics on DKK2 and DKK4 orthologs.Int J Mol Med.2005;16(3) :477-81．
24． Ugolini F,Charafe-Jauffret E,Bardou VJ,et al.WNT pathway and mammary carcinogenesis:loss of expression of candidate tumor suppressor gene SFRP4 in most invasive carcinomas except of the medullary type.Oncogene.2001;20(41) :5810-7．
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27． Takada T,Yagi Y,Maekita T,et al.Methylation-associated silencing of the Wnt antagonist SFRP4 gene in human ovarian cancers.Cancer Sci.2004;95(9) :741-4．
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21． Verras M,Brown J,Li X,Nusse R,Sun Z.Wnt3a growth factor induces androgen receptor-mediated transcription and enhances cell growth in human prostate cancer cells.Cancer Res.2004;64(24) :8860-6．
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28． Zou H,Molina JR,Harrington JJ,et al.Aberrant methylation of secreted frizzled-related protein genes in esophageal adenocarcinoma and Barrett's esophagus.Int J Cancer.2005;116(4) :584-91．
29． Lodygin D,Epanchintsev A,Menssen A,et al.Functional epigenomics identifies genes frequently silenced in prostate cancer.Cancer Res.2005;65(10) :4218-27．
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31． Caldwell GM,Jones CE,Taniere P,et al.The Wnt antagonist sFRP4 is downregulated in premalignant large bowel adenomas.Br J Cancer.2006;94(6) :922-7．
32． Wong SC,Lo SF,Lee KC,et al.Expression of frizzled-related protein and Wnt-signalling molecules in invasive human breast tumours.J Pathol.2002;196(2) :145-53．
33． Klopocki E,Kristiansen G,Wild PJ,et al.Loss of SFRP4 is associated with breast cancer progression and poor prognosis in early stage tumors.Int J Oncol.2004;25(3) :641-9．
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35． Stoehr R,Wissmann C,Suzuki H,et al.Deletions of chromosome 8p and loss of Sfrp4 expression are progression markers of papillary bladder cancer.Lab Invest.2004;84(4) :465-78．