WIF1在胆囊癌中恢复表达可抑制肿瘤生长并促进肿瘤细胞凋亡以及可调控EpCAM等蛋白质的表达
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摘要
研究背景
胆囊癌发病率在消化道恶性肿瘤中位居第五位，是胆道系统最常见的恶性肿瘤。早期诊断、早期治疗是目前唯一治愈胆囊癌的有效手段，但是多数患者就医时，已处于晚期，无法接受手术。影响胆囊癌患者预后的主要原因有胆囊癌发展迅速，易侵犯周围脏器发生转移等。因此对胆囊癌生长侵袭转移机制方面的研究，寻找控制胆囊癌侵袭转移的治疗方法对提高胆囊癌患者生存期具有重要意义。
Wnt信号通路作为体内高度保守的代谢通路，广泛参与了胚胎分化，细胞增殖等总要过程，正常细胞内此通路一般处于关闭状态，而它的异常激活，往往与肿瘤的发生发展相关。以往的研究表明部分肿瘤中， WIF-1作为Wnt代谢通路的抑制因子之一，可通过阻断Wnt通路从而抑制肿瘤的生长。但是WIF-1在胆囊癌中的表达情况以及对于胆囊癌的生物学行为的影响以及其作用机制目前仍尚无报道。
本研究在相关前期文献报导的基础上首次探讨WIF-1基因在胆囊癌的表达及其临床、病理和预后意义；研究其在胆囊癌增殖、侵袭、转移等过程中的作用；构建裸鼠胆囊癌原位移植瘤模型；研究WIF-1对于Wnt代谢通路蛋白质表达及胆囊癌细胞超微结构的影响。从而深化对胆囊癌发病和进展机制的了解，探索胆囊
癌新的治疗靶点。第一部分：WIF-1在胆囊癌中的表达及其临床病理和预后意义
目的:本研究旨在探讨WIF-1的表达与胆囊癌的临床病理特点及预后的关系。
方法:用免疫组化评价WIF-1蛋白在40例胆囊癌以及45例慢性胆囊炎中的表达情况，结合临床数据进行分析。采用SPSS13.0软件包进行统计分析,计数资料采用χ~2检验，WIF-1表达与临床病理学特征的关系用四格表确切概率法，P<0.05视为有统计学意义。
结果：WIF-1在40例胆囊癌标本中，仅9例WIF-1表达阳性，表达率为22.5%（9/40），而在45例胆囊炎中，WIF1的表达率为100%（45/45），两者之间的阳性率具有显著差异性（P<0.001）。胆囊癌组织中WIF-1基因表达与年龄、性别、肿瘤大小、有无伴有胆囊结石、组织学类型、分化程度等临床病理学资料均未见相关性（P>0.05），提示WIF-1的缺失表达可能是胆囊癌发生过程中的早期事件。
第二部分：WIF-1基因在胆囊癌细胞中表达情况及WIF-1过表达胆囊癌细胞株的构建鉴定
目的：检测WIF-1在三株胆囊癌细胞系中的表达情况并初步探讨造成这种情况的原因：进而构建过表达WIF-1胆囊癌GBC-SD细胞株，并对其进行检测WIF-1mRNA和蛋白质表达变化情况。
方法：采用RTPCR、Western-Blot、启动子甲基化特异性PCR技术分析GBC-SD、NOZ以及SGC-996三株细胞WIF-1mRNA、WIF-1蛋白及WIF-1基因启动子区域甲基化状态；并通过质粒转染胆囊癌细胞系GBC-SD，应用RT-PCR及Weatern-Blot技术检测转染后人胆囊癌细胞GBC-SD的WIF-1的mRNA及蛋白表达。
结果：GBC-SD、NOZ、SGC-996细胞系中WIF-1均缺失表达，三株细胞中WIF-1启动子区域均存在甲基化状况，这可能是造成WIF-1在三株细胞中缺失表达的原因。过表达WIF-1质粒作用于人胆囊癌细胞株GBC-SD后，WIF-1的mRNA表达恢复， WIF-1蛋白表达恢复。
第三部分：过表达WIF-1基因对人胆囊癌细胞增殖、侵袭、转移及凋亡能力的影响
目的：研究过表达WIF-1基因对人胆囊癌细胞增殖、侵袭、转移及凋亡能力的影响。
方法：胆囊癌GBC-SD细胞分为三组：GBC-SD未处理组，GBC-SD空质粒组（GBC-SD-MOCK）以及GBC-SD过表达组（GBC-SD-WIF-1）。CCK-8法检测三组细胞增殖能力；Transwell小室运动侵袭试验检测细胞迁移和侵袭能力；流式细胞仪检测WIF-1诱导肿瘤细胞凋亡能力；明胶酶谱法检测细胞上清MMPs分泌变化情况。
结果：过表达WIF-1基因能抑制GBC-SD细胞增殖、迁移及侵袭，并可促进GBC-SD细胞凋亡（P<0.01）。空质粒组及未处理组细胞间增殖、侵袭、转移凋亡能力无明显差异（P>0.05）。过表达WIF-1能抑制MMP-2及MMP-9的分泌（P<0.01），GBC-SD-MOCK组及GBC-SD组分泌MMP2，9能力无明显差异（P>0.05）。
第四部分：过表达WIF-1基因对人胆囊癌细胞裸鼠皮下移植瘤侵袭和转移的影响
目的：研究过表达WIF-1基因对人胆囊癌细胞裸鼠皮下移植瘤生长侵袭的影响。
方法：胆囊癌GBC-SD细胞分为三组：GBC-SD未处理组，GBC-SD空质粒组（GBC-SD-MOCK）以及GBC-SD过表达组（GBC-SD-WIF-1），15只裸鼠随机分为3组，每组5只。收集对数生长期的三组细胞，注射100μl，1×106细胞/鼠细胞悬液于实验裸鼠左前腋皮下，观察五周后处死裸鼠，测量称重体积，收集标本制做病理切片。
结果：3组裸鼠成瘤率100%，过表达WIF-1组裸鼠肿瘤生长速度及成瘤体积重量均明显小于其余2组（P<0.001）；空质粒组及未处理阴性组成瘤无明显差异（P>0.05）；三组细胞成瘤均未见腹腔转移及肝脏转移，表明过表达WIF-1基因抑制人胆囊癌细胞在裸鼠体内生长转移。
第五部分过表达WIF-1对于下游蛋白质的调控以及细胞微观结构的影响
目的：研究过表达WIF-1对于GBC-SD细胞内蛋白质表达的影响，分析WIF-1对于WNT代谢通路的作用机制及观察过表达WIF-1蛋白的GBC-SD细胞系超微结构变化。
方法：采用Raybiotech蛋白芯片半定量检查过表达WIF-1蛋白的GBC-SD细胞系内部分蛋白质表达变化；采用扫描电镜观察其GBC-SD-WIF-1、GBC-SD-MOCK、GBC-SD三株细胞超微结构变化。
结果：WIF-1可导致EpCAM等多种蛋白质的表达发生上调或下降，这些蛋白质参与了细胞内诸多重要生理病理过程，提示WIF-1可能通过调节这些蛋白质的表达而参与了肿瘤细胞上皮间质化过程；扫描电镜结果提示GBC-SD-WIF-1细胞内溶酶体分泌显著增多，而GBC-SD细胞与GBC-SD-MOCK细胞内则未见这一变化。
结论：
1、多数胆囊癌组织中缺失表达WIF-1，WIF-1表达情况与肿瘤大小、分化程度和TNM分期无明显相关，表明WIF-1的失活可能为胆囊癌发生发展过程中的早期事件。
2、胆囊癌细胞系GBC-SD、NOZ-996及SGC-996三株细胞系中WIF-1mRNA均缺失表达，不表达WIF-1蛋白，这可能与其启动子区域存在甲基化相关；构建过表达WIF-1质粒作用于人胆囊癌细胞株GBC-SD后，WIF-1mRNA表达恢复， WIF-1蛋白表达恢复。
3、过表达WIF-1基因能抑制GBC-SD细胞增殖、迁移及侵袭能力，并可促进GBC-SD细胞凋亡，与之相对应MMP2，9在过表达WIF-1蛋白GBC-SD细胞中表达降低，证明了WIF-1对于GBC-SD的侵袭转移具有抑制效应可能与MMPs分泌相关。
4、人胆囊癌细胞GBC-SD皮下接种裸鼠可产生移植瘤；过表达WIF-1基因可抑制人胆囊癌细胞在裸鼠体内生长转移。
5、过表达WIF-1可导致GBC-SD细胞内部分蛋白质的表达情况发生改变，这些蛋白质广泛的参与了细胞的增殖、分化等多个生理病理过程，并可能与肿瘤细胞上皮间质化过程相关。扫描电镜结果表明细胞内溶酶体显著增多，这可能与肿瘤胞内蛋白质的分泌变化相关。
Background
The incidence of the gallbladder ranked fifth in the digestive tract malignant tumor, isthe most common malignant tumor of the biliary system. Early diagnosis andtreatment was the only effective mean to cure gallbladder at present, but most patientswas to late to go to a doctor so that was inoperable. The main reasons affecting theprognosis of patients with gallbladder were the rapidity of the gallbladderdevelopment, the easy to occurrence of the invasion of adjacent tissues and metastasis,etc. So, to research the mechanisms of invasion of the gallbladder and the metastasisof gallbladder growth, to find the cure for controlling the invasion and metastasis ofgallbladder in improving the survival of the patients with gallbladder was of greatsignificance.
Wnt signaling pathway is a conservative signaling pathway, playing veryimportant roles in diverse physiological processes such as embryonic differentiation,cell proliferation,etc.
The pathway normally closed in the normal cells, and its abnormal activation, is oftenassociated with the development of tumor. Previous studies have shown that the WIF-
1could inhibit tumor growth by blocking the Wnt pathways as one of the inhibitors ofWnt metabolic pathway in some tumors. But WIF-1expression in the gallbladderand gallbladder for the biological behavior of the influence of the mechanism of itsaction, and there was still no report.
This study on the basis of the relevant previous literature reports for the first time todiscuss WIF-1gene in expression and its clinical significance, pathology andprognosis of gallbladder; Research in gallbladder proliferation, invasion andmetastasis process function. Gallbladder in situ nude mice transplantation tumormodel was build. Research the Influence of for WNT WIF-1metabolic pathway to the protein expression. So as to deepen the understanding of the mechanisms ofgallbladder disease and progress, and exploring the gallbladder new therapeutictargets.
The first part: WIF-1expression and its significance in clinical pathology andprognosis in the gallbladder
Objective: this study was to explore the Relationship of the WIF-1expression andthe clinical pathological characteristics and prognosis of gallbladder.Method: used immunohistochemical to evaluate the expression of the WIF-1proteinin40cases of gallbladder and45cases of chronic cholecystitis.combined with clinicaldata to analyze. used SPSS13.0software package to statistical analysis, count dataused chi-square test, WIF-1expression and clinical pathological features of relationsused chi-square test, P <0.05was statistically significant.
Result: WIF-1in the specimens of40cases of gallbladder, only9cases of WIF-1expressed positive,the expression rate was22.5%(9/40), and in50cases ofcholecystitis, the expression of WIF1rate was100%(45/45), the positive ratebetween the two has the remarkable difference (P <0.001). In gallbladder tissue, theWIF-1gene expression and age, gender, tumor size, with or without associated withgallbladder stone, histological type, differentiation degree and clinical pathology datacorrelation was not found (P>0.05), prompted lack of expression may be WIF-1gallbladder occurred early in the process of the event.
The second part: The WIF-1gene expression in gallbladder cells and the buildidentification of the overexpression of gallbladder WIF-1cell line
Objective: to test the expression of the WIF–1in cell lines in three gallbladder cellsand to preliminary discuss on the cause of this situation, and then build a gallbladderGBC-SD express WIF-1cell lines, and carried on the test.Method: using RT-PCR, Western Blot, promoter methylation specific PCR to analysis theWIF-1mRNA of the GBC-SD, NOZ and SGC–996cells, the promoter regionmethylation status of the protein WIF-1and WIF-1gene.And through the plasmidtransfection GBC–SD of the gallbladder cell line, used RT-PCR and Weatern-Blotto detect the GBC-SD WIF-1mRNA and protein expression of the gallbladder cellafter transfection.
Result:The WIF-1of the GBC-SD, NOZ and SGC–996were missing expression, inthree cells,there were all the promoter methylation status of the WIF-1, this may bethe reason of the missing expression of the WIF-1. after the overexpression plasmidWIF-1functing in gallbladder cell lines GBC-SD, WIF-1mRNA expression wassignificantly raised, WIF1protein expression was restore.
The third part: The influence of overexpression WIF1gene to the cellproliferation, invasion, metastasis and apoptosis of the human gallbladdercarcinoma cell
Objective: to study the overexpression WIF–1gene on the cell proliferation,invasion, metastasis and apoptosis of the human gallbladder carcinoma cell.
Method: the gallbladder GBC-SD cells were divided into three groups: the GBC-SD untreated group, the GBC-SD empty plasmid group (GBC-SD-MOCK) andthe GBC-SD overexpression group (GBC-SD-WIF-1). Used the CCK method todetect the cell proliferation ability of the three groups; Transwell Chambersmovement attacking tested cell migration and invasion ability; Flow cytometryinstrument detected the ability to induce apoptosis of the WIF-1; the change of theMMPs of the Cell supernatant was measured by gelatin zymography.
Result: The overexpression WIF-1gene could inhibit the cell proliferation, migration,and invasion of the GBC-SD, and can promote the apoptosis of the GBC-SD (P <0.01). The empty plasmid group and untreated group between cell proliferation andapoptosis of invasion and metastasis ability has no obvious difference (P>0.05).Overexpression WIF1could inhibit the secretion of MMP-2and MMP-9(P <0.01),the ability of the GBC-SD-MOCK and GBC-SD group secreting MMP2,9were no obvious difference (P>0.05).
The fourth part:The influence on the overexpression WIF1gene to humangallbladder cells in nude mice subcutaneously transplanted tumor invasion andmetastasis
Objective: to study The influence of overexpression WIF-1gene on gallbladder cellsin nude mice subcutaneously transplanted tumor growth and invasion.Method: the gallbladder GBC-SD cells were divided into three groups: the GBC-SD untreated group, the GBC-SD empty plasmid group (GBC-SD-MOCK) andthe GBC-SD overexpression group (GBC-SD-WIF-1),15nude mice wererandomly divided into3groups, each group5only. Collected three groups of cells inlogarithmic phase, and injected100mu l,1x106cells/mouse cells suspension intothe left anterior axillary subcutaneous nude mice in the experiments, mice wereeuthanized at observation after five weeks, measuring weighing volume, collectedspecimen made pathological section.
Result:
1, successfully build gallbladder subcutaneous cells GBC-SD nude micetransplantation tumor model;
2, The tumor rate of3groups of nude mice was100%, overexpression WIF-1set ofnude mice tumor growth and into tumors had accumulated weight were significantlyless than the rest of the other two groups (P <0.001); The empty plasmid group anduntreated negative tumor without obvious difference (P>0.05); showed thatoverexpression WIF-1inhibit the growth of human gallbladder cells in nude mice invivo.
The fifth part:The regulation of the overexpression WIF-1for the downstreamprotein and the influence of the micro structure of the cell
Objective: to study the overexpression of WIF-1for the influence of the GBC-SD protein expression in the cell, to analysis the WNT mechanism of action of metabolicpathway of the WIF-1and to observe the GBC-SD cell ultrastructure change of theoverexpression WIF1proteinMethod: used qualitative Raybiotech protein chip to check the GBC-SD part changesin protein expression of the overexpression WIF1protein.Observed the ultrastructuralchange by the scanning electron microscopy.Result: WIF-1could lead to DKK-1, and other protein expression change, theseproteins were involved in many important physiological and pathological processes inthe cell, prompted WIF-1might affect the GBC-SD qualitative process betweenepithelial cells; the Scanning electron microscopy (sem) results suggested the GBC-SD-WIF-1lysosomes in the cell secretion was significantly increased, the GBC-SD cells and the GBC-SD-MOCK, you did not see the change.
Conclusion:
1, The majority of gallbladder tissues were lack of the WIF–1expression, there werenot significant correlation between the WIF-1expression and the tumor size, degreeof differentiation and TNM staging, suggested that the deactivation of the WIF-1might be the early event in the process of occurence and development of thegallbladder.
2,The expression were missing of the WIF-1mRNA in the GBC-SD, NOZ-996andSGC-996of the gallbladder cell line, WIF1protein wasnot expressed, this might berelated to its promoter region methylation.Build overexpression WIF-1plasmid to acton human gallbladder GBC-SD cell line, the WIF-1mRNA expression level, WIF1protein expression was restored after it.
3, Overexpression WIF-1gene could inhibit the ability of the proliferation,migration and invasion of the GBC-SD cell, and could improve the apoptosis of theGBC-SD,, and at the same time MMP2,9protein expressed in overexpression WIF-1the GBC-SD cells decreased, also proved the inhibitory effect of WIF-1to theGBC-SD.
4, The GBC gallbladder cells-SD could be induced by subcutaneous to inoculate the nude mice to produce the transplantation tumor; Overexpression WIF-1gene couldinhibit the growth of human gallbladder cells in nude mice in vivo.
5, Overexpression WIF-1could lead to GBC-SD part intracellular protein expressionchange, these proteins involved in cell proliferation, differentiation, and otherphysiological and pathological process, and might be associated with qualitativeprocess between epithelial tumor cells. Scanning electron microscopy (sem) resultsshowed that the lysosomes in the cell significantly increased, which may be related toits protein secretion change.
胆囊癌发病率在消化道恶性肿瘤中位居第五位，是胆道系统最常见的恶性肿瘤。早期诊断、早期治疗是目前唯一治愈胆囊癌的有效手段，但是多数患者就医时，已处于晚期，无法接受手术。影响胆囊癌患者预后的主要原因有胆囊癌发展迅速，易侵犯周围脏器发生转移等。因此对胆囊癌生长侵袭转移机制方面的研究，寻找控制胆囊癌侵袭转移的治疗方法对提高胆囊癌患者生存期具有重要意义。
Wnt信号通路作为体内高度保守的代谢通路，广泛参与了胚胎分化，细胞增殖等总要过程，正常细胞内此通路一般处于关闭状态，而它的异常激活，往往与肿瘤的发生发展相关。以往的研究表明部分肿瘤中， WIF-1作为Wnt代谢通路的抑制因子之一，可通过阻断Wnt通路从而抑制肿瘤的生长。但是WIF-1在胆囊癌中的表达情况以及对于胆囊癌的生物学行为的影响以及其作用机制目前仍尚无报道。
本研究在相关前期文献报导的基础上首次探讨WIF-1基因在胆囊癌的表达及其临床、病理和预后意义；研究其在胆囊癌增殖、侵袭、转移等过程中的作用；构建裸鼠胆囊癌原位移植瘤模型；研究WIF-1对于Wnt代谢通路蛋白质表达及胆囊癌细胞超微结构的影响。从而深化对胆囊癌发病和进展机制的了解，探索胆囊
癌新的治疗靶点。第一部分：WIF-1在胆囊癌中的表达及其临床病理和预后意义
目的:本研究旨在探讨WIF-1的表达与胆囊癌的临床病理特点及预后的关系。
方法:用免疫组化评价WIF-1蛋白在40例胆囊癌以及45例慢性胆囊炎中的表达情况，结合临床数据进行分析。采用SPSS13.0软件包进行统计分析,计数资料采用χ~2检验，WIF-1表达与临床病理学特征的关系用四格表确切概率法，P<0.05视为有统计学意义。
结果：WIF-1在40例胆囊癌标本中，仅9例WIF-1表达阳性，表达率为22.5%（9/40），而在45例胆囊炎中，WIF1的表达率为100%（45/45），两者之间的阳性率具有显著差异性（P<0.001）。胆囊癌组织中WIF-1基因表达与年龄、性别、肿瘤大小、有无伴有胆囊结石、组织学类型、分化程度等临床病理学资料均未见相关性（P>0.05），提示WIF-1的缺失表达可能是胆囊癌发生过程中的早期事件。
第二部分：WIF-1基因在胆囊癌细胞中表达情况及WIF-1过表达胆囊癌细胞株的构建鉴定
目的：检测WIF-1在三株胆囊癌细胞系中的表达情况并初步探讨造成这种情况的原因：进而构建过表达WIF-1胆囊癌GBC-SD细胞株，并对其进行检测WIF-1mRNA和蛋白质表达变化情况。
方法：采用RTPCR、Western-Blot、启动子甲基化特异性PCR技术分析GBC-SD、NOZ以及SGC-996三株细胞WIF-1mRNA、WIF-1蛋白及WIF-1基因启动子区域甲基化状态；并通过质粒转染胆囊癌细胞系GBC-SD，应用RT-PCR及Weatern-Blot技术检测转染后人胆囊癌细胞GBC-SD的WIF-1的mRNA及蛋白表达。
结果：GBC-SD、NOZ、SGC-996细胞系中WIF-1均缺失表达，三株细胞中WIF-1启动子区域均存在甲基化状况，这可能是造成WIF-1在三株细胞中缺失表达的原因。过表达WIF-1质粒作用于人胆囊癌细胞株GBC-SD后，WIF-1的mRNA表达恢复， WIF-1蛋白表达恢复。
第三部分：过表达WIF-1基因对人胆囊癌细胞增殖、侵袭、转移及凋亡能力的影响
目的：研究过表达WIF-1基因对人胆囊癌细胞增殖、侵袭、转移及凋亡能力的影响。
方法：胆囊癌GBC-SD细胞分为三组：GBC-SD未处理组，GBC-SD空质粒组（GBC-SD-MOCK）以及GBC-SD过表达组（GBC-SD-WIF-1）。CCK-8法检测三组细胞增殖能力；Transwell小室运动侵袭试验检测细胞迁移和侵袭能力；流式细胞仪检测WIF-1诱导肿瘤细胞凋亡能力；明胶酶谱法检测细胞上清MMPs分泌变化情况。
结果：过表达WIF-1基因能抑制GBC-SD细胞增殖、迁移及侵袭，并可促进GBC-SD细胞凋亡（P<0.01）。空质粒组及未处理组细胞间增殖、侵袭、转移凋亡能力无明显差异（P>0.05）。过表达WIF-1能抑制MMP-2及MMP-9的分泌（P<0.01），GBC-SD-MOCK组及GBC-SD组分泌MMP2，9能力无明显差异（P>0.05）。
第四部分：过表达WIF-1基因对人胆囊癌细胞裸鼠皮下移植瘤侵袭和转移的影响
目的：研究过表达WIF-1基因对人胆囊癌细胞裸鼠皮下移植瘤生长侵袭的影响。
方法：胆囊癌GBC-SD细胞分为三组：GBC-SD未处理组，GBC-SD空质粒组（GBC-SD-MOCK）以及GBC-SD过表达组（GBC-SD-WIF-1），15只裸鼠随机分为3组，每组5只。收集对数生长期的三组细胞，注射100μl，1×106细胞/鼠细胞悬液于实验裸鼠左前腋皮下，观察五周后处死裸鼠，测量称重体积，收集标本制做病理切片。
结果：3组裸鼠成瘤率100%，过表达WIF-1组裸鼠肿瘤生长速度及成瘤体积重量均明显小于其余2组（P<0.001）；空质粒组及未处理阴性组成瘤无明显差异（P>0.05）；三组细胞成瘤均未见腹腔转移及肝脏转移，表明过表达WIF-1基因抑制人胆囊癌细胞在裸鼠体内生长转移。
第五部分过表达WIF-1对于下游蛋白质的调控以及细胞微观结构的影响
目的：研究过表达WIF-1对于GBC-SD细胞内蛋白质表达的影响，分析WIF-1对于WNT代谢通路的作用机制及观察过表达WIF-1蛋白的GBC-SD细胞系超微结构变化。
方法：采用Raybiotech蛋白芯片半定量检查过表达WIF-1蛋白的GBC-SD细胞系内部分蛋白质表达变化；采用扫描电镜观察其GBC-SD-WIF-1、GBC-SD-MOCK、GBC-SD三株细胞超微结构变化。
结果：WIF-1可导致EpCAM等多种蛋白质的表达发生上调或下降，这些蛋白质参与了细胞内诸多重要生理病理过程，提示WIF-1可能通过调节这些蛋白质的表达而参与了肿瘤细胞上皮间质化过程；扫描电镜结果提示GBC-SD-WIF-1细胞内溶酶体分泌显著增多，而GBC-SD细胞与GBC-SD-MOCK细胞内则未见这一变化。
结论：
1、多数胆囊癌组织中缺失表达WIF-1，WIF-1表达情况与肿瘤大小、分化程度和TNM分期无明显相关，表明WIF-1的失活可能为胆囊癌发生发展过程中的早期事件。
2、胆囊癌细胞系GBC-SD、NOZ-996及SGC-996三株细胞系中WIF-1mRNA均缺失表达，不表达WIF-1蛋白，这可能与其启动子区域存在甲基化相关；构建过表达WIF-1质粒作用于人胆囊癌细胞株GBC-SD后，WIF-1mRNA表达恢复， WIF-1蛋白表达恢复。
3、过表达WIF-1基因能抑制GBC-SD细胞增殖、迁移及侵袭能力，并可促进GBC-SD细胞凋亡，与之相对应MMP2，9在过表达WIF-1蛋白GBC-SD细胞中表达降低，证明了WIF-1对于GBC-SD的侵袭转移具有抑制效应可能与MMPs分泌相关。
4、人胆囊癌细胞GBC-SD皮下接种裸鼠可产生移植瘤；过表达WIF-1基因可抑制人胆囊癌细胞在裸鼠体内生长转移。
5、过表达WIF-1可导致GBC-SD细胞内部分蛋白质的表达情况发生改变，这些蛋白质广泛的参与了细胞的增殖、分化等多个生理病理过程，并可能与肿瘤细胞上皮间质化过程相关。扫描电镜结果表明细胞内溶酶体显著增多，这可能与肿瘤胞内蛋白质的分泌变化相关。
Background
The incidence of the gallbladder ranked fifth in the digestive tract malignant tumor, isthe most common malignant tumor of the biliary system. Early diagnosis andtreatment was the only effective mean to cure gallbladder at present, but most patientswas to late to go to a doctor so that was inoperable. The main reasons affecting theprognosis of patients with gallbladder were the rapidity of the gallbladderdevelopment, the easy to occurrence of the invasion of adjacent tissues and metastasis,etc. So, to research the mechanisms of invasion of the gallbladder and the metastasisof gallbladder growth, to find the cure for controlling the invasion and metastasis ofgallbladder in improving the survival of the patients with gallbladder was of greatsignificance.
Wnt signaling pathway is a conservative signaling pathway, playing veryimportant roles in diverse physiological processes such as embryonic differentiation,cell proliferation,etc.
The pathway normally closed in the normal cells, and its abnormal activation, is oftenassociated with the development of tumor. Previous studies have shown that the WIF-
1could inhibit tumor growth by blocking the Wnt pathways as one of the inhibitors ofWnt metabolic pathway in some tumors. But WIF-1expression in the gallbladderand gallbladder for the biological behavior of the influence of the mechanism of itsaction, and there was still no report.
This study on the basis of the relevant previous literature reports for the first time todiscuss WIF-1gene in expression and its clinical significance, pathology andprognosis of gallbladder; Research in gallbladder proliferation, invasion andmetastasis process function. Gallbladder in situ nude mice transplantation tumormodel was build. Research the Influence of for WNT WIF-1metabolic pathway to the protein expression. So as to deepen the understanding of the mechanisms ofgallbladder disease and progress, and exploring the gallbladder new therapeutictargets.
The first part: WIF-1expression and its significance in clinical pathology andprognosis in the gallbladder
Objective: this study was to explore the Relationship of the WIF-1expression andthe clinical pathological characteristics and prognosis of gallbladder.Method: used immunohistochemical to evaluate the expression of the WIF-1proteinin40cases of gallbladder and45cases of chronic cholecystitis.combined with clinicaldata to analyze. used SPSS13.0software package to statistical analysis, count dataused chi-square test, WIF-1expression and clinical pathological features of relationsused chi-square test, P <0.05was statistically significant.
Result: WIF-1in the specimens of40cases of gallbladder, only9cases of WIF-1expressed positive,the expression rate was22.5%(9/40), and in50cases ofcholecystitis, the expression of WIF1rate was100%(45/45), the positive ratebetween the two has the remarkable difference (P <0.001). In gallbladder tissue, theWIF-1gene expression and age, gender, tumor size, with or without associated withgallbladder stone, histological type, differentiation degree and clinical pathology datacorrelation was not found (P>0.05), prompted lack of expression may be WIF-1gallbladder occurred early in the process of the event.
The second part: The WIF-1gene expression in gallbladder cells and the buildidentification of the overexpression of gallbladder WIF-1cell line
Objective: to test the expression of the WIF–1in cell lines in three gallbladder cellsand to preliminary discuss on the cause of this situation, and then build a gallbladderGBC-SD express WIF-1cell lines, and carried on the test.Method: using RT-PCR, Western Blot, promoter methylation specific PCR to analysis theWIF-1mRNA of the GBC-SD, NOZ and SGC–996cells, the promoter regionmethylation status of the protein WIF-1and WIF-1gene.And through the plasmidtransfection GBC–SD of the gallbladder cell line, used RT-PCR and Weatern-Blotto detect the GBC-SD WIF-1mRNA and protein expression of the gallbladder cellafter transfection.
Result:The WIF-1of the GBC-SD, NOZ and SGC–996were missing expression, inthree cells,there were all the promoter methylation status of the WIF-1, this may bethe reason of the missing expression of the WIF-1. after the overexpression plasmidWIF-1functing in gallbladder cell lines GBC-SD, WIF-1mRNA expression wassignificantly raised, WIF1protein expression was restore.
The third part: The influence of overexpression WIF1gene to the cellproliferation, invasion, metastasis and apoptosis of the human gallbladdercarcinoma cell
Objective: to study the overexpression WIF–1gene on the cell proliferation,invasion, metastasis and apoptosis of the human gallbladder carcinoma cell.
Method: the gallbladder GBC-SD cells were divided into three groups: the GBC-SD untreated group, the GBC-SD empty plasmid group (GBC-SD-MOCK) andthe GBC-SD overexpression group (GBC-SD-WIF-1). Used the CCK method todetect the cell proliferation ability of the three groups; Transwell Chambersmovement attacking tested cell migration and invasion ability; Flow cytometryinstrument detected the ability to induce apoptosis of the WIF-1; the change of theMMPs of the Cell supernatant was measured by gelatin zymography.
Result: The overexpression WIF-1gene could inhibit the cell proliferation, migration,and invasion of the GBC-SD, and can promote the apoptosis of the GBC-SD (P <0.01). The empty plasmid group and untreated group between cell proliferation andapoptosis of invasion and metastasis ability has no obvious difference (P>0.05).Overexpression WIF1could inhibit the secretion of MMP-2and MMP-9(P <0.01),the ability of the GBC-SD-MOCK and GBC-SD group secreting MMP2,9were no obvious difference (P>0.05).
The fourth part:The influence on the overexpression WIF1gene to humangallbladder cells in nude mice subcutaneously transplanted tumor invasion andmetastasis
Objective: to study The influence of overexpression WIF-1gene on gallbladder cellsin nude mice subcutaneously transplanted tumor growth and invasion.Method: the gallbladder GBC-SD cells were divided into three groups: the GBC-SD untreated group, the GBC-SD empty plasmid group (GBC-SD-MOCK) andthe GBC-SD overexpression group (GBC-SD-WIF-1),15nude mice wererandomly divided into3groups, each group5only. Collected three groups of cells inlogarithmic phase, and injected100mu l,1x106cells/mouse cells suspension intothe left anterior axillary subcutaneous nude mice in the experiments, mice wereeuthanized at observation after five weeks, measuring weighing volume, collectedspecimen made pathological section.
Result:
1, successfully build gallbladder subcutaneous cells GBC-SD nude micetransplantation tumor model;
2, The tumor rate of3groups of nude mice was100%, overexpression WIF-1set ofnude mice tumor growth and into tumors had accumulated weight were significantlyless than the rest of the other two groups (P <0.001); The empty plasmid group anduntreated negative tumor without obvious difference (P>0.05); showed thatoverexpression WIF-1inhibit the growth of human gallbladder cells in nude mice invivo.
The fifth part:The regulation of the overexpression WIF-1for the downstreamprotein and the influence of the micro structure of the cell
Objective: to study the overexpression of WIF-1for the influence of the GBC-SD protein expression in the cell, to analysis the WNT mechanism of action of metabolicpathway of the WIF-1and to observe the GBC-SD cell ultrastructure change of theoverexpression WIF1proteinMethod: used qualitative Raybiotech protein chip to check the GBC-SD part changesin protein expression of the overexpression WIF1protein.Observed the ultrastructuralchange by the scanning electron microscopy.Result: WIF-1could lead to DKK-1, and other protein expression change, theseproteins were involved in many important physiological and pathological processes inthe cell, prompted WIF-1might affect the GBC-SD qualitative process betweenepithelial cells; the Scanning electron microscopy (sem) results suggested the GBC-SD-WIF-1lysosomes in the cell secretion was significantly increased, the GBC-SD cells and the GBC-SD-MOCK, you did not see the change.
Conclusion:
1, The majority of gallbladder tissues were lack of the WIF–1expression, there werenot significant correlation between the WIF-1expression and the tumor size, degreeof differentiation and TNM staging, suggested that the deactivation of the WIF-1might be the early event in the process of occurence and development of thegallbladder.
2,The expression were missing of the WIF-1mRNA in the GBC-SD, NOZ-996andSGC-996of the gallbladder cell line, WIF1protein wasnot expressed, this might berelated to its promoter region methylation.Build overexpression WIF-1plasmid to acton human gallbladder GBC-SD cell line, the WIF-1mRNA expression level, WIF1protein expression was restored after it.
3, Overexpression WIF-1gene could inhibit the ability of the proliferation,migration and invasion of the GBC-SD cell, and could improve the apoptosis of theGBC-SD,, and at the same time MMP2,9protein expressed in overexpression WIF-1the GBC-SD cells decreased, also proved the inhibitory effect of WIF-1to theGBC-SD.
4, The GBC gallbladder cells-SD could be induced by subcutaneous to inoculate the nude mice to produce the transplantation tumor; Overexpression WIF-1gene couldinhibit the growth of human gallbladder cells in nude mice in vivo.
5, Overexpression WIF-1could lead to GBC-SD part intracellular protein expressionchange, these proteins involved in cell proliferation, differentiation, and otherphysiological and pathological process, and might be associated with qualitativeprocess between epithelial tumor cells. Scanning electron microscopy (sem) resultsshowed that the lysosomes in the cell significantly increased, which may be related toits protein secretion change.
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74、 Wnt inhibitory factor1decreases tumorigenesis and metastasis in osteosarcoma.Mol Cancer Ther.2010Mar;9(3):731-41.
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85、Chim CS, Fung TK, Wong KF, Lau JS, Liang R. Infrequent Wnt inhibitoryfactor-1(WIF-1) methylation in chronic lymphocytic leukemia. Leuk Res.2006Sep;30(9):1135-9.
86、Lin YC, You L, Xu Z, He B, Mikami I, Thung E, Chou J, Kuchenbecker K, Kim J,Raz D, Yang CT, Chen JK, Jablons DM. Wnt signaling activation and WIF-1silencing in nasopharyngeal cancer cell lines. Biochem Biophys Res Commun.2006Mar10：341(2):635-40.
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