【作者】 金京春；

【导师】 林贞花；

【作者基本信息】 延边大学 ， 病理学与病理生理学， 2012， 博士

【摘要】 目的：探讨p12-LOX,NF-κB和Ezrin基因在上皮细胞源性肿瘤演进过程中的作用及机制,为上皮细胞源性肿瘤的诊断、预后评估及临床治疗提供可能的新分子靶点。方法：培养处于不同阶段的3种小鼠表皮细胞,即正常的JB6P-、已启动化(initiated)的JB6P+、黑色素瘤B16细胞和7种人胃癌细胞,即高分化的MKN-1、SNU-216,中分化的MKN-28、MKN-74、NCI-N87(N87)和低分化的MKN-45、AGS细胞。提取3种小鼠表皮细胞(JB6P-、JB6P+和B16细胞)的总RNA,进行miRNA基因芯片检测,明确肿瘤演进过程中差异miRNA表达,并通过Targetscan网站检索miR17、miR-19和miR-31的作用靶点。用p12-LOX的选择性抑制剂黄芩素(Baicalein,BAI)、NF-κB的选择性抑制剂BAY11-7082(BAY)以及NF-κB和Ezrin的特异性siRNA (NF-κB siRNA和Ezrin siRNA)处理细胞,用RT-PCR法检测p12-LOX和Ezrin基因mRNA的表达,并进行细胞增殖、划痕、软琼脂克隆形成、单个细胞克隆形成和细胞凋亡检测等功能实验,并对Ezrin蛋白进行免疫荧光染色,同时利用蛋白印迹法检测7种胃癌细胞的Ezrin蛋白表达水平。此外,还对335例胃病变组织标本(包括癌旁正常胃粘膜26例、不典型增生32例和胃癌277例)进行Ezrin蛋白的免疫组化染色,结合临床资料进行相关性分析。结果：1、小鼠表皮细胞miRNA基因芯片检测结果：共检测722种miRNA。与正常JB6P-细胞相比,在JB6P+和(或)B16中有46种miRNA表达上调(变化最明显的有miR-138、miR-130b、miR-378、miR-31、miR-19b和miR-17等),35种miRNA表达下调(变化最明显的有miR-214、miR-486、miR-143和miR-145等)。Targescan网站检索发现：其中miR-17、miR-19和miR-31等与MAPK、PI3K/Akt、NF-κB等信号转导通路有相关性。2、RT-PCR结果：(1)p12-LOX mRNA的表达：①小鼠表皮正常JB6P-细胞的表达最低,其次为已启动化的JB6P+细胞,黑色素瘤B16细胞的表达最高,提不p12-LOX与黑色素瘤的发生发展有密切相关性；②胃癌细胞中的p12-LOXmRNA表达水平有明显差异,在高分化的MKN-1、SNU-216细胞中表达最低,其次为中分化的MKN-74、MKN-28和N87细胞,在低分化的MKN45细胞中表达最高,提示p12-LOX高表达可促进胃癌的发展。(2)Ezrin mRNA的表达：①与JB6P+细胞相比较,B16细胞的Ezrin mRNA表达明显增高,提示Ezrin可促进小鼠黑色素瘤的发生；②胃癌细胞中的Ezrin mRNA表达水平也有明显差异,在低侵袭性的MKN-1细胞中表达较低,而在高侵袭性的MKN-74、MKN-28、MKN-45和高迁移性SNU-216等细胞中表达较高,提示Ezrin高表达可促进人胃癌的发展及侵袭。(3)BAI和BAY处理对胃癌细胞Ezrin mRNA表达的影响：BAI和BAY处理可明显抑制MKN-74和N87细胞Ezrin mRNA的表达,反向提示p12-LOX和NF-κB可参与调控Ezrin的表达。3、MTT细胞增殖实验结果：经BAI和BAY处理,B16和人胃癌细胞均呈浓度和时间依赖性地抑制细胞增殖,与对照组比较均有显著差异(P<0.01),反向提示p12-LOX和NF-κB可促进上皮细胞源性肿瘤细胞的增殖。4、细胞迁移(划痕)实验结果：BAI呈浓度依赖性的地抑制B16细胞的迁移,BAI和BAY以及NF-κB siRNA和Ezrin siRNA均明显抑制B16和MKN-1、SNU-216、AGS等细胞的迁移,反向提示p12-LOX、NF-κB和Ezrin基因可促进肿瘤细胞迁移。5、Ezrin蛋白的免疫荧光染色结果：(1)Ezrin蛋白表达在静止的细胞中主要分布于细胞膜和细胞浆,而在迁移的细胞中主要分布于细胞伸出伪足的部位；(2)BAI和Ezrin siRNA处理可明显下调B16和MKN-1细胞的Ezrin蛋白表达水平,反向提示p12-LOX可通过上调Ezrin蛋白的表达促进细胞迁移；(3)BAY及NF-κB siRNA处理也明显抑制了细胞迁移,但Ezrin蛋白的荧光表达没有受抑制,提示NF-κB信号转导通路可以促进肿瘤细胞迁移,但不是通过调控Ezrin蛋白发生作用的。6、软琼脂克隆形成实验结果：BAI处理呈浓度依赖性地明显抑制B16细胞的软琼脂克隆形成,提示p12-LOX抑制剂可杀伤小鼠B16细胞。7、单个细胞克隆形成实验结果：BAI呈浓度依赖性的明显抑制小鼠B16细胞的单个细胞克隆形成,提示p12-LOX可促进单个细胞增殖,其抑制剂可杀伤小鼠黑色素瘤细胞。8、细胞凋亡检测结果：BAI和BAY以及Ezrin siRNA处理明显促进细胞凋亡,提示p12-LOX、 NF-κB和Ezrin可通过抑制细胞凋亡促进肿瘤的演进。9、胃组织Ezrin蛋白免疫组化染色结果：(1)Ezrin蛋白染色强阳性率在正常胃粘膜组为0%,不典型增生组和胃癌组中分别为37.5%和60.6%,与正常组比较均有显著性差异(分别P<0.05和P<0.01)；(2)Ezrin蛋白染色强阳性率与胃癌分型、淋巴转移、临床分级和无病生存期呈正相关,与对照组比较均有显著性差异(分别P<0.01,P<0.01,P<0.05和P<0.01),而与年龄、性别和组织学类型等均无关。提示,Ezrin蛋白可作为胃癌临床诊断和预后评估的有效分子指标,Ezrin有望成为胃癌治疗的新分子靶点。结论：1、在上皮源性肿瘤的演进过程中,不同发展阶段存在明显的差异miRNA表达,其中上调表达变化明显的有miR-138、miR-130b、miR-378、miR-31、miR-19b和miR-17等,而下调表达明显的有miR-214、miR-486、miR-143和miR-145等。2、p12-LOX可通过上调肿瘤细胞Ezrin mRNA和蛋白表达促进小鼠黑色素瘤和人胃癌细胞的增殖和侵袭。3、NF-κB抑制剂可明显抑制细胞迁移、促进细胞凋亡,但没有影响Ezrin蛋白的表达,提示NF-κB促进肿瘤演进的作用不是通过调控Ezrin蛋白实现的。4、Ezrin基因可促进体外小鼠黑色素瘤和人胃癌细胞的迁移,抑制细胞凋亡,具有促进肿瘤发展和侵袭的作用；Ezrin蛋白过表达与胃癌Lauren分型、淋巴转移、临床分期和无病生存期有明显的正相关,提示Ezrin蛋白可作为胃癌早期诊断和预后评估的有效分子指标之一。更多还原

【Abstract】 AIM:This study is aimed to investigate the roles and mechanism of p12-LOX, NF-κB and Ezrin genes in epithelial tumorigenesis, and to provide the possible new targets for the diagnosis, prognosis and target therapy of epithelial cancers.Methods:The different stages of three kinds of mouse epidermal cells, including normal JB6P-, initiated JB6P+and melanoma B16cells, and seven of human gastric cancer cells, including well differentiated MKN-1, SNU-216, moderately differentiated MKN-28, MKN-74, NCI-N87(N87), and poor differentiated MKN-45, AGS, were cultured. Total RNA extract from mouse JB6P-, JB6P+and B16cells, was analyzed by using miRNA gene microarray and defined the abnormal expression of miRNA in tumorigenesis, and the biological targets of miR-17, miR-19and miR-31were searched by using Targetscan website. Above cultured cells were treated by p12-LOX selective inhibitor baicalein (BAI), NF-κB specific inhibitor BAY11-7082(BAY), and specific siRNA of NF-κB and Ezrin (NF-κB siRNA and Ezrin siRNA), and were performed RT-PCR and Western blotting to detect mRNA and protein expression in above treated cells and their control groups, respectively. The untreated and treated cells were analyzed by MTT, scratches and apoptosis assay, soft agar colony formation, single cell cloning efficiency, and immunofluorescence staining for Ezrin protein. Additionally, total335cases of gastric lesions, including26of normal gastric mucosa,32of atypical hyperplasia, and277of gastric cancer, were examined by immunohistochemical staining for Ezrin protein, and the significance to clinical data of gastric cancer was further analyzed.Results:1. miRNA gene chip results in mouse epidermal cell:Total722of miRNAs were detected by gene chip, and the results showed that there are46kinds of miRNA expression were up-regulated (miR-138, miR-130b, miR-378, miR-31, miR-19b and miR-17, et al) and35kinds of miRNA expression were downregulated (miR-214, miR-486, miR-143and miR-145, et al) in JB6P+and B16cells compared with normal JB6P-cells. Furthermore, it was found that miR-17, miR-19, and miR-31were targeted to MAPK、 PI3K/Akt、NF-κB signaling pathways by searching Targetscan.2. RT-PCR:(1)p12-LOX mRNA expression:①The p12-LOX mRNA expressions levels was highest in B16cell, followed by initiated JB6P+cell, then the normal JB6P-cell, suggesting that p12-LOX overexpression might promote the tumor development.②The p12-LOX mRNA expressions were different in various gastric cancer cell lines, and the levels was highest in poorly differentiated MKN45cells, followed by moderate differentiated MKN-74, MKN-28and N87cells, then well differentiated MKN-1and SNU-216cells, suggesting that p12-LOX overexpression might predict the poor prognosis of gastric cancer.(2) Ezrin mRNA expression:①Ezrin mRNA expression was significantly increased in B16cells compared with the JB6P+cells, suggesting that Ezrin overexpression closely related with tumor development.②Ezrin mRNA expression levels in various gastric cancer cell lines were different. It was lower in MKN-1weak invasive cells, than in MKN-74, MKN-28and MKN-45cells with high invasive ability, and SNU-216cells with high migration ability. It suggested that Ezrin overexpression might promote tumor metastasis.(3) BAI and BAY treatment inhibited Ezrin mRNA expression:Ezrin mRNA expression were significantly decreased in BAI treated MKN-74and N87cells compared with the untreated above cells, suggesting p12-LOX and NF-κB could up-regulated Ezrin gene expression.3. Cell proliferation assay:BAI and BAY treatments showed that BAY could significantly inhibit the proliferation of various above gastric cancer cell lines compared with each of the control groups (P<0.01), respectively, and it showed the dose and time dependent manner, suggesting that p12-LOX and NF-κB could promote the cell proliferation of epithelial cancer.4. Cell migration (scratch) assay:BAI could significantly inhibited B16cell migration compared with the control group, and it showed the dose dependent manner, suggesting that p12-LOX and NF-κB could also promote the cell proliferation of epithelial cancer. BAI, BAY, NF-κB siRNA and NF-κB siRNA treatments significantly inhibited the cell migration of MKN-1, SNU-216and AGS cells, suggesting that p12-LOX, NF-κB and Ezrin gene could promote tumor metastasis and progression.5. Immunofluorescence staining for Ezrin protein:(1) Ezrin protein located at the cytoplasm and membrane in resting cells, however, its location mainly concentrated at the membranous protrusion site in the migrating cells.(2) BAI and Ezrin siRNA treatments could decrease the Ezrin protein expression levels in B16and MKN-1cells, suggesting that the p12-LOX regulates Ezrin protein expression and promotes cell migration and invasion.(3) BAY and NF-κB siRNA treatments could not inhibit Ezrin protein expression in B16cells by immunofluorescence staining, however, BAY and NF-κB siRNA treatments could significantly inhibited migration of B16and MKN-1cells, suggesting that NF-κB signaling pathway might promote the tumorigenesis but not through Ezrin protein regulation.6. Soft agar colony formation:BAI treatments significantly inhibited soft agar colony formation in B16cells by concentration-dependent manner, suggesting that the inhibitor of p12-LOX could kill the mouse melanoma cells in vitro.7. Single cell coloning efficiency:BAI treatment significantly inhibited one cell colony formation in B16cells at the concentration-dependent manner, suggesting that inhibition of p12-LOX could decrease the cell proliferation and effectively killed the melanoma cells in vitro.8. Apoptosis analysis:BAI, BAY and Ezrin siRNA treatments could significantly induce apoptosis of JB6P+and MKN-74cells, indicating that p12-LOX, NF-κB and Ezrin could promote tumorigenesis by inhibiting apoptosis.9. The results of immunohistochemical staining for Ezrin protein:(1) Ezrin positive rate were significantly higher in dysplasia and gastric cancer compared with normal mucosa, respectively60.6%,37.5%and0%(P<0.05and P<0.01).(2) Ezrin staining strong positive rate are positively correlated with Lauren Types, lymph metastasis, clinical stage and disease free survival (respectively P<0.01, P<0.01,<0.05and P<0.01), however, Ezrin protein expression level was not correlated with the patient age, gender, WHO’s histological type status of gastric carcinomas (P>0.05). It suggested that Ezrin protein could be a new molecular target for the diagnosis, prognosis, and therapy of gastric cancer.Conclusions:1. The differential miRNA expression was existed in the different stage of epithelial tumorigenesis, and the significantly up-regulated miRNAs were miR-138, miR-130b, miR-378,miR-31, miR-19b and miR-17, et al and the significantly down-regulated miRNAs were miR-214, miR-486, miR-143and miR-145, et al.2.p12-LOX could promote the cell proliferation and invasiveness, and inhibit apoptosis in murine melanoma and human gastric cancer cells by up-regulating Ezrin expression.3. The specific inhibitor of NF-κB could significantly inhibit cell migration and induce cell apoptosis, but couldn’t change the Ezrin protein expression level, suggesting that NF-κB could promote the progression of gastric cancer but not through Ezrin protein regulation.4. Ezrin gene could promote the cell migration and inhibit apoptosis of murine melanoma and human gastric cancer in vitro, indicating that Ezrin could promote the progression of epithelial tumorigenesis. Additionally, Ezrin overexpression showed the significant positive correlation with the Lauren type, lymph node metastasis, clinical staging and disease-free survival of gastric cancers, suggesting that Ezrin might be an effective molecular marker for early diagnosis and prognostic evaluation of gastric cancer.更多还原