ILK与RI的相互作用通过ILK信号通路调节膀胱癌EJ细胞EMT的分子机制研究

发布时间：2018-06-19 02:48

本文选题：整合素连接激酶 + 核糖核酸酶抑制因子　；参考：《重庆医科大学》2016年硕士论文

【摘要】：目的研究整合素连接激酶(ILK)与核糖核酸酶抑制因子(RI)蛋白的相互作用,探讨两者相互作用对膀胱癌EJ细胞的ILK信号通路及EMT影响的分子机制。方法1.根据ILK基因序列设计引物,经PCR法扩增获得ILK基因片段后,分别连接到真核表达载体pEYFP-N1和pCMV-3xflag-CMVTM-10上。通过酶切和DNA测序鉴定载体,并分别命名为p EYFP-N1-ILK and pCMV-3×Flag-ILK。将pCMV-3×Flag-ILK载体和对照空白载体,以及LV5-RNH1和LV5NC分别稳定转染EJ细胞株,命名为EJ-ILK,EJ-FLAG,EJ-RI和EJ-LV5,经Western blot和细胞免疫荧光检测蛋白过表达情况。2.在EJ和HEK293细胞内进行ILK与RI的免疫共沉淀实验,荧光共振能量转移实验和免疫荧光共定位实验,用以研究体内的相互作用。在细胞外应用GST pulldown实验研究ILK与RI在体外的相互作用。3.CCK8检测各组细胞增殖能力;流式细胞术检测细胞周期;细胞免疫荧光检测蛋白ri,ilk,p-akt和p-gsk3β表达水平;小管形成实验检测ilk和ri相互作用对体外血管生成的影响。3.收集的稳定表达细胞株ej-ilk,ej-flag,ej-ri,ej-lv5和ej,用westernblot检测ilk与ri相互作用对ej细胞emt及ilk信号通路的影响。5.构建balb/c裸鼠移植瘤模型,观察移植瘤生长,肿瘤微血管生长以及肺转移情况;应用免疫组化和组织免疫荧光检测肿瘤组织中emt相关蛋白及ilk信号通路关键蛋白的表达水平。另外,在临床人膀胱癌及癌旁组织标本中同时检测了ilk及ri蛋白的表达。结果1.测序结果显示:重组真核表达质粒peyfp-n1-ilk和pcmv-3×flag-ilk构建正确。细胞免疫荧光和westernblot结果显示ilk和ri在ej细胞中成功过表达。2.实验结果显示:ilk与ri在体内和体外均存在相互作用。4.cck8结果显示ej-ilk细胞增值能力较对照组明显增强,而ej-ri细胞则明显减弱;流式细胞分析结果表明ej-ri细胞与对照组相比,明显阻滞于s期;小管形成实验提示ri和ej相互作用能够抑制体外血管的形成。3.通过检测各组稳定表达细胞株,显示emt相关蛋白和ilk信号通路的一些关键靶蛋白呈现显著的变化。5.balb/c裸鼠注射各组细胞后,与2个对照组相比,ej-ilk组瘤重明显增加;而EJ-RI组与对照组比较,瘤重明显降低。组织免疫荧光和HE染色结果提示EJ-ILK组微血管明显增加,EJ-RI组则明显减少。肺组织HE染色实验表明,EJ-ILK组有明显肺转移,而EJ-RI组未见明显转移。免疫组织化学和免疫荧光检测结果显示:EJ-ILK组中EMT及ILK信号通路蛋白变化明显;EJ-RI组中EMT相关蛋白和ILK通路关键蛋白与对照组比较有显著变化,且与EJ-ILK组成反相关。结论1.ILK和RI在体内外均存在相互作用。2.过表达ILK促进细胞增殖,EMT以及转移。3.上调RI抑制ILK信号通路以及EMT。4.上调RI抑制血管生成,肿瘤形成以及转移。
[Abstract]:Objective to study the interaction between integrin linked kinase (ILK) and ribonuclease inhibitor (Ri) protein, and to explore the effect of integrin linked kinase (ILK) and ribonuclease inhibitor (Ri) on the ilk signaling pathway and the molecular mechanism of EMT in EJ cells of bladder cancer. Method 1. The ilk gene fragment was amplified by PCR and cloned into eukaryotic expression vectors pEYFP-N1 and pCMV-3xflag-CMVTM-10, respectively. The vector was identified by restriction endonuclease digestion and DNA sequencing and named as pEYFP-N1-ilk and pCMV-3 脳 Flag-ILK. The pCMV-3 脳 Flag-ilk vector and the control vector, LV5-RNH1 and LV5NC were stably transfected into EJ cell line, named EJ-ILKPER-FLAGN EJ-RI and EJ-LV5, respectively. The overexpression of protein was detected by Western blot and cellular immunofluorescence. The immunoprecipitation of ilk and RI, fluorescence resonance energy transfer and immunofluorescence co-localization were carried out in EJ and HEK293 cells to study the interaction in vivo. The interaction of ilk and RI in vitro was studied by pulldown assay in vitro. 3. The proliferative ability of each group was detected by CCK8, the cell cycle was detected by flow cytometry, and the expression of rifikophane p-akt and p-gsk3 尾 were detected by cellular immunofluorescence. The effect of ilk and ri interaction on angiogenesis in vitro was detected by tubulogenesis assay. The stable expression cell lines ej-ilkine ej-flagej-rij-lv5 and ejj-lv5 were collected. The effects of ilk and ri interaction on emt and ilk signaling pathway were detected by westernblot. The balb/c xenograft tumor model was established to observe the tumor growth, tumor microvessel growth and lung metastasis. The expression of emt related proteins and key proteins of ilk signaling pathway in tumor tissues were detected by immunohistochemistry and tissue immunofluorescence. In addition, the expression of ilk and ri protein were detected in clinical specimens of bladder cancer and adjacent tissues. Result 1. Sequencing results showed that the recombinant eukaryotic expression plasmids peyfp-n1-ilk and pcmv-3 脳 flag-ilk were constructed correctly. The results of cellular immunofluorescence and westernblot showed that ilk and ri were overexpressed in EJ cells. The results showed that there was interaction between in vivo and in vitro. 4. Cck8 results showed that the proliferation of ej-ilk cells was significantly higher than that of the control group, while that of ej-ri cells was significantly decreased, and the flow cytometry analysis showed that ej-ri cells were significantly higher than those of the control group. The results showed that the interaction of ri and EJ could inhibit the formation of blood vessels in vitro. By detecting the stable expression cell lines of each group, it was found that emt related protein and some key target proteins of ilk signal pathway showed significant changes. 5. After injected with each group of cells, the tumor weight of the control group was significantly increased compared with that of the two control groups. The tumor weight in EJB-RI group was significantly lower than that in control group. The results of tissue immunofluorescence and HE staining showed that the increase of microvessels in EJB-ilk group was significantly decreased in EJ- RI group. Lung tissue HE staining showed that there were significant lung metastasis in EJ-ilk group, but no obvious metastasis in EJ-RI group. The changes of EMT and ilk signaling pathway proteins in the EJ-ILK group were significantly different from those in the control group, and were inversely correlated with the composition of EJ-ilk in the EJ-RI group. Ilk and RI have interaction in vivo and in vitro. 2. Overexpression of ilk promotes proliferation and metastasis of EMT. Upregulation of RI inhibits ilk signaling pathway and EMT.4. Upregulation of RI inhibits angiogenesis, tumor formation and metastasis.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.14

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