USP7稳定Ki-67蛋白促进NSCLC细胞增殖的机制与意义

发布时间：2018-05-14 01:42

本文选题：USP7 + Ki-67　；参考：《上海交通大学》2015年硕士论文

【摘要】：研究背景和目的USP7作为一个去泛素酶在肿瘤的发生发展进程中发挥着一定的作用;Ki-67作为一个细胞增殖的标记分子,同时也参与促进肿瘤的发展与转移。我们在非小细胞肺癌中发现USP7能够调控Ki-67促进癌症的发展。研究方法1.应用免疫组织化学方法检测正常肺组织和NSCLC组织中USP7表达;应用Western blot、Real-time PCR方法检测人正常肺上皮细胞Beas2B和NSCLC细胞中USP7蛋白和m RNA;通过在NSCLC细胞中用USP7活性抑制剂P22077及转染USP7 si RNA质粒干预后,运用CCK8和AO-EB双荧光染色法检测细胞的增殖和存活状态。2.在NSCLC细胞中用P22077及转染USP7 si RNA质粒干预后,通过Western blot检测MCM2、PCNA蛋白表达,通过细胞免疫荧光(CIF)检测Ki-67蛋白表达;运用CIF检测人正常肺上皮细胞Beas2B和NSCLC细胞中Ki-67蛋白表达;运用免疫组织化学方法检测正常肺组织和NSCLC组织中Ki-67表达;通过裸鼠皮下移植瘤实验检测转染USP7 si RNA质粒的A549细胞增殖成瘤体积。3.通过Real-time PCR检测人正常肺上皮细胞Beas2B和NSCLC细胞中Ki-67m RNA表达和在NSCLC细胞中用P22077及转染USP7 si RNA质粒干预后,Ki-67m RNA的表达情况;应用CIF,在NSCLC细胞中检测USP7与Ki-67的定位情况;应用MG132及转染USP7 C223A突变质粒后,进一步研究USP7对Ki-67的调控机制;通过CCK8实验,检测转染USP7 si RNA质粒后细胞对药物敏感性影响。研究结果1.在NSCLC组织中USP7在核内高表达,并且分化程度越低的组织表达越高;与Beas2B细胞相比NSCLC细胞中USP7的蛋白和m RNA均高表达;应用P22077及转染USP7 si RNA质粒检测NSCLC细胞的增殖效率降低,细胞保持存活状态。2.在NSCLC细胞中应用P22077和转染USP7 si RNA质粒后MCM2、PCNA的蛋白表达不变,Ki-67蛋白表达减少;与Beas2B细胞相比NSCLC细胞中Ki-67的蛋白高表达;在NSCLC组织中Ki-67也在核内高表达,并且分化程度越低的组织表达越高;在A549细胞中转染USP7 si RNA质粒后,细胞裸鼠皮下移植瘤与对照组和NC组比瘤体体积减少显著。3.在Beas2B和NSCLC细胞Ki-67的m RNA基本一致,在NSCLC细胞中应用P22077、转染USP7 si RNA质粒后,Ki-67 m RNA保持不变;在NSCLC细胞中USP7和Ki-67有共定位现象;MG132拮抗P22077所诱导对Ki-67蛋白表达下调作用,应用MG132后在转染USP7 si RNA细胞中Ki-67蛋白表达恢复;过表达USP7 C223A后,Ki-67蛋白表达减少;转染USP7 si RNA质粒的细胞对药物敏感性无变化,甚至还有所提高。研究结论1.USP7在NSCLC组织和细胞中过表达,并且抑制USP7活性或者下调USP7后均可以减慢细胞增殖,但细胞仍存活。2.USP7可以调控Ki-67的蛋白表达,并且Ki-67在NSCLC组织和细胞中过表达,下调USP7后,裸鼠皮下移植瘤与对照组和NC组比瘤体体积减少显著,同时Ki-67表达也下调。3.在NSCLC细胞中,USP7通过去泛素作用调节Ki-67的蛋白稳定性。
[Abstract]:Background and objective USP7, as a diubiquitin enzyme, plays a role in tumorigenesis and progression. Ki-67 is a marker of cell proliferation, and it is also involved in promoting tumor development and metastasis. We have found that USP7 regulates the development of non-small cell lung cancer by Ki-67. Method 1. Immunohistochemical method was used to detect the expression of USP7 in normal lung tissues and NSCLC tissues. USP7 protein and mRNAs in Beas2B and NSCLC cells of normal human lung epithelial cells were detected by Western blotreal-time PCR method, and then treated with USP7 activity inhibitor P22077 and USP7 si RNA plasmid transfection in NSCLC cells. CCK8 and AO-EB double fluorescence staining were used to detect the proliferation and survival state of the cells. After the intervention of P22077 and transfected USP7 si RNA plasmid in NSCLC cells, the expression of MCM2PCNA protein was detected by Western blot, the expression of Ki-67 protein was detected by cellular immunofluorescence assay, and the expression of Ki-67 protein was detected by CIF in Beas2B and NSCLC cells of normal human lung epithelial cells. Immunohistochemical method was used to detect the expression of Ki-67 in normal lung tissues and NSCLC tissues, and the tumor volume of A549 cells transfected with USP7 si RNA plasmid was detected by subcutaneous tumor transplantation in nude mice. Real-time PCR was used to detect the expression of Ki-67m RNA in Beas2B and NSCLC cells, and the expression of Ki-67m RNA in NSCLC cells treated with P22077 and USP7 si RNA plasmid, and the localization of USP7 and Ki-67 in NSCLC cells. The regulation mechanism of USP7 on Ki-67 was further studied by MG132 and USP7 C223A mutant plasmid, and the effect of USP7 si RNA plasmid transfection on drug sensitivity was detected by CCK8 experiment. Results 1. The higher the expression of USP7 in the nucleus and the lower the degree of differentiation, the higher the expression of USP7 protein and m RNA in NSCLC cells compared with Beas2B cells. The proliferation efficiency of NSCLC cells was detected by P22077 and USP7 si RNA plasmid transfection. Cells remain alive. In NSCLC cells, the expression of Ki-67 in NSCLC cells was significantly higher than that in Beas2B cells, and the expression of Ki-67 in the nucleus was also higher in NSCLC cells than in NSCLC cells, and the expression of Ki-67 protein in NSCLC cells was decreased after pP22077 and USP7 si RNA plasmids were transfected into Beas2B cells, and the expression of Ki-67 in the nucleus of NSCLC cells was also higher than that of Beas2B cells. After transfection of USP7 si RNA plasmid in A549 cells, the tumor volume in nude mice decreased significantly compared with control group and NC group. The m RNA of Ki-67 in Beas2B and NSCLC cells was basically the same, and P22077 was used in NSCLC cells. After transfection of USP7 si RNA plasmid, Ki-67 m RNA remained unchanged, and USP7 and Ki-67 co-located in NSCLC cells. MG132 antagonized the down-regulation of Ki-67 protein expression induced by P22077. The expression of Ki-67 protein recovered after transfection of MG132 in USP7 si RNA cells and decreased after overexpression of USP7 C223A. The sensitivity of the cells transfected with USP7 si RNA plasmid was not changed or even increased. Conclusion 1.USP7 overexpression in NSCLC tissues and cells, and inhibition of USP7 activity or down-regulation of USP7 can slow down cell proliferation, but cell survival. 2. USP7 can regulate the expression of Ki-67 protein, and Ki-67 overexpression in NSCLC tissues and cells. After down-regulation of USP7, the volume of subcutaneous transplanted tumor in nude mice was significantly lower than that in control group and NC group, and the expression of Ki-67 was also down-regulated. In NSCLC cells, Ki-67 protein stability is regulated by deubiquitin.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R730.2

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