FOXC2及MUC4促进多形性胶质母细胞瘤增殖、迁移与侵袭作用及其分子机制研究

发布时间：2018-09-09 18:38

【摘要】：第一部分FOXC2促进多形性脑胶质母细胞瘤增殖、迁移与侵袭作用及其机制研究目的：揭示FOXC2(forkhead box C2,叉头框C2)在GBM (Glioblastoma,多形性胶质母细胞瘤)组织及GBM细胞系中的表达情况并利用GBM细胞系构建FOXC2高表达细胞模型与FOXC2基因沉默细胞模型。检测FOXC2对于GBM细胞增殖，迁移及侵袭能力的影响并研究该影响在GBM细胞内的调节机制。方法：利用RT-PCR、Western blot以及免疫组织化学实验测定GBM人脑组织样本、对照人脑组织样本以及六种GBM细胞系中FOXC2的表达情况。根据所得六种GBM细胞系中FOXC2表达结果，进一步通过质粒转染与RNA基因沉默实验构建FOXC2高表达的SNB19-FOXC2细胞模型、FOXC2基因沉默的T98G-shFOXC2细胞模型及EGFR(epidermal growth factor receptor,表皮生长因子)沉默的SNB19-FOXC2-siEGFR细胞模型。应用MTT和细胞克隆形成实验测定上述所构建细胞系中FOXC2表达对SNB19和T98G细胞增殖能力的影响并通过细胞伤口愈合实验和Boyden小室模型测定SNB19-FOXC2细胞与T98G-shFOXC2的迁移及侵袭能力变化。通过RT-PCR、Western blot和免疫组化实验测定FOXC2对EGFR在GBM细胞系中表达量的影响。同时在SNB19-FOXC2细胞中构建EGFR基因沉默模型SNB19-FOXC2-siEGFR1,2。通过MTT实验，Boyden小室模型实验，细胞伤口愈合实验，研究EGFR对于FOXC2促GBM细胞增殖，迁移以及侵袭能力的调控作用。结果：分别将10份GBM病人大脑样本与10份对照病人大脑样本的RT-PCR和Western blot实验结果比较得出所有GBM病人大脑样本中FOXC2的表达量都明显高于对照病人大脑样本。免疫组化实验结果中也发现GBM病人大脑样本中的FOXC2阳性细胞数也同样高于对照病人脑组织样本。在U87, T98G, SNB19,SW1088, SF767和SW1783六种GBM细胞系中，T98G中FOXC2表达量最高而SNB19中FOXC2的表达量最低。根据此结果，进一步将T98G细胞用于FOXC2基因沉默实验构建T98G-shFOXC2细胞模型，而将SNB19细胞用于FOXC2基因转染实验，构建FOXC2基因高表达SNB19-FOXC2模型。MTT和细胞克隆形成实验结果显示，SNB19-FOXC2细胞相对与对照组SNB19细胞增殖能力大幅提高，克隆形成总数明显增多，克隆形成范围更广。而T98G-shFOXC2细胞相对与对照组T98G细胞克隆形成数目明显减少，克隆形成范围也明显缩小。细胞伤口愈合实验和Boyden小室实验结果显示，SNB19-FOXC2细胞能够显著加快细胞伤口愈合速度并且具有2倍于SNB19细胞穿透Transwell膜的能力。另一方面，FOXC2基因沉默后，T98G-shFOXC2细胞迁移与侵袭的能力显著降低并且T98G-shFOXC2细胞对于细胞伤口愈合的促进作用以及透过Transwell膜的能力都明显小于T98G细胞。RT-PCR及Western blot实验结果显示，FOXC2基因沉默能够明显降低T98G细胞中EGFR在RNA转录和蛋白水平的表达。利用RNA干扰技术，进一步沉默SNB19-FOXC2细胞中EGFR相关基因，构建SNB19-FOXC2-siEGFR细胞模型。MTT实验，，Boyden小室模型实验及细胞伤口愈合实验结果显示，SNB19-FOXC2-siEGFR细胞的增殖、迁移与侵袭的能力都较SNB19-FOXC2细胞有大幅的下滑。该结果说明EGFR基因在FOXC2促进GBM细胞的增殖、迁移与侵袭能力的发挥过程中起着重要的调控作用。结论：（1）FOXC2在GBM病人组织样本及GBM细胞系中为显著高表达。（2）FOXC2在GBM细胞增殖、迁移与侵袭过程中起到至关重要的作用。（3）FOXC2调控EGFR在GBM细胞中的表达（4）EGFR是FOXC2调控GBM细胞增殖、迁移与侵袭的重要调控因子。第二部分MUC4粘蛋白上调表皮生长因子受体表达及其对多型性胶质母细胞瘤增殖、迁移与侵袭促进作用研究目的：探究MUC4(mucin4,4型粘蛋白)在GBM病人组织与GBM细胞系中表达情况。构建MUC4高表达与MUC4基因沉默细胞模型。揭示MUC4对于GBM细胞增殖、迁移及侵袭活性的影响及其分子机制。方法：通过RT-PCR、免疫组化以及Western blot实验测定对照病人脑组织样品、GBM病人脑组织样品与六种GBM细胞系中MUC4表达量高低。根据六种GBM细胞中MUC4的表达情况进而通过基因转染与RNA干扰实验构建MUC4过表达SNB19-MUC4细胞、MUC4沉默T98G-shMUC4细胞模型及EGFR (epidermal growthfactor receptor,表皮生长因子)沉默SNB19-MUC4-siEGFR细胞模型。利用MTT和细胞克隆形成实验测定MUC4对GBM细胞增殖能力影响。通过伤口愈合实验和Boyden小室模型测定SNB19-MUC4细胞以及T98G-shMUC4细胞的迁移及侵袭能力。通过RT-PCR、Western blot和免疫组化实验测定MUC4对EGFR表达的影响。同时构建EGFR基因沉默SNB19-MUC4-siEGFR1,2,3细胞模型。通过MTT实验，Boyden小室模型实验，细胞愈合实验，研究EGFR沉默后MUC4促GBM细胞增殖，迁移以及侵袭能力的变化情况。结果： RT-PCR实验测定11份对照病人大脑样本与11份GBM病人大脑样本中MUC4的表达发现，11份GBM病人大脑样本中MUC4的表达明显高于对照大脑样本并且在免疫组织化学实验结果中GBM病人大脑样本中的MUC4阳性细胞数也同样明显多于对照样本。利用RT-PCR及Western blot实验测定了六种GBM细胞系中MUC4的表达量，结果显示其中T98G细胞系中MUC4表达量最高而SNB19细胞系中MUC4的表达量最低，进而将T98G细胞用于构建MUC4基因沉默T98G-shMUC4细胞模型而将SNB19细胞用于MUC4基因转染实验构建MUC4基因过表达SNB19-MUC4细胞模型。在细胞增殖实验中，MTT和细胞克隆形成实验结果显示SNB19-MUC4细胞相对于对照组SNB19细胞增殖能力大幅提高，克隆数量更多且克隆范围更广。另一方面，T98G-shMUC细胞相对与照组T98G细胞克隆数量明显下降，范围上也有所缩小。细胞伤口愈合实验证明SNB19-MUC4细胞能够显著加快细胞伤口愈合速度。Boyden小室模型结果显示SNB19-MUC4细胞具有3倍与对照组细胞透过Transwell膜的能力。然而，细胞伤口愈合实验与Boyden小室模型结果说明经MUC4基因沉默后T98G-shMUC4细胞对于伤口愈合的促进作用以及透过Transwell膜的能力都明显小于未沉默组细胞。RT-PCR及Western blot实验结果显示，MUC4基因沉默能够明显降低T98G细胞中EGFR在RNA和蛋白水平的表达。利用RNA干扰技术，进一步将SNB19-MUC4细胞中EGFR相关基因EGFR-1,2,3沉默。MTT实验，Boyden小室模型实验，细胞愈合实验结果显示，SNB19-MUC4细胞的增殖、迁移与侵袭的能力都较沉默前有大幅的下降。说明EGFR基因在MUC4促进GBM细胞的增殖、迁移与侵袭能力的过程中发挥重要的调控作用。结论：（1）MUC4在GBM细胞系中显著表达并且在GBM细胞的增殖、迁移与侵袭过程中发挥重要作用。（2）MUC4能够调控EGFR在GBM细胞中的表达，EGFR对于MUC4促进GBM细胞的增殖、迁移与侵袭能力有重要的影响。
[Abstract]:Part one: FOXC2 promotes the proliferation, migration and invasion of glioblastoma multiforme and its mechanism.
AIM: To investigate the expression of FOXC2 (forkhead box C2) in GBM (Glioblastoma multiforme glioblastoma) tissues and GBM cell lines, and to construct FOXC2 high expression cell model and FOXC2 gene silencing cell model using GBM cell lines. The regulation mechanism in GBM cells.
METHODS: The expression of FOXC2 in GBM human brain tissue samples was detected by RT-PCR, Western blot and immunohistochemical assay, and compared with human brain tissue samples and six GBM cell lines. 2 cell model, T98G-shFOXC2 cell model with FOXC2 gene silencing and SNB19-FOXC2-siEGFR cell model with EGFR (epidermal growth factor receptor) silencing. MTT and clone formation assay were used to determine the effect of FOXC2 expression on the proliferation of SNB19 and T98G cells and cell injury. The migration and invasiveness of SNB19-FOXC2 cells and T98G-shFOXC2 cells were measured by oral healing test and Boyden cell model. The effect of FOXC2 on EGFR expression in GBM cell lines was determined by RT-PCR, Western blot and immunohistochemistry. The EGFR gene silencing model SNB19-FOXC2-siEGFR1,2 was constructed in SNB19-FOXC2 cells by MTT. The effects of EGFR on FOXC2-induced proliferation, migration and invasion of GBM cells were studied by Boyden cell model and wound healing test.
Results: The expression of FOXC2 in brain samples of 10 GBM patients was significantly higher than that of 10 control patients by RT-PCR and Western blot. The number of FOXC2 positive cells in brain samples of GBM patients was also found by immunohistochemistry. In U87, T98G, SNB19, SW1088, SF767 and SW1783 GBM cell lines, the expression of FOXC2 was the highest in T98G and the lowest in SNB19. According to this result, T98G cells were further used in FOXC2 gene silencing experiment to construct T98G-shFOXC2 cell model, while SNB19 cells were used in FOXC2 gene silencing experiment. The results of MTT and cell cloning showed that the proliferation ability of SNB19-FOXC2 cells was greatly improved, the total number of cloning formation was significantly increased, and the range of cloning formation was wider. Cell wound healing test and Boyden chamber test showed that SNB19-FOXC2 cells significantly accelerated wound healing and had the ability to penetrate Transwell membrane twice as fast as SNB19 cells. On the other hand, after FOXC2 gene was silenced, T98G-shFOXC2 cells showed significant migration and invasion ability. The results of RT-PCR and Western blot showed that FOXC2 gene silencing could significantly reduce the expression of EGFR at RNA transcription and protein levels in T98G cells. EGFR-related genes in B19-FOXC2 cells were used to construct SNB19-FOXC2-siEGFR cell model. MTT, Boyden chamber and wound healing experiments showed that the proliferation, migration and invasion of SNB19-FOXC2-siEGFR cells were significantly lower than those of SNB19-FOXC2 cells. It plays an important regulatory role in the process of proliferation, migration and invasion.
CONCLUSION: (1) FOXC2 is highly expressed in GBM tissues and GBM cell lines. (2) FOXC2 plays an important role in the proliferation, migration and invasion of GBM cells. (3) FOXC2 regulates the expression of EGFR in GBM cells. (4) EGFR is an important regulator of GBM cell proliferation, migration and invasion.
Part 2 MUC4 mucin up-regulates the expression of epidermal growth factor receptor and promotes the proliferation, migration and invasion of glioblastoma multiforme
Objective: To investigate the expression of MUC4 (mucin 4,4 mucin) in GBM tissues and GBM cell lines, and to construct a cell model of MUC4 overexpression and MUC4 gene silencing.
METHODS: The expression of MUC4 in brain tissue samples of GBM patients and six GBM cell lines was determined by RT-PCR, immunohistochemistry and Western blot. According to the expression of MUC4 in six GBM cells, the over-expressed SNB19-MUC4 cells were constructed by gene transfection and RNA interference, and T98G-shM was silenced by MUC4. UC4 cell model and EGFR (epidermal growth factor receptor) silencing SNB19-MUC4-siEGFR cell model. MTT and cell clone formation assays were used to determine the effect of MUC4 on the proliferation of GBM cells. The effect of MUC4 on EGFR expression was determined by RT-PCR, Western blot and immunohistochemical staining. The SNB19-MUC4-siEGFR 1,2,3 cell model with EGFR gene silencing was constructed. The proliferation, migration and invasiveness of GBM cells after EGFR silencing were studied by MTT test, Boyden cell model test and cell healing test.
Results: The expression of MUC4 in 11 brain samples of GBM patients and 11 brain samples of GBM patients was detected by RT-PCR. The expression of MUC4 in 11 brain samples of GBM patients was significantly higher than that in control brain samples, and the number of MUC4 positive cells in brain samples of GBM patients was also significantly higher than that in control brain samples. The expression of MUC4 in six GBM cell lines was measured by RT-PCR and Western blot. The results showed that MUC4 was the highest in T98G cell line and the lowest in SNB19 cell line. T98G cells were used to construct mutant T98G-shMUC4 cell model and SNB19 cells were used to construct MUC4 gene transfection experiment. In cell proliferation experiments, MTT and cell clone formation assays showed that SNB19-MUC4 cells had significantly higher proliferation capacity, more clones and a wider range of clones than the control group. On the other hand, T98G-shMUC cells had significantly lower number of clones than the control group T98G cells. Cell wound healing experiments showed that SNB19-MUC4 cells significantly accelerated wound healing. Boyden's compartment model showed that SNB19-MUC4 cells had three times the ability of control and control cells to penetrate Transwell membranes. The results of RT-PCR and Western blot showed that MUC4 gene silencing could significantly reduce the expression of EGFR at RNA and protein levels in T98G cells. SNB19-MUC4 cells were further treated with RNA interference technique. EGFR-1,2,3 gene was silenced. MTT test, Boyden chamber model test and cell healing test showed that the proliferation, migration and invasion of SNB19-MUC4 cells were significantly decreased compared with those before silencing.
Conclusion: (1) MUC4 is highly expressed in GBM cells and plays an important role in the proliferation, migration and invasion of GBM cells. (2) MUC4 can regulate the expression of EGFR in GBM cells. EGFR plays an important role in the proliferation, migration and invasion of GBM cells.
【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

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