乳腺癌中p100蛋白高表达对其生物学行为的影响及其机制探讨
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摘要
目的：
p100蛋白是一种多功能蛋白,它既作为共激活因子介导STAT6和基本转录调控元件之间的作用,又参与pre-mRNA剪接加工过程。此外,p100蛋白作为RISC的组份之一可能参与调控microRNA的代谢。最近研究表明p100在结肠癌和前列腺癌中表达异常,这些表象提示我们p100蛋白与肿瘤的发生发展之间可能存在某种联系。本课题的主要目的是在已有的p100蛋白研究基础上,发现p100蛋白异常表达对乳腺肿瘤形成等生物学行为的影响并探讨其可能的分子机制。
方法：
第一部分采用免疫组化方法检测人乳腺癌组织和正常乳腺组织中p100蛋白的表达情况。
第二部分我们将p100I质粒转染乳腺癌MDA-MB-231细胞并建立了稳定低表达p100蛋白的细胞株,利用此细胞株进行体外Transwell小室侵袭实验和划痕实验,观察p100低表达对乳腺癌细胞侵袭性和迁移能力的影响,从而了解p100蛋白低表达对乳腺癌细胞生物学行为的影响。
第三部分我们进行了裸鼠乳腺肿瘤移植实验,将对照组和p100低表达的乳腺癌MDA-MB-231细胞株接种至裸鼠第二对乳腺脂肪垫,观察并测定肿瘤体积变化,分析在体内p100低表达对乳腺肿瘤生长速率和侵袭性有何作用。
第四部分采用ChIP-GLAS启动子芯片技术筛选出启动子能与p100蛋白高亲和力结合的靶基因一-TGF-β信号通路成员,RT-PCR进行验证。利用mRNA表达谱基因芯片技术筛选出与p100低表达密切相关的基因,同时利用分析软件将筛选出的差异表达基因按照其所在的信号通路归类。选取其中差异表达较大的TGF-β通路成员进行Real-time PCR和Western blot,检测裸鼠乳腺原位肿瘤组织,瞬时转染siRNA-p100细胞和p100I稳定株乳腺癌MDA-MB-231细胞中几种Smad蛋白的mRNA水平和蛋白表达水平。
结果：
第一部分：我们观察到p100蛋白在人乳腺癌组织中p100的表达显著高于正常乳腺组织。
第二部分：我们成功地建立了稳定表达p100I的乳腺癌MDA-MB-231稳定株细胞。体外Transwell小室实验结果显示,p100蛋白提高乳腺癌MDA-MB-231细胞的侵袭能力,p100蛋白低表达降低231稳定株细胞的侵袭性。用231稳定株细胞进行的划痕实验得到了与侵袭实验相似的结果：p100蛋白过表达提高231稳定株细胞的迁移能力。
第三部分：我们进行的裸鼠体内实验表明,p100低表达对乳腺原位肿瘤生长具有明显的抑制作用,p100蛋白过表达促进肿瘤细胞的体内增殖。此外,p100蛋白低表达组裸鼠肿瘤向周围组织侵袭的能力低于对照组。
第四部分：在前期完成的ChIP-GLAS启动子芯片结果中,我们发现TGF-β信号通路相关的十几个转录因子或调节因子的启动子与p100蛋白具有较高的亲和力。我们选取其中七个在HeLa细胞和乳腺癌MDA-MB-231细胞中进行mRNA水平的验证实验。结果与芯片结果基本一致：当p100蛋白过表达时,HeLa和MDA-MB-231细胞中Smad1, Smad2, TGIF1, Smad4, TGFβ1I1和Smurf2的mRNA水平显著高于对照组。分析mRNA表达谱基因芯片结果时我们发现,TGF-β信号通路中的某些成员的表达水平随p100蛋白低表达而发生明显的改变。Western blot检测裸鼠乳腺原位肿瘤组织中TGF-β通路蛋白的表达水平时,我们发现p100低表达肿瘤组织中p-Smadl, Smad2/3和p-Smad2蛋白的表达均低于HK组和对照组。瞬转siRNA-p100组细胞中Smad6和Smad9的mRNA相对表达量明显低于对照组,而Smad3和Smurf2的mRNA水平高于对照组。稳定表达p100I组细胞只有Smad6的相对表达量显著低于对照组,其余被检测蛋白均无明显变化。
结论：
1.p100蛋白在乳腺癌组织中高表达。
2.p100蛋白提高乳腺癌MDA-MB-231细胞在体外的侵袭和迁移能力。p100蛋白促进裸鼠体内乳腺原位肿瘤的生长、侵袭和增殖能力。活体内p100蛋白低表达影响TGF-β两个信号转导通路的活性。
3.细胞内p100蛋白异常表达可能通过调节TGF-β信号通路中的一条或两条途径的活性进而影响细胞的生物学行为。
Objectives:
p100is a multifunctional protein. It has been identified as a bridging factor between STAT6and the basal transcription machinery and also participates in pre-mRNA splicing. It is reported recently p100is one of components of RNAi-induced silencing complex (RISC) and its overexpression alter the expression level of some miRNA in cells. Meanwhile, alterations in p100protein expression are found in prostate cancer and colon cancers. Therefore we suppose that p100protein may have function in tumorigenesis and progression. The main purpose of this study is to clarify the effect of p100protein on breast cancer biological behaviours and find out its potential molecular mechanism.
Methods:
Part Ⅰ:To investigate p100expression in different human breast cancer specimens by. immunohistochemical appoach.
Part Ⅱ:Plasmid p100I were transfected into breast cancer cell line MDA-MB-231and then to construct a stable transfectant cell line which stably suppress the expression of p100protein. Cell invasion and mobility assays were performed with stable transfectant231cells to evaluate the effect of low expression of p100on its biological behaviours in vitro.
Part Ⅲ:in vivo assay. Parental and stable transfectant231cells were injected into the second mammary fat pads of athymic nude mice. Observing and noting the volume of tumor till the mean diameter of tumor reached to1.0-1.5cm. Evaluate the effect of low expressed p100on tumor growth velocity and invasion to neighbour tissues.
Part Ⅳ:Target genes (TGFβ signal pathway members) whose promoter had high affinity binding with p100proteins were selected from ChIP-GLAS results, and verified by RT-PCR and real-time PCR in HeLa and MDA-MB-231cells, respectively. P100low expression related genes were also selected and classified into the signal pathway they involved in. Several components of TGFβ signal pathway were subject to real-time PCR and Western blot in tumor tissues, siRNA-p100 transient transfection231cells and stable transfectant MDA-MB-231cell line.
Results
Part Ⅰ:The expression of p100protein is significantly higher in human breast cancer tissues than that of normal breast tissues.
Part Ⅱ:We have successfully constructed a stable transfectant MDA-MB-231cell line whose p100expression were suppressed persistently.
Part Ⅲ:In vivo assay shows that low expression of p100decrease the growth velocity of breast tumor in nude mice. The invasiveness of tumor in p100I group is lower than that of parental group.
Part Ⅳ:The RT-PCR experiments in HeLa and MDA-MB-231cells verified that mRNA level of Smadl, Smad2, TGIF1, Smad4, TGFB1I1and Smurf2increased significantly when p100protein was overexpressed. After analysing the results of gene expression profiles in stable transfectant231cell line we found altered expression of some members of TGFβ signal pathway in p100I group compared with parental group. The protein expression of p-Smadl, Smad2/3and p-Smad2decreased with p100protein low expressed in tumor tissues. There were no significant mRNA differences in vitro between the parental231cells and transient transfectant siRNA-p100except Smad6and Smad9.
Conclusion:
Our study show that p100protein are high expressed in breast cancer tissues, p100protein enhanced the invasion and mobility of breast cancer cell line MDA-MB-231in vitro and in vivo. Abnormal expression of p100may affect the breast cancer cell biological behaviour by regulating the TGFp signal pathway activity.
p100蛋白是一种多功能蛋白,它既作为共激活因子介导STAT6和基本转录调控元件之间的作用,又参与pre-mRNA剪接加工过程。此外,p100蛋白作为RISC的组份之一可能参与调控microRNA的代谢。最近研究表明p100在结肠癌和前列腺癌中表达异常,这些表象提示我们p100蛋白与肿瘤的发生发展之间可能存在某种联系。本课题的主要目的是在已有的p100蛋白研究基础上,发现p100蛋白异常表达对乳腺肿瘤形成等生物学行为的影响并探讨其可能的分子机制。
方法：
第一部分采用免疫组化方法检测人乳腺癌组织和正常乳腺组织中p100蛋白的表达情况。
第二部分我们将p100I质粒转染乳腺癌MDA-MB-231细胞并建立了稳定低表达p100蛋白的细胞株,利用此细胞株进行体外Transwell小室侵袭实验和划痕实验,观察p100低表达对乳腺癌细胞侵袭性和迁移能力的影响,从而了解p100蛋白低表达对乳腺癌细胞生物学行为的影响。
第三部分我们进行了裸鼠乳腺肿瘤移植实验,将对照组和p100低表达的乳腺癌MDA-MB-231细胞株接种至裸鼠第二对乳腺脂肪垫,观察并测定肿瘤体积变化,分析在体内p100低表达对乳腺肿瘤生长速率和侵袭性有何作用。
第四部分采用ChIP-GLAS启动子芯片技术筛选出启动子能与p100蛋白高亲和力结合的靶基因一-TGF-β信号通路成员,RT-PCR进行验证。利用mRNA表达谱基因芯片技术筛选出与p100低表达密切相关的基因,同时利用分析软件将筛选出的差异表达基因按照其所在的信号通路归类。选取其中差异表达较大的TGF-β通路成员进行Real-time PCR和Western blot,检测裸鼠乳腺原位肿瘤组织,瞬时转染siRNA-p100细胞和p100I稳定株乳腺癌MDA-MB-231细胞中几种Smad蛋白的mRNA水平和蛋白表达水平。
结果：
第一部分：我们观察到p100蛋白在人乳腺癌组织中p100的表达显著高于正常乳腺组织。
第二部分：我们成功地建立了稳定表达p100I的乳腺癌MDA-MB-231稳定株细胞。体外Transwell小室实验结果显示,p100蛋白提高乳腺癌MDA-MB-231细胞的侵袭能力,p100蛋白低表达降低231稳定株细胞的侵袭性。用231稳定株细胞进行的划痕实验得到了与侵袭实验相似的结果：p100蛋白过表达提高231稳定株细胞的迁移能力。
第三部分：我们进行的裸鼠体内实验表明,p100低表达对乳腺原位肿瘤生长具有明显的抑制作用,p100蛋白过表达促进肿瘤细胞的体内增殖。此外,p100蛋白低表达组裸鼠肿瘤向周围组织侵袭的能力低于对照组。
第四部分：在前期完成的ChIP-GLAS启动子芯片结果中,我们发现TGF-β信号通路相关的十几个转录因子或调节因子的启动子与p100蛋白具有较高的亲和力。我们选取其中七个在HeLa细胞和乳腺癌MDA-MB-231细胞中进行mRNA水平的验证实验。结果与芯片结果基本一致：当p100蛋白过表达时,HeLa和MDA-MB-231细胞中Smad1, Smad2, TGIF1, Smad4, TGFβ1I1和Smurf2的mRNA水平显著高于对照组。分析mRNA表达谱基因芯片结果时我们发现,TGF-β信号通路中的某些成员的表达水平随p100蛋白低表达而发生明显的改变。Western blot检测裸鼠乳腺原位肿瘤组织中TGF-β通路蛋白的表达水平时,我们发现p100低表达肿瘤组织中p-Smadl, Smad2/3和p-Smad2蛋白的表达均低于HK组和对照组。瞬转siRNA-p100组细胞中Smad6和Smad9的mRNA相对表达量明显低于对照组,而Smad3和Smurf2的mRNA水平高于对照组。稳定表达p100I组细胞只有Smad6的相对表达量显著低于对照组,其余被检测蛋白均无明显变化。
结论：
1.p100蛋白在乳腺癌组织中高表达。
2.p100蛋白提高乳腺癌MDA-MB-231细胞在体外的侵袭和迁移能力。p100蛋白促进裸鼠体内乳腺原位肿瘤的生长、侵袭和增殖能力。活体内p100蛋白低表达影响TGF-β两个信号转导通路的活性。
3.细胞内p100蛋白异常表达可能通过调节TGF-β信号通路中的一条或两条途径的活性进而影响细胞的生物学行为。
Objectives:
p100is a multifunctional protein. It has been identified as a bridging factor between STAT6and the basal transcription machinery and also participates in pre-mRNA splicing. It is reported recently p100is one of components of RNAi-induced silencing complex (RISC) and its overexpression alter the expression level of some miRNA in cells. Meanwhile, alterations in p100protein expression are found in prostate cancer and colon cancers. Therefore we suppose that p100protein may have function in tumorigenesis and progression. The main purpose of this study is to clarify the effect of p100protein on breast cancer biological behaviours and find out its potential molecular mechanism.
Methods:
Part Ⅰ:To investigate p100expression in different human breast cancer specimens by. immunohistochemical appoach.
Part Ⅱ:Plasmid p100I were transfected into breast cancer cell line MDA-MB-231and then to construct a stable transfectant cell line which stably suppress the expression of p100protein. Cell invasion and mobility assays were performed with stable transfectant231cells to evaluate the effect of low expression of p100on its biological behaviours in vitro.
Part Ⅲ:in vivo assay. Parental and stable transfectant231cells were injected into the second mammary fat pads of athymic nude mice. Observing and noting the volume of tumor till the mean diameter of tumor reached to1.0-1.5cm. Evaluate the effect of low expressed p100on tumor growth velocity and invasion to neighbour tissues.
Part Ⅳ:Target genes (TGFβ signal pathway members) whose promoter had high affinity binding with p100proteins were selected from ChIP-GLAS results, and verified by RT-PCR and real-time PCR in HeLa and MDA-MB-231cells, respectively. P100low expression related genes were also selected and classified into the signal pathway they involved in. Several components of TGFβ signal pathway were subject to real-time PCR and Western blot in tumor tissues, siRNA-p100 transient transfection231cells and stable transfectant MDA-MB-231cell line.
Results
Part Ⅰ:The expression of p100protein is significantly higher in human breast cancer tissues than that of normal breast tissues.
Part Ⅱ:We have successfully constructed a stable transfectant MDA-MB-231cell line whose p100expression were suppressed persistently.
Part Ⅲ:In vivo assay shows that low expression of p100decrease the growth velocity of breast tumor in nude mice. The invasiveness of tumor in p100I group is lower than that of parental group.
Part Ⅳ:The RT-PCR experiments in HeLa and MDA-MB-231cells verified that mRNA level of Smadl, Smad2, TGIF1, Smad4, TGFB1I1and Smurf2increased significantly when p100protein was overexpressed. After analysing the results of gene expression profiles in stable transfectant231cell line we found altered expression of some members of TGFβ signal pathway in p100I group compared with parental group. The protein expression of p-Smadl, Smad2/3and p-Smad2decreased with p100protein low expressed in tumor tissues. There were no significant mRNA differences in vitro between the parental231cells and transient transfectant siRNA-p100except Smad6and Smad9.
Conclusion:
Our study show that p100protein are high expressed in breast cancer tissues, p100protein enhanced the invasion and mobility of breast cancer cell line MDA-MB-231in vitro and in vivo. Abnormal expression of p100may affect the breast cancer cell biological behaviour by regulating the TGFp signal pathway activity.
引文
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