RNA结合蛋白QKI-5在乳腺癌中的初步功能研究
摘要
雌激素与雌激素受体结合,形成雌激素-雌激素受体复合物,启动含有雌激素应答元件的基因的转录,在组织再生和骨代谢中具有重要的生理意义。然而雌激素信号通路的异常亦可导致乳腺细胞表型及生物学特性的改变,从而诱发乳腺癌的生成。ERβ是1996年kuiper等从大鼠前列腺和卵巢cDNA文库中克隆得到的一种新的雌激素受体。随着ERβ的发现,人们积极开展了ERβ与乳腺癌的发生发展和预后以及其在指导乳腺癌的内分泌治疗中的价值研究,但截至目前ERβ在乳腺癌中的作用尚不明确。尽管如此,大量的研究还是提示ERβ对乳腺癌来说是一个预后良好的指标。
QKI是属于STAR(signal transduction and activation of RNA)家族的一种RNA结合蛋白,在进化过程中高度保守,在神经系统发育过程中参与髓鞘发育。qki基因不同突变体小鼠可以表现在出生前死于心血管肌肉系统发育障碍,或于出生后10天表现为快速的震颤以及到了成年强直性惊厥的发作。
除了在神经系统中对于髓鞘的形成发挥重要功能以外,QKI蛋白在血管发生、细胞凋亡、细胞黏附、细胞生长及形态形成和器官发生等方面具有举足轻重的功能。根据RNA结合蛋白QKI与RNA序列的结合序列的特异性,对整个RNA进行筛选发现,QKI可能调控多达1430个下游靶基因,而其中有24%的基因参与细胞的增殖,转移,高度提示QKI可能在肿瘤的增殖,转移中发挥作用。此外,我们对qki基因上游启动子区进行分析,发现该基因具有保守的ERE的结合位点,提示qki也可能受到雌激素的调节。本研究通过RT-PCR、Western Blot、启动子荧光报告系统等实验验证了雌激素对QKI的表达调控,通过分析生物信息学预测的两个潜在的QKI靶基因cdc42和par1在乳腺肿瘤中的表达以及改变QKI表达对该基因的影响,探讨QKI对该基因的表达调控及其在肿瘤发生发展中的作用。本研究的初步结论如下:
1.利用Adeasy系统成功构建了可表达QKI-5的腺病毒表达载体。通过该载体可以实现在转染效率低的细胞中过表达QKI-5的目的。
2.分别观察了几种ER受体表达不同的乳腺癌细胞系中,QKI都有表达,表达水平高低与受体ER亚型之间无明确相关性;在肿瘤侵袭性高的、ERβ阳性的细胞中QKI表达水平最高;
3.细胞经过无酚红和吸附炭预处理后的血清培养两天后,再加入雌激素作用不同时间,通过RT-PCR,western-blot检测,qki基因的转录水平和蛋白表达水平呈升高的趋势。相反在ERβ的拮抗剂tamoxifen作用之后,RT-PCR检测其qki基因的转录水平下降。初步证明QKI可能受ER的转录调控。通过荧光素酶报告系统实验表明qki基因启动子的活性在雌激素处理之后有所上调,在tamoxifen作用后下调。表达调控可能参与乳腺肿瘤中QKI的表达异常,而加剧肿瘤的发生发展。
4.通过人为升高和降低QKI的表达,我们发现cdc42,par1与QKI的表达呈现负相关,腺病毒载体感染乳腺癌细胞后,观察到乳腺癌细胞发生形态和功能的改变。划痕实验显示过表达QKI的细胞移动能力降低。提示QKI可能通过抑制cdc42和par1的表达实现抑制乳腺癌细胞移动的功能。
综上所述,我们的实验初步说明,在乳腺肿瘤发生发展过程中,雌激素受体信号的紊乱可能参与了qki表达的降低,进而引起cdc42和par1下游靶基因的表达增加参与肿瘤的发生发展。
Estrogen receptor(ER) consisting of ERαand ERβcan modulate the target gene expression when bind to ligand mainly through binding the estrogen response element (ERE). The complex biological effects mediated by ER involve communication between many proteins and signaling pathways. The activity of these transcription factors is regulated by a variety of factors, including ligand binding, phosphorylation, coregulators and the effector pathways. The end result of target gene transcription is to modulate physiological processes, such as reproductive organ development and function, bone density, and unfortunate contribution to the growth and development of breast cancer. ERβwas first cloned from rat prostate using degenerate PCR. The role of ERβin breast cancer growth and development is not as clear as the role of ERα. ERβmight have a modulating effect in breast cancer. Overall, the majority of studies suggest that the presence of ERβis a good prognostic marker for breast cancer.
The quaking gene (qki) encodes a highly conserved RNA binding protein (QKI), which is a member of the“STAR”(signal transduction and activation of RNA) family of RNA binding proteins. Quaking has a critical role in myelination. Some homozygous mutant quaking mice died prior to birth becanse of the defect of cardiovascular and muscle development. Others have severe central nervous system dysmyelination, causing quaking movements during normal motor activities from10 days postnatal and tonic convulsion when growing up.
In addition to its fundamental role in myelination, QKI also participated in vascular development, apoptosis, cell adhesion, cell growth, morphogenesis and organogenesis. In light of the validated target motif and RNA binding specificity of QKI targets, there are about 1430 putative QKI targets and about 24% putative mRNA targets having function in proliferation and migration. Further, the promotor region of QKI contains a conserved ERE, suggesting a role of QKI in breast cancer. In this study, we analyzed the expression of QKI with or without the treatment of estrogen or tamoxifen in multiple breast cancer cell lines both at mRNA and protein level. In addition, we validate the response of QKI promoter to estrogen through reporter assay. Furthermore, through construction of adenovirus of QKI, we compared the expression of CDC42 and Par1 both under forced QKI expression and control. The main findings of our study are as follows:
1.The recombinant adenovirus of Qki-5 has been successfully constructed and expressed in the MDA-MB-231 cells of breast cancer.
2.RT-PCR and Western-blot results showed that the mRNA and protein level of qki increased under the stimulation of 17β-estradiol. The activity of qki promoter was upregulated under the stimulation of17β-estradiol.RT-PCR results showed the transcriptional level of qki and activity of qki promoter was downregulated under the stimulation of tamoxifen. In other words, ERE in the QKI promoter region might mediate the QKI response to estrogen. We also observed that transcriptional level of cyclinD1 was downregulated under the stimulation of 17β-estradiol in MDA-MB-231(ERα-/ERβ+).
3.Upon overexpression and specifically silencing the expression of QKI, endogenous expression of cdc42 and par1 were significantly changed. Consistently, wounding assay shows that forced expression of QKI nearly abrogate the migration of breast cancer cells.
In conclusion, our results primarily set a model that during the breast cancer development, ER signal aberration leads to downregulation of QKI, which in turn enhances the expression of CDC42 and Par1, and thus facilitate the process of tumor metastasis.
QKI是属于STAR(signal transduction and activation of RNA)家族的一种RNA结合蛋白,在进化过程中高度保守,在神经系统发育过程中参与髓鞘发育。qki基因不同突变体小鼠可以表现在出生前死于心血管肌肉系统发育障碍,或于出生后10天表现为快速的震颤以及到了成年强直性惊厥的发作。
除了在神经系统中对于髓鞘的形成发挥重要功能以外,QKI蛋白在血管发生、细胞凋亡、细胞黏附、细胞生长及形态形成和器官发生等方面具有举足轻重的功能。根据RNA结合蛋白QKI与RNA序列的结合序列的特异性,对整个RNA进行筛选发现,QKI可能调控多达1430个下游靶基因,而其中有24%的基因参与细胞的增殖,转移,高度提示QKI可能在肿瘤的增殖,转移中发挥作用。此外,我们对qki基因上游启动子区进行分析,发现该基因具有保守的ERE的结合位点,提示qki也可能受到雌激素的调节。本研究通过RT-PCR、Western Blot、启动子荧光报告系统等实验验证了雌激素对QKI的表达调控,通过分析生物信息学预测的两个潜在的QKI靶基因cdc42和par1在乳腺肿瘤中的表达以及改变QKI表达对该基因的影响,探讨QKI对该基因的表达调控及其在肿瘤发生发展中的作用。本研究的初步结论如下:
1.利用Adeasy系统成功构建了可表达QKI-5的腺病毒表达载体。通过该载体可以实现在转染效率低的细胞中过表达QKI-5的目的。
2.分别观察了几种ER受体表达不同的乳腺癌细胞系中,QKI都有表达,表达水平高低与受体ER亚型之间无明确相关性;在肿瘤侵袭性高的、ERβ阳性的细胞中QKI表达水平最高;
3.细胞经过无酚红和吸附炭预处理后的血清培养两天后,再加入雌激素作用不同时间,通过RT-PCR,western-blot检测,qki基因的转录水平和蛋白表达水平呈升高的趋势。相反在ERβ的拮抗剂tamoxifen作用之后,RT-PCR检测其qki基因的转录水平下降。初步证明QKI可能受ER的转录调控。通过荧光素酶报告系统实验表明qki基因启动子的活性在雌激素处理之后有所上调,在tamoxifen作用后下调。表达调控可能参与乳腺肿瘤中QKI的表达异常,而加剧肿瘤的发生发展。
4.通过人为升高和降低QKI的表达,我们发现cdc42,par1与QKI的表达呈现负相关,腺病毒载体感染乳腺癌细胞后,观察到乳腺癌细胞发生形态和功能的改变。划痕实验显示过表达QKI的细胞移动能力降低。提示QKI可能通过抑制cdc42和par1的表达实现抑制乳腺癌细胞移动的功能。
综上所述,我们的实验初步说明,在乳腺肿瘤发生发展过程中,雌激素受体信号的紊乱可能参与了qki表达的降低,进而引起cdc42和par1下游靶基因的表达增加参与肿瘤的发生发展。
Estrogen receptor(ER) consisting of ERαand ERβcan modulate the target gene expression when bind to ligand mainly through binding the estrogen response element (ERE). The complex biological effects mediated by ER involve communication between many proteins and signaling pathways. The activity of these transcription factors is regulated by a variety of factors, including ligand binding, phosphorylation, coregulators and the effector pathways. The end result of target gene transcription is to modulate physiological processes, such as reproductive organ development and function, bone density, and unfortunate contribution to the growth and development of breast cancer. ERβwas first cloned from rat prostate using degenerate PCR. The role of ERβin breast cancer growth and development is not as clear as the role of ERα. ERβmight have a modulating effect in breast cancer. Overall, the majority of studies suggest that the presence of ERβis a good prognostic marker for breast cancer.
The quaking gene (qki) encodes a highly conserved RNA binding protein (QKI), which is a member of the“STAR”(signal transduction and activation of RNA) family of RNA binding proteins. Quaking has a critical role in myelination. Some homozygous mutant quaking mice died prior to birth becanse of the defect of cardiovascular and muscle development. Others have severe central nervous system dysmyelination, causing quaking movements during normal motor activities from10 days postnatal and tonic convulsion when growing up.
In addition to its fundamental role in myelination, QKI also participated in vascular development, apoptosis, cell adhesion, cell growth, morphogenesis and organogenesis. In light of the validated target motif and RNA binding specificity of QKI targets, there are about 1430 putative QKI targets and about 24% putative mRNA targets having function in proliferation and migration. Further, the promotor region of QKI contains a conserved ERE, suggesting a role of QKI in breast cancer. In this study, we analyzed the expression of QKI with or without the treatment of estrogen or tamoxifen in multiple breast cancer cell lines both at mRNA and protein level. In addition, we validate the response of QKI promoter to estrogen through reporter assay. Furthermore, through construction of adenovirus of QKI, we compared the expression of CDC42 and Par1 both under forced QKI expression and control. The main findings of our study are as follows:
1.The recombinant adenovirus of Qki-5 has been successfully constructed and expressed in the MDA-MB-231 cells of breast cancer.
2.RT-PCR and Western-blot results showed that the mRNA and protein level of qki increased under the stimulation of 17β-estradiol. The activity of qki promoter was upregulated under the stimulation of17β-estradiol.RT-PCR results showed the transcriptional level of qki and activity of qki promoter was downregulated under the stimulation of tamoxifen. In other words, ERE in the QKI promoter region might mediate the QKI response to estrogen. We also observed that transcriptional level of cyclinD1 was downregulated under the stimulation of 17β-estradiol in MDA-MB-231(ERα-/ERβ+).
3.Upon overexpression and specifically silencing the expression of QKI, endogenous expression of cdc42 and par1 were significantly changed. Consistently, wounding assay shows that forced expression of QKI nearly abrogate the migration of breast cancer cells.
In conclusion, our results primarily set a model that during the breast cancer development, ER signal aberration leads to downregulation of QKI, which in turn enhances the expression of CDC42 and Par1, and thus facilitate the process of tumor metastasis.
引文
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2.Chen T, Cote J, Carvajal HV, Richard S. Identification of Sam68 arginine glycine-rich sequences capable of conferring nonspecific RNA binding to the GSG domain. J Biol Chem. 2001, 276(33):30803-30811.
3.Lakiza O, Frater L, Yoo Y, Villavicencio E, Walterhouse D, Goodwin EB, Iannaccone P. STAR proteins quaking-6 and GLD-1 regulate translation of the homologues GLI1 and tra-1 through a conserved RNA 3'UTR-based mechanism. Dev Biol. 2005, 287(1):98-110.
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2.Chen T, Cote J, Carvajal HV, Richard S. Identification of Sam68 arginine glycine-rich sequences capable of conferring nonspecific RNA binding to the GSG domain. J Biol Chem. 2001, 276(33):30803-30811.
3.Lakiza O, Frater L, Yoo Y, Villavicencio E, Walterhouse D, Goodwin EB, Iannaccone P. STAR proteins quaking-6 and GLD-1 regulate translation of the homologues GLI1 and tra-1 through a conserved RNA 3'UTR-based mechanism. Dev Biol. 2005, 287(1):98-110.
4.Ebersole TA, Chen Q, Justice MJ, Artzt K. The quaking gene product necessary in embryogenesis and myelination combines features of RNA binding and signal transduction proteins. Nat Genet. 1996, 12(3):260-265.
5.Sidman RL, Dickie MM, Appel SH. Mutant mice (quaking and jimpy) with deficient myelination in the central nervous system. Science. 1964, 144:309-11.
6.Ebersole T, Rho O, Artzt K. The proximal end of mouse chromosome 17: new molecular markers identify a deletion associated with quakingviable. Genetics. 1992,131(1):183-190.
7.Kondo T, Furuta T, Mitsunaga K, Ebersole TA, Shichiri M, Wu J, Artzt K, Yamamura K, Abe K. Genomic organization and expression analysis of the mouse qkI locus. Mamm Genome. 1999, 10(7):662-669.
8.King TR, Dove WF. Pleiotropic action of the murine quaking locus: structure of the qkv allele. amm Genome. 1991, 1(1):47-52.
9.Larocque D, Richard S. QUAKING KH domain proteins as regulators of glial cell fate and myelination. RNA Biol. 2005, 2(2):37-40.
10.Hardy RJ, Loushin CL, Friedrich VL Jr, Chen Q, Ebersole TA, Lazzarini RA, Artzt K. Neural cell type-specific expression of QKI proteins is altered in quakingviable mutant mice. J Neurosci. 1996, 16(24):7941-7949.
11.Wu HY, Dawson MR , Reynolds R , Hardy RJ. Expression of QKI proteins and MAP1B identifies actively myelinating oligodendrocytes in adult rat brain. Mol Cell Neurosci , 2001 ,17:292-302.
12.Pilotte J , Larocque D , Richard S. Nuclear translocation controlled by alternatively spliced isoforms inactivates the QUAKING apoptotic inducer. Genes Dev , 2001 , 15:845-858.
13.Chen T, Richard S. Structure-function analysis of Qk1: a lethal point mutation in mouse quaking prevents homodimerization. Mol Cell Biol. 1998, 18(8):4863-7481.
14.Haroutunian V, Katsel P, Dracheva S, Davis KL. The human homolog of the QKI gene affected in the severe dysmyelination "quaking" mouse phenotype: downregulated in multiple brain regions in schizophrenia. Am J Psychiatry. 2006, 163(10):1834-1837.
15.Lu Z, Zhang Y, Ku L, Wang H, Ahmadian A, Feng Y. The quakingviable mutation affects qkI mRNA expression specifically in myelin-producing cells of the nervous system. Nucleic Acids Res. 2003, 31(15):4616-4624.
16.Zhao L, Tian D, Xia M, Macklin WB, Feng Y. Rescuing qkV dysmyelination by a single isoform of the selective RNA-binding protein QKI. J Neurosci. 2006, 26(44):11278-11286.
17.Hardy RJ. Molecular defects in the dysmyelinating mutant quaking. J Neurosci Res. 1998, 51(4):417-422.
18.Wu J, Zhou L, Tonissen K, Tee R, Artzt K. The quaking I-5 protein (QKI-5) has a novel nuclear localization signal and shuttles between the nucleus and the cytoplasm. J Biol Chem. 1999, 274(41):29202-2910.
19.Pilotte J, Larocque D, Richard S. Nuclear translocation controlled by alternatively spliced isoforms inactivates the QUAKING apoptotic inducer. Genes Dev. 2001, 15(7):845-858.
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