新型辐射相关基因的筛选及功能研究

【作者】 王朝云；

【导师】 叶棋浓；

【作者基本信息】 中国人民解放军军事医学科学院 ， 遗传学， 2008， 硕士

【摘要】 雌激素类化合物是防治急性放射病的有效药物之一,如国内研制的523、E838等,因此我们推测雌激素信号途径中的组成成分应与辐射防护相关。目前公认的是,雌激素通过与雌激素受体(ER)起作用,调节ER的转录活性,这一过程受一类称为ER共调节因子的蛋白质的平衡调节,因此我们推测ER共调节因子在辐射防护中可能起重要作用,我们尝试了多种ER共调节因子与辐射应答相关的可能性,如FHL1、FHL3、RPBMS、PES1、MEMO及HPIP等,在众多候选蛋白中我们发现HPIP和FHL1与辐射相关。HPIP是2000年Abramovich C[1]等以PBX1作为诱饵,利用酵母双杂交技术从胎儿肝脏cDNA文库中筛选得到的。因而,这个蛋白被命名为造血相关的PBX相互作用蛋白质(hematopoietic PBX-interacting protein HPIP)。我们用酵母双杂交技术以ERβAF2为诱饵从乳腺文库中也分离到了与ER相互作用的蛋白质HPIP,免疫沉淀实验证实ER与HPIP相互作用。我们在研究HPIP与辐射敏感性关系的过程中,通过不同剂量的γ射线辐射发现10 Gy条件下HPIP蛋白水平升高最明显,采用10 Gy辐射后HPIP蛋白表达水平随时间变化而逐渐升高。RT-PCR实验表明HPIP在mRNA水平具有与蛋白水平一致的变化趋势,说明HPIP的启动子区域可能包含辐射敏感序列,因此我们检测了一系列长度不同的启动子在辐射前后的活性,发现-782～-1及-1282～-782区域辐射后启动子活性明显升高,推测这两部分区域可能对辐射后HPIP转录水平的升高发挥重要作用。为了进一步研究HPIP与辐射敏感性的关系,我们建立了过表达HPIP及敲低内源性HPIP的乳腺癌细胞株MCF-7。生长和密度曲线实验表明,与空载体相比,辐射前HPIP稳定表达的细胞株生长速度较快,而辐射后HPIP促进细胞生长的功能消失。克隆形成实验进一步验证了这一结果,辐射后HPIP导致细胞的克隆形成率大幅度下降,说明辐射后HPIP使细胞的生存能力降低,即HPIP具有辐射增敏作用。为了深入探讨HPIP介导的辐射增敏的分子机制,我们分别在p53阴性、p53阳性、ATM阴性、BRCA1阴性的细胞株中检测辐射对HPIP的影响,发现辐射前后ATM缺陷细胞株ATBV5及BRCA1阴性的细胞株HCC1937中HPIP蛋白水平几乎不发生改变;野生型P53功能缺失的细胞株如MDA-MB231、MDA-MB-453和p53功能正常的细胞株如MCF-7、ZR75-1,其内源性HPIP表达水平在辐射后随时间延长而逐渐增加,提示HPIP的辐射应答可能与p53无关。利用HPIP过表达和敲减的稳定转染细胞株,检测辐射前后与DNA损伤修复和周期阻滞相关的蛋白如p53、p21、pERK的变化。实验结果表明,在不辐射情况下HPIP能升高p21水平,辐射后升高的倍数明显下降即辐射使HPIP升高p21的能力下降,通过克隆形成实验进行验证,当转染外源p21使辐射后HPIP升高p21的程度与辐射前一致时,能够基本恢复其克隆形成能力,说明辐射后p21表达下降可能是HPIP辐射致敏的机理之一,但HPIP与p21并不存在直接的结合,因此推测有其它成分发挥中介作用。当不辐射时HPIP升高pERK,辐射后稳定细胞株都升高了pERK,但升高幅度基本一致,克隆形成实验验证,pERK的抑制剂PD98059使细胞的克隆形成能力下降,但由HPIP导致的辐射敏感性仍然存在,说明pERK并不参与HPIP的辐射增敏作用。辐射前后HPIP对p53均没有显著影响,基本可以排除p53参与HPIP辐射后的作用。FHL1作为FHL家族成员包含4个半LIM结构域,是我们筛选的与辐射相关的另一个蛋白。生长曲线实验结果是FHL1辐射前抑制细胞生长,辐射后FHL1使细胞保持原来水平;克隆形成实验结果与生长曲线一致,辐射前FHL1使细胞的克隆形成能力下降,辐射后克隆形成率并没有进一步降低,初步说明FHL1可能有抵抗辐射的作用,但需要进一步验证。综上所述,我们在雌激素信号通路中发现了两个与辐射相关的雌激素受体共调节因子HPIP和FHL1。实验结果表明,HPIP具有辐射增敏的作用,FHL1具有辐射抵抗的作用。这是目前在雌激素信号通路中发现的新型辐射相关的雌激素受体共调节因子,它们在辐射后出现的不同生物学效应,因此深入研究其分子机理为将来开发不同用途的辐射药物奠定了基础。更多还原

【Abstract】 Estrogenic chemicals, such as 523 and E838, are the active drugs used to prevent acute radiation diseases, so we hypothesized that some proteins in estrogen signal pathway may be associated with radiation sensitivity. It is well accepted that estrogen plays its role via estrogen receptors (ERs) and regulates the transcription of estrogen-responsive genes. The process is modulated by the proteins called ER co-regulators. Thus, we assumed that ER co-regulators may be involved in radioprotection. We screened many ER co-regulators, such as FHL1, FHL3, RPBMS, MEMO and HPIP. Fortunately, we identified HPIP and FHL1 as the targets.HPIP was originally identified from the fetal liver cDNA library through yeast two-hybrid-system with PBX1 as bait. Consequently, the protein was named hematopoietic PBX-interacting protein (HPIP). With ERβAF2 as a bait protein we found out HPIP by the same system. Immunoprecipitaion assays confirmed the interaction between ER and HPIP. When studying the relationship of HPIP with radiation, we know that HPIP protein level is highest after 10Gy of gamma irradiation, and using the same dose of irradiation, HPIP levels gradually increased with time. RT-PCR experiment indicated that the change tendency of HPIP mRNA was similar to that of the protein, suggesting that there may be a promoter sequence sensitive to irradiation. For this reason we detect a series of promoter fragments of different length. The results showed that the -782～-1 bp and -1282～-782area of the HPIP promoter was sensitive to irradiation. To research the relation of HPIP and radiosensitivity, we established the HPIP overexpression and knockdown stable MCF-7 cell lines. Growth curve assays showed that HPIP promoted the cell growth compared with empty vector, but after irradiation the function disappears. Colony formation also displayed the same conclusion. All above results suggest that HPIP is radiosensitive. With the purpose of investigating the molecular mechanism of HPIP, we detecte the changes in p53-positive cell lines, p53-negative cell lines, AT-defective cell lines, and BRCA1-defective cell lines. The results showed that in AT-defective and BRCA1-defective cell lines there was no obvious change in HPIP levels after irradiation. However, both in p53-positive and–negative cell lines, HPIP was still irradiation-inducible, indicating that radiation inducibility of HPIP has nothing to do with p53. We detected the p53, p21, pERK in the HPIP overexpression and knockdown stable MCF7 cell lines before and after radiation. The results demonstrated that HPIP could increase p21 level even without irradiation and p21 could be radiation-inducible. However, the magnitude of the p21 inducibility with HPIP overexpression was lower than that without HPIP overexpression. Regardless of HPIP overexpression, irradiation increased pERK. HPIP could not influence p53 before and after radiation. In order to confirm the mechanism, by colony formation we found that, through transfection of exogenous p21 into HPIP overexpression stable cell lines, the ability of colony formation is approximately equivalent to that of control cells. At the same time, using the pERK inhibitor PD98059 to inactivate pERK, we showed that it did not affect the ability of colony formation at different HPIP levels.As a member of the FHL family, FHL1 is the other target we chose. The growth curve assays indicated that FHL1 inhibited the cell proliferation without irradiation; while after radiation FHL1 could protect the cells from irradiation. Colony formation assays also confirmed that FHL1 is an anti-radiation gene.In conclusion, we identified two irradiation-associated genes, HPIP and FHL1, in estrogen signal pathway. The experiment results indicate that HPIP may function as a radiosensitizer and FHL1 a radioresister. These are the novel radio-associated genes isolated from ER coregulators and have different effects on radiosensitivity. Further research on their mechanisms will establish a solid foundation for us to develop radiation drugs of various purpose.更多还原