节点文献
睾丸高表达基因Fank1和RSB66的功能研究
【作者】 王海龙；
【作者基本信息】 北京协和医学院 ， 生物化学与分子生物学， 2010， 博士
【摘要】 哺乳动物的精子发生是由精原细胞增殖分化最终形成精子的复杂而有序的过程,历经有丝分裂、减数分裂和变形三个阶段。在精子发生的每一个过程中都需要维持细胞增殖、细胞分化和细胞凋亡的精密平衡,而这些生命活动是由众多时间和空间特异性基因的转录和表达来精确调控的。因此,揭示睾丸高表达蛋白质的功能特征,对阐明精子发生的分子机制具有十分重要的意义。本研究对睾丸高表达蛋白质Fank1和RSB-66的表达特征和功能进行了初步研究。1.睾丸特异表达蛋白质Fank1的功能研究Fank1基因是我室根据NCBI数据库中的一个人睾丸特异性EST,通过电子克隆的方法获得的具有完整读码框的一个新基因。其开放阅读框编码330个氨基酸的蛋白质,氨基端是一个FN3结构域,羧基端由六个ANK结构域组成。Northern blot、RT-PCR和Western blotting分析结果表明Fank1特异表达于睾丸组织。以Fank1全长序列作为诱饵,筛选人胎脑cDNA表达文库,最终得到6条不同的基因序列,其中一个是JAB1。我们在体外和体内两种体系中确证了Fank1和JAB1的相互作用。首先,利用原核表达系统表达和纯化了融合蛋白GST-Fank1以及GST-FN3,与等量的HEK 293T细胞抽提液进行孵育后进行GST pulldown实验,结果发现细胞内源的JAB1蛋白质与GST-Fank1和GST-FN3能够同时被沉淀。同时我们将V5-JAB1和Flag-Fank1、Flag-FN3、Flag-ANK的表达质粒分别共同转染HEK293T细胞,用抗Flag的抗体进行沉淀,结果显示过表达的Fank1能与JAB1蛋白质相结合,FN3结构域和ANK结构域也能与JAB1相互作用。此外我们应用细胞免疫荧光的方法证明过表的JAB1与Fank1在空间上有共同的定位。为研究Fank1的功能,我们构建了pEGFP-N1-Fankl/CHO稳定细胞株和对照pEGFP-N1/CHO稳定细胞株。利用Mercury系统检测Fank1对细胞信号通路的影响,发现Fank1可以明显激活HSE和AP-1信号通路。已知JAB1参与AP-1信号通路的调节,我们通过双荧光素酶报告基因分析的方法验证Fank1在AP-1信号通路中的作用,结果发现加入Fank1可以明显增加AP-1的转录活性,并且其转录活性随着Fank1剂量的增加而增加。同时也证明了Fank1的两个结构域FN3和ANK也可以增加AP-1活性,但与Fank1全长相比,它们的激活作用较弱。通过小RNA干扰技术将JAB1有效干扰后,Fank1激活AP-1转录活性的能力明显下降,这说明Fankl对AP-1转录活性的激活是依赖JAB1的。为了了解Fank1激活AP-1信号通路的可能机制,我们检测了Fank1对c-Jun在细胞中的分布和活化水平。实验发现,Fank1可以增加c-Jun在细胞核内的聚集以及可以增加c-Jun的磷酸化水平,而过表达Fank1并没有引起JNK和磷酸化的JNK变化,说明c-Jun磷酸化水平的增加不是依赖于JNK的磷酸化增加。当JAB1被小RNA干扰后,细胞核内的c-Jun以及c-Jun的磷酸化水平都明显降低,说明JAB1对于介导Fankl调节AP-1活性是必要的。进一步应用电泳迁移率实验(EMSA)证实,与对照组相比,过表Fank1可以增加AP-1的DNA结合能力,增加其转录活性。AP-1在细胞凋亡中发挥重要作用,为了研究Fank1激活AP-1信号通路对细胞凋亡的影响,我们构建了过表Fank1的HeLa稳定细胞和对照HeLa细胞。在40J/m2的UV照射过表Fank1的HeLa细胞和对照HeLa细胞后,流式细胞术检测发现照射3小时以后两组细胞均有凋亡发生,与对照组相比,过表Fank1组在不同时间所发生的细胞凋亡数比对照组明显减少。同时发现在UV刺激后的不同时间点与对照组比较,c-Jun和磷酸化的c-Jun都有明显增加,但JNK和磷酸化的JNK并没有变化,而JAB1在两组细胞不同时间点之间没有变化。检测中发现具有抗凋亡作用的AP-1的靶基因Bcl-3表达升高,说明Fank1通过Bcl-3途径发挥抗凋亡作用。为了验证这种作用,我们通过RNA干扰JAB1后发现,Fank1发挥的这种抗凋亡作用可以被逆转。最后我们发现小鼠睾丸组织中有Bcl-3的表达,而文献报告c-Jun和JAB1在睾丸中均有表达。这样上述体细胞的实验结果就进一步提示,Fank1在精子发生的减数分裂和变形阶段可能也通过Bcl-3发挥抗凋亡作用。2、睾丸高表达蛋白质RSB66的功能研究RSB66基因是我们从RSB文库分离到的一条新基因,Northern blot显示RSB66只在大鼠睾丸中特异性表达。通过酵母双杂交、细胞内免疫共沉淀和GST pull down已经证实RSB66和INCA1 (Inhibitor of Cdk interacting with Cyclin A1)可以相互作用。为了研究RSB66的功能,我们利用原核表达系统分别对小鼠源的RSB66、INCA1和RSB66的人源的同源蛋白质C9orf9和INCA1进行原核表达,期望能够获得RSB66/INCA1复合物的晶体结构,对RSB66的功能进行提示。通过优化表达条件,我们获得了RSB66的可溶性表达并且得到纯的蛋白质,为晶体的生长奠定基础；但INCA1只能以GST-INCAl的融合形式在Rossetta感受态细胞中进行表达,并且以包涵体的形式出现,这为晶体的生长带来不便。我们试图利用双表达载体RSFDuet-1同时表达这两个蛋白质,以益于形成复合物。但经过多种条件的摸索均未成功,致使从晶体结构提示蛋白质功能的探索暂时终止鉴于c9orf9与肿瘤的发生有关,我们应用荧光定量PCR技术检测了良性前列腺增生组织与前列腺癌组织中c9orf9的表达以及不同乳腺癌细胞系中c9orf9的表达差异。结果发现,前列腺癌组织中c9orf9 mRNA含量明显低于良性前列腺增生组织。同时发现c9orf9在正常肾细胞中表达,而在肾癌细胞系中均未检测到表达,这一结果提示c9orf9可能作为一种候选的抑癌基因在前列腺癌的发生发展中发挥作用,而在多种乳腺癌细胞系中只有HCC38中c9orf9高表达。我们还筛选出针对c9orf9的有效RNA干扰靶点,这就为采用此靶点对c9orf9高表达的HCC38乳腺癌细胞进行表达干扰,进而研究c9orf9的功能奠定了基础。同时我们构建并筛选出稳定表达c9orf9的HeLa稳定细胞株,并通过双胸苷同步化的方法对过表c9orf9的HeLa稳定细胞进行细胞周期研究,发现过表c9orf9可以明显延迟G1/S期进程。最后我们筛选出可以有效干扰RSB66的干扰靶点,成功构建并筛选出RSB66RNA干扰的稳定GC-1细胞克隆,为利用我组建立的小鼠睾丸曲细精管显微注射平台对其在动物水平进行功能探索奠定了基础。
【Abstract】 Spermatogenesis is a complex developmental process including mitosis of spermatogonial stem cells, meiosis of spermatocytes and spermiogenesis. Many testis highly expressed genes involve in the regulation of spermatogenesis, such as cell proliferation, cell differentiation and cell apoptosis. Therefore, identification and characterization of these genes is of great value to delineate the mechanism of spermatogenesis. In the present study, we show some primary results from exploring the expression and function of Fankl and RSB66 in mammalian spermatogenesis.1. The functional study of testis highly expressed protein, FanklFankl is a novel gene obtained from a human testis-specific EST by in silicon cloning. Fankl encodes a 330-amino-acid consisted protein which contains a fibronectin type III domain in N-terminal and six ankyrin repeats in C-terminal. The result of Northern blot, RT-PCR and Western blotting showed that Fankl is expressed highly in testis.Using yeast two-hybrid system with the Fankl full length gene as bait to screen the human fetus brain expression library, six potential interaction proteins with Fankl were obtained. One nucleotide sequence among these clones was JAB1. In vitro and in vivo systems had been used to comfirm the interaction between Fankl and JAB1. GST-Fank1 and GST-FN3 fusion protein had been expressed in E coli cells, and purified by affinity chromatography. GST pull-down assay was performed by incubating GST-Fankl and GST-FN3 fusion protein or GST with HEK 293T cell lysate. The results showed that endogenous JAB1 protein can be co-precipitated with GST-Fankl and GST-FN3 using Glutathione Sepharose 4B agarose, but not with GST alone. To further examine whether Fankl and FN3, ANK domain can interact with JAB1 in physiological condition, pcDNA6-HisB-V5-JAB1 and p3xFlag-CMV-14-Fankl, p3xFlag-CMV-14-FN3, p3xFlag-CMV-14-ANK plasmids were co-transfected into HEK 293T cells. Lysates of transfected cells had been immunoprecipitated by anti-Flag and anti-V5 antibody respectively. The results showed that overexpressed Fankl protein could interact with JAB1 in vivo, moreover, we found FN3 domain strongly interacted with JAB1, while the ANK domain only exhibited a slight interaction with JAB1. Besides, we showed overexpressed Fank1 and JAB1 co-localised to both the cytoplasm and nucleus in MCF-7 cells and HEK 293T cells using immunocytochemistry and confocal microscopy.To study the function of Fank1, we constructed pEGFP-N1-Fank1/CHO and pEGFP-N1/CHO stable cell lines. Using CLONTECH’s Mercury pathway profiling SEAP system, we found Fank1 overexpression significantly activated the HSE and AP-1 signalling pathways. Because JAB1 has been reported to activate an AP-1-dependent promoter and our results showed that Fank1 could interact with JAB1. we used dual luciferase reporter gene assay to testify the effect of Fank1 on AP-1 transcriptional activity. HEK 293T cells were transfected with an AP-1 promoter-driven luciferase reporter gene in the presence of c-Jun, JAB1 and Fank1. The results indicated that Fank1 overexpression increased the relative activation levels of the AP-1 reporter and Fank1 enhanced AP-1 activity in a concentration-dependent manner. In addition, we found that either the FN3 domain or ankyrin repeats of Fank1 could increase AP-1 activity, but full-length Fank1 enhanced this activity more than either domain alone. Furthermore, we found knockdown of JAB1 inhibited the Fank1-dependent activation of the AP-1 reporter gene. Taken together, these results suggested that Fank1 could activate the AP-1 pathway and that JAB1 is involved in Fank1-mediated activation of AP-1.To further define the mechanism of AP-1 activation by Fank1, the distribution and phosphorylation status of endogenous c-Jun was examined. HEK 293T cells were transfected with Flag-tagged Fank1 or with a control expression vector. Cytoplasmic and nuclear proteins were isolated and analysed by Western blotting. We found Fank1 increased c-Jun expression in the nucleus and Phosphorylation of both Ser-63 and Ser-73 of c-Jun was elevated. We also found Fank1 did not alter JNK activity compared to control. To further confirm the effect of Fank1 on c-Jun activation, we performed EMSA analysis and found Fank1 overexpression increased the binding capacity of c-Jun to DNA. To investigate the role of JAB1 in endogenous c-Jun activation by Fank1, we suppressed the expression of JAB1 by RNA interference method and found the increase in c-Jun expression and activation by Fank1 was visibly inhibited when JAB1 was knocked down. Taken together, these results suggested that Fank1 could enhance endogenous c-Jun activity and that this regulation was JAB1 dependent.AP-1 transcription factors are involved in regulation of apoptosis. To observe the effect of Fank1 on cell apoptosis, we generated HeLa/Flag-Fank1 and HeLa/Con. We measured apoptosis by FACS analysis and observed that apoptosis decreased significantly at different times in stable HeLa/Flag-Fank1 cells in comparison to control stable cells after UV exposure. To evaluate the degree of anti-apoptosis in stable HeLa/Flag-Fank1 cells after UV stimulation, we measured the cleavage of caspase 7 and poly(ADP-ribose) polymerase (PARP) and found the level of cleaved caspase 7 and PARP decreased in stable HeLa/Flag-Fank1 cells compared with the control stable cells. Furthermore, we found that overexpressed Fank1 could enhance the total expression and phosphorylation levels of c-Jun when compared with the control group after UV stimulation and Fank1 did not affect the total expression and phosphorylation levels of JNK. We detected the anti-apoptotic proteins that were the AP-1 target genes such as Bcl-2, Bcl-xL and Bcl-3, and found Bcl-3 was significantly upregulated compared with the control vector after UV exposure. Taken together, these results suggest that Fank1 exerted its anti-apoptotic effect by activating AP-1 activity, which then upregulated the expression of Bcl-3 after UV stimulation. Meanwhile, we investigated the functional linkage between Fank1 and JAB1 in the regulation of anti-apoptosis by knock down of JAB1. In all groups in which JAB1 was silenced, we found that Fank1 could not exert its anti-apoptotic effect after UV exposure and the upregulation of c-Jun and Bcl-3 by Fank1 was significantly suppressed. Taken together, these results indicate that the regulation of anti-apoptosis by Fankl is JAB1 dependent.Fank1 is a testis highly expressed gene, it is reported that c-Jun and JAB1 are expressed in testis, meanwhile our results confirmed Bcl-3 was expressed in testis. We supposed that Fank1 could function as a anti-apoptotis protein by upregulation of Bcl-3 through AP-1 pathway in Spermatogenesis.2. The functional study of testis highly expressed protein, RSB66rsb66 was a new gene obtained from RSB subtracted library. Northern blot showed that rsb66 was specifically expressed in rat testis. In previous studies, the interaction between RSB66 and INCA1 had been confirmed by a Yeast Two-Hybrid system, co- immunoprecipitation and GST-pull down assay respectively. We expect to analyze the Crystal structure of RSB66/INCA1 complex to reveal the function of RSB66. We expressed soluble recombinant GST-mRSB66 protein and His-RSB66 protein in E.coli and purified mRSB66 protein was obtained by affinity chromatography and gelfiltration chromatography method. However it was difficult to gain soluble INCA1 protein in different expressin vectors and different competent cells in prokaryotic expression system. And this caused a pause on analyzing the crystal structure of RSB66/INCA1 complex that would reveal the function of RSB66.It was reported that c9orf9, the human homologue of RSB66 was expressed lowly in bladder and lung carcinoma. We investigated the c9orf9 expression levels in the benign prostatic hyperplasia and prostate carcinoma clinical samples. The expression level of c9orf9 was measured by real-time PCR. The average expression of c9orf9 mRNA in prostate carcinoma was lower than that in benign prostatic hyperplasia. Meanwhile, the c9orf9 level in several breast cancer cell lines was detected by real-time PCR and Western blotting, highly expressed c9orf9 was found in HCC38 cell line. In addition, we screened the efficent RNA interference sequence for c9orf9. To further study the function of RSB66, HeLa-pMSCVpuro and HeLa-pMSCVpuro-c9orf9 stable cells were established and the cell cycle was analysed by flow cytometry. Compared with HeLa-pMSCVpuro stable cells, obvious prolonged G1/S phase was observed in HeLa-pMSCVpuro-c9orf9 stable cells occurred at 4,8,9h after release.Based on the technique of microinjection into seminiferous tubules of mouse and the germ cell transplantation system, we screened the efficient RNA interference target sequence for mRSB66 and constructed mRSB66(-)/GC1 and negative/GC1 stable cell lines. Next we will transplant mRSB66(-)/GC1 and negative/GC1 stable cell lines into mouse seminiferous tubules and observe the possible function of RSB66 in animal model.