蛋白酶体亚基PSMD7在纤毛/鞭毛解聚及食管癌发生发展中的作用
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摘要
泛素-蛋白酶体系统(ubiquitin-proteasome system, UPS)参与真核生物的细胞周期、凋亡和信号传导等多个生物过程的调节,肿瘤细胞尤其依赖UPS来清除细胞内由于快速增殖而产生的有害的不成熟和不规则蛋白,以增强细胞的抗凋亡能力。此外已有研究证实,泛素存在于模式生物衣藻和盐藻的鞭毛中,并且在线虫、小鼠和人类的纤毛蛋白质组中都已被鉴定出来,因此我们推断UPS可能参与纤毛/鞭毛的生物学功能。
纤毛和鞭毛是进化上非常保守的细胞器,结构基本相同,它们都来源于细胞质膜下的基体,向上突出于细胞表面。几乎人类所有类型的细胞都有纤毛,而鞭毛存在于低等真核生物,其基因组结构简单便于进行研究,其中鞭毛内运输机制最早就是在模式生物衣藻中发现的,随后在高等生物的纤毛中也被证实。纤毛/鞭毛的解聚和细胞周期以及外界信号有关,细胞增殖时纤毛发生解聚,基体转变为中心体参与纺锤体的形成,待细胞完成有丝分裂后,细胞又会长出新的纤毛。肿瘤的发生常常伴随着纤毛的异常,且纤毛内存在多个和肿瘤相关信号通路,但二者之间的分子机制尚不完全清楚。纤毛在解聚过程中会有大量的纤毛蛋白被降解,虽然泛素结合体系在纤毛和鞭毛中已被鉴定出来,但蛋白酶体是否参与纤毛/鞭毛的解聚,以及UPS能否通过影响纤毛/鞭毛的功能来参与肿瘤的发生发展未见报道。
然而纤毛长度较短不易被观察,且在体外培养容易丢失。鞭毛长度较长,通过简单的理化条件即可诱导其解聚。所以利用模式生物来进行研究可以帮助我们了解UPS和纤毛解聚之间的分子机制。杜氏盐藻和衣藻都为真核生物绿藻,二者的不同之处在于衣藻细胞外有一层细胞壁,其鞭毛上也有细胞壁的延伸,而盐藻则没有,在结构上和哺乳动物更加接近,故本研究选取盐藻的鞭毛作为模式生物器来研究UPS和鞭毛解聚之间的关系,为进一步研究纤毛在肿瘤发生发展中的分子机制提供依据。
蛋白酶体由20S核心颗粒(core particle, CP)和19S调节颗粒(regulatory particle, RP)组成。20S CP是蛋白酶体的降解部位,包含水解活性位点；19S RP作为多聚泛素化蛋白的受体,负责将蛋白底物去泛素化并将蛋白解折叠,并运送至20S CP。目前20SCP的抑制剂硼替佐米已经在临床上用于治疗多发性骨髓瘤和淋巴瘤,但易产生耐药性,且对实体瘤的治疗效果差。研究显示,19S RP中的一些去泛素化酶参与肿瘤的发生发展并可以作为潜在的肿瘤治疗靶点。
PSMD7是19S蛋白酶体lid中的一个去泛素化酶亚基,对19S RP结构组织的分析表明,在组成lid亚复合体的9个亚基中,PSMD7位于中央核心部位,同时和其他4个亚基相结合,说明PSMD7是19S蛋白酶体lid结构中的重要组成部分,但它在内的具体功能还不完全清楚。为了了解蛋白酶体在纤毛/鞭毛中的分子机制,并寻找肿瘤治疗的有效靶点,本课题从两个方面对PSMD7在胞内可能发挥的功能进行了研究：1.PSMD7是否参与纤毛/鞭毛的解聚?2.它是否可以影响肿瘤的发生发展?
第一部分蛋白酶体亚基PSMD7参与纤毛/鞭毛蛋白kinesin的降解
目的
了解鞭毛解聚过程中蛋白酶体活性的变化,以及PSMD7与纤毛/鞭毛蛋白之间的相互作用,初步研究PSMD7是否参与纤毛/鞭毛的解聚,为进一步研究UPS影响纤毛/鞭毛功能的分子机制打下基础。
方法
1泛素-蛋白酶体系统在鞭毛解聚后的激活状态
用免疫荧光和Western blotting方法检测所提取鞭毛中的泛素结合体系。用IBMX诱导鞭毛解聚后,观察鞭毛中泛素化蛋白的聚集情况；甘油密度梯度离心纯化细胞蛋白酶体,用荧光多肽底物检测蛋白酶体活性的变化；实时荧光定量PCR检测盐藻PSMD7(DsPSMD7)的表达变化。
2PSMD7和纤毛/鞭毛kinesin的结合
酵母双杂交技术分别检测盐藻中DsPSMD7和鞭毛kinesin(KCBP)及293T细胞中PSMD7(HsPSMD7)和纤毛kinesin(Kif3a)的相互作用,并进一步用GST pulldown和免疫共沉淀验证二者之间的相互作用。用蛋白酶体抑制剂分别处理盐藻和293T细胞,观察KCBP和Kif3a在胞内的降解情况。
结果
1泛素-蛋白酶体系统在鞭毛解聚后的激活状态
鞭毛发生解聚后,鞭毛内泛素化蛋白的量增多,胞内蛋白酶体的活性增加了将近2倍,说明泛素-蛋白酶体系统体参与了盐藻鞭毛解聚过程。DsPSMD7在鞭毛解聚过程中的mRNA水平表达量增高,在鞭毛解聚后30min表达量达到最高。
2PSMD7和纤毛／鞭毛kinesin的结合
酵母双杂交显示DsPSMD7和KCBP,HsPSMD7和Kif3a可以相互作用,且GST pulldown和免疫共沉淀进一步验证了二者之间的结合。用蛋白酶体抑制剂分别处理细胞后,KCBP和Kif3a两个kinesin在胞内的聚集增多,说明纤毛／鞭毛kinesin的降解由蛋白酶体参与。
结论
鞭毛解聚后蛋白酶体活性增强,同时PSMD7的表达量增加。纤毛或鞭毛kinesin都可以和PSMD7相互作用,并被蛋白酶体降解,说明PSMD7参与纤毛／鞭毛的解聚。
第二部分蛋白酶体亚基PSMD7在食管鳞癌发生中的作用
目的
鉴于去泛素化酶对肿瘤细胞的生存能力是必需的,我们探讨了PSMD7对蛋白酶体活性的影响,以及在食管鳞癌发生发展中的作用,为深入研究PSMD7在肿瘤细胞中作用的分子机制,以及UPS能否通过影响纤毛/鞭毛的功能来参与肿瘤的发生发展提供实验依据。
方法
1蛋白酶体在正常食管上皮细胞和食管鳞癌细胞中的表达差异
分别提取正常食管上皮细胞Het-1A和食管鳞癌细胞EC9706和Eca109的细胞核、细胞质和中心体,Western blotting检测PSMD7在各亚细胞结构中的表达。免疫荧光观察PSMD7在正常食管上皮细胞和食管鳞癌细胞中的定位。
2慢病毒介导的PSMD7基因干扰对食管鳞癌细胞增殖、周期和凋亡影响的体外研究
检测感染PSMD7RNAi慢病毒的EC9706细胞内泛素化蛋白的聚集情况和蛋白酶体活性的变化,分别用CCK-8实验和流式细胞仪检测细胞的增殖、周期和凋亡的变化。并用Western blotting检测PSMD7基因干扰后对p53表达的影响。同时将PSMD7RNAi慢病毒联合硼替佐米处理食管鳞癌细胞,观察细胞蛋白酶体功能和凋亡的变化。
3慢病毒介导的PSMD7基因干扰对移植瘤生长的影响
建立裸鼠移植瘤模型,观察PSMD7基因干扰后对食管鳞癌细胞EC9706的致瘤能力的影响。同时用硼替佐米对荷瘤裸鼠治疗两周,测量肿瘤大小以绘制生长曲线,并计算抑瘤率。病理组织学观察成瘤细胞的形态变化,免疫组化检测细胞泛素化蛋白的表达,TUNEL法分析不同处理组的裸鼠移植瘤组织细胞凋亡的变化。
结果
1蛋白酶体在正常食管上皮细胞和食管鳞癌细胞中的表达差异
PSMD7在两株食管鳞癌细胞EC9706和Eca109中的表达均高于正常食管上皮细胞Het-IA,在三种细胞的细胞核中的表达没有明显变化,但在食管鳞癌细胞质和中心体中的表达明显高于正常食管上皮细胞。
2慢病毒介导的PSMD7基因干扰对食管鳞癌细胞增殖、周期和凋亡影响的体外研究
PSMD7RNAi可以使EC9706胞内多泛素化蛋白增加,蛋白酶体的活性降低,抑制细胞的增殖,促进细胞的凋亡,还可以影响胞内p53的功能。此外PSMD7基因干扰后还可以增强硼替佐米对蛋白酶体功能的抑制和肿瘤细胞的凋亡。
3慢病毒介导的PSMD7基因干扰对移植瘤生长的影响
PSMD7基因干扰后的EC9706细胞在裸鼠体内生长缓慢,肿瘤体积只有对照的组的1/2。其联合蛋白酶体抑制剂硼替佐米治疗对肿瘤抑制率最高,肿瘤体积是对照组的40%,病理组织学检查发现成瘤细胞的异质性降低。免疫组化结果显示PSMD7基因干扰组、硼替佐米治疗组和联合治疗组细胞中的泛素化蛋白均增多,TUNEL显示治疗组的细胞都有不同程度的凋亡,其中联合治疗组凋亡的比例最大。
结论
PSMD7在食管鳞癌细胞中的高表达,且定位发生改变。PSMD7RNAi可以降低食管鳞癌细胞蛋白酶体的活性,使细胞增殖能力降低,并诱导细胞的凋亡,对肿瘤生长有明显的抑制作用,还可以增强食管鳞癌细胞对硼替佐米的敏感性。抑制PSMD7的表达能降低蛋白酶体的功能来诱导肿瘤细胞的凋亡,提示它可能是食管鳞癌的一个有效治疗靶点。
The ubiquitin proteasome system (UPS) is the major protein degradation system in eukaryotic cells and regulates a variety of cellular functions, including removal of damaged proteins, cell cycle regulation, apoptosis and signal transduction. Cancer cells depend on high proteasome activity to remove harmful immature and irregular proteins generated by rapid proliferation and to strength the anti-apoptosis ability. Studies have shown that the ubiquitin-UNTjunction system are present in flagella of model organism of algae Chlamydomonas reinhardtii and Dunaliella salina, and recently it has also been identified in the ciliome of Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Homo sapiens. So we infer that UPS may be involved in the biological function of cilia/flagella.
Cilia and flagella are UNTserved organelles during evolution with similar structure and origin from basal body protruding from cell surface. Flagella exist in lower eukaryotes whose genome structures are more simple and easy to study. The intraflagellar transport mechanism was first identified in the model organisms Chlamydomonas reinhardtii, and then was UNTfirmed in the cilia of higher organelle. Nearly all kinds of human cells have cilia. Cilia disassembly is closely related to cell-cycle Control, which releases the centrosome to form the mitotic spindle and cilia are regenerated after the completion of cell cycle. Tumor cells usually have abnormal cilia and there are multiple tumor-related signaling pathways in cilia, but the molecular mechanism between them is not clear. A large number of ciliary proteins will be degraded during disassembly. Although the ubiquitin UNTjunction system has been identified in cilia/flagella, whether proteasome is involved in the ciliary/flagellar disassembly and whether UPS could be involved in the tumorigenesis remains unclear.
However, cilia are hard to be observed and disappeared during cell culture in vitro. Flagella are longer and can be induce to disassembly by simple operation which could be used as model organelle to help us to better understand the molecular mechanism between UPS and ciliary disassembly. Dunaliella salina and Chlamydomonas reinhardtii are eukaryotic green algae with the difference that the former has a layer of cell wall but the latter not. The flagellar structure of Dunaliella salina is more similar with mammalian cilia. So the flagella of Dunaliella salina were used as a model organelle in this study to provide evidence for the research of cilia and tumorigenesis.
Based on both structure and function the proteasome can be further divided into two sub-complexes, a central20S proteolytic core particle (CP) that is capped at either end by one or two19S regulatory particles (19S RP). The20S CP is the degradation unit and UNTtains the active sites enquired to hydrolyze proteins into peptides. The19S RP Controls the degradation of ubiquitin-tagged substrates by acting as a receptor for polyubiquitinated proteins and facilitating their ATP-dependent translocation into the catalytic chamber of the20S CP. Inhibitors of20S CP bortezomib is used for treatment of multiple myeloma and lymphoma, but these cancer cells are easily to produce resistance to bortezomib. Besides, bortezomib has poor effect in solid tumor treatment. Several19S RP associated deubiquitinases have been shown to be involved in cancer progression and as such are emerging targets for anti-cancer therapies.
PSMD7is a deubiquitinase subunit in the lid of19S RP proteasome and occupies a central position within this subcomplex showed by structural organizational analysis. PSMD7is centered by other4subunits forming the heterotetrameric core structure, suggesting the essential role of PSMD7in the lid subcomplex of19S RP. However, the cellular function of it remains unknown. In this study, in order to provide an evidence for the molecular mechanism of proteasome in the ciliary/flagellar disassembly and to find therapy targets for cancer, we investigate the possible function of PSMD7:1. whether is it involved in the ciliary/flagellar disassembly?2. whether does it play a role in tumorigenesis?
Part1Proteasome subunit PSMD7is involved in the degradation of the ciliary/flagellar protein kinesin
Objective
The proteasome activity during flagellar disassembly and interactions of PSMD7with ciliary/flagellar proteins were observed to preliminary study whether PSMD7participated in ciliary/flagellar disassembly and provide basis for further research on the molecular mechanism of UPS in ciliary/flagellar function.
Methods
1The activity of ubiquitin-proteasome system after flagellar disassembly
Immunofluorescence and Western blotting methods were used to examine the ubiquitin-UNTjunction system in the isolated flagella. After flagellum disassembly induced by IBMX, ubiquitin protein aggregation was observed, the proteasome was purified by glycerol density gradient centrifugation and its activity was determined using fluorescence peptide substrate, and the mRNA expression of PSMD7of D.salina (DsPSMD7) was analyzed by real-time fluorescent quantitative PCR.
2The binding of PSMD7with ciliary/flagellar kinesin
Interactions of DsPSMD7with KCBP in D.salina cells and HsPSMD7with Kif3a in293T cells were detected by yeast two hybrid technology, which were further UNTfirmed by GST pulldown and co-immunoprecipitation. The degradations of KCBP and Kif3a by proteasome were determined by proteasome inhibitor treatment.
Results
1The activity of ubiquitin-proteasome system after flagellar disassembly
After flagellar disassembly, ubiquitinated proteins were accumulated and the proteasome activity was increased nearly2times, indicating that the ubiquitin-proteasome system is involved in the flagellar disassembly of D.salina. The mRNA level of DsPSMD7was upregulated during flagella disassembly, and reached the highest at30min after disassembly.
2The binding of PSMD7with ciliary/flagellar kinesin
Yeast two hybrid showed that DsPSMD7interacted with KCBP and HsPSMD7interacted with Kif3a, which were UNTfirmed by GST pulldown and co-immunoprecipitation. Two kinesin, KCBP and Kif3a became accumulated in cells after proteasome inhibitor treatment, suggesting that these cilia/flagella kinesin were degraded by proteasome.
UNTclusion
Expression level of PSMD7, which mediates degradation of the ciliary or flagellar kinesin, is elevated during flagellar disassembly suggesting that PSMD7may participate in ciliary or flagellar disassembly.
Part2Functional study of proteasome subunit PSMD7in esophageal squamous cell carcinomas
Objective
1Differential expression of proteasome between normal esophageal epithelium cells and esophageal squamous cancer cells
The nucleus, cytoplasm and centrosome were respectively extracted from normal esophageal epithelium and esophageal squamous cancer cells. The translational expressions of PSMD7were detected in various subcellular structures by Western blotting. The localizations of PSMD7were observed by immunofluorescence.
2Effect of PSMD7gene knockdown mediated by lentiviral mediated RNAi on the cell proliferation, cycle and apoptosis of esophageal squamous cancer cells
The accumulation of ubiquitinated proteins and proteasome activity were examined in EC9706cells infected by PSMD7RNAi lentiviral particles. Cell proliferation was analyzed by CCK-8, cell cycle and apoptosis were tested by flow cytometry instrument. The p53level was inspected after PSMD7gene knockdown using Western blotting. The proteasome function and apoptosis of EC9706cells were investigated after PSMD7RNAi lentiviral particles and bortezomib treatment.
3The inhibition of xenografted tumor cell growth by PSMD7gene knockdown mediated by lentiviral mediated RNAi
Xenograft tumor nude mice models were development to explore the tumorigenic capacity of EC9706cells with PSMD7gene knockdown. At the same time, xenograft tumor nude mice were treated by bortezomib for two weeks.The tumor volume was measured and the tumor growth curves were drawn and tumor inhibition rates were calculated. The pathological morphology of tumor cell was observed, the accumulation of ubiquitinated proteins in the tumor cells were surveyed by immunohistochemical,and tumor cell apoptosis in tumor tissues were analyzed by TUNEL method.
Results
1Differential expression of proteasome between normal esophageal epithelium cells and esophageal squamous cancer cells
The expression of PSMD7in two of esophageal squamous cancer cells, EC9706and Eca109, were higher than that in normal esophageal epithelium. There was no obvious change in the expressed of it in the nucleus of these three cells, but the esophageal squamous cancer cells had significantly higher expression in the cytoplasm and centrosome than normal esophageal epithelial cells.
2Effect of PSMD7gene knockdown mediated by lentiviral mediated RNAi on the cell proliferation, cycle and apoptosis of esophageal squamous cancer cells
The ubiquitinated proteins were accumulated and the proteasome activity was reduced in EC9706cells after the PSMD7gene knockdown by lentiviral mediated RNAi. PSMD7RNAi also inhibited the proliferation, promoted the apoptosis and affected the function of intracellular p53in EC9706cells. Moreover, the proteasomal functional inhibition and apoptosis induced by bortezomib were enhanced by PSMD7RNAi.
3The inhibition of xenografted tumor cell growth by PSMD7gene knockdown mediated by lentiviral mediated RNAi
PSMD7RNAi EC9706cells showed lower growth rate in nude mice and the tumor volume of it is only the1/2of the Control group. It had the highest tumor inhibition rate combined with bortezomib therapy with40%tumor volume of the Control group. Pathological histology examination showed reduced tumor cell heterogeneity and increased accumulation of ubiquitinated proteins after PSMD7RNAi and bortezomib treatment. The treatment groups have more apoptosis cells than the Control, in which the combined treatment group showed the best effect in apoptotic induction.
UNTclusion
PSMD7RNAi can decrease the proliferation, and induce apoptosis of esophageal squamous cancer cells. Also, PSMD7knockdown can effectively inhibit the growth of transplantable tumors in nude mice through inducing apoptosis of tumor cells and raising sensitivity of cancer cells to bortezomib, suggesting that it is likely an effective therapeutic target for esophageal squamous cell carcinoma.
纤毛和鞭毛是进化上非常保守的细胞器,结构基本相同,它们都来源于细胞质膜下的基体,向上突出于细胞表面。几乎人类所有类型的细胞都有纤毛,而鞭毛存在于低等真核生物,其基因组结构简单便于进行研究,其中鞭毛内运输机制最早就是在模式生物衣藻中发现的,随后在高等生物的纤毛中也被证实。纤毛/鞭毛的解聚和细胞周期以及外界信号有关,细胞增殖时纤毛发生解聚,基体转变为中心体参与纺锤体的形成,待细胞完成有丝分裂后,细胞又会长出新的纤毛。肿瘤的发生常常伴随着纤毛的异常,且纤毛内存在多个和肿瘤相关信号通路,但二者之间的分子机制尚不完全清楚。纤毛在解聚过程中会有大量的纤毛蛋白被降解,虽然泛素结合体系在纤毛和鞭毛中已被鉴定出来,但蛋白酶体是否参与纤毛/鞭毛的解聚,以及UPS能否通过影响纤毛/鞭毛的功能来参与肿瘤的发生发展未见报道。
然而纤毛长度较短不易被观察,且在体外培养容易丢失。鞭毛长度较长,通过简单的理化条件即可诱导其解聚。所以利用模式生物来进行研究可以帮助我们了解UPS和纤毛解聚之间的分子机制。杜氏盐藻和衣藻都为真核生物绿藻,二者的不同之处在于衣藻细胞外有一层细胞壁,其鞭毛上也有细胞壁的延伸,而盐藻则没有,在结构上和哺乳动物更加接近,故本研究选取盐藻的鞭毛作为模式生物器来研究UPS和鞭毛解聚之间的关系,为进一步研究纤毛在肿瘤发生发展中的分子机制提供依据。
蛋白酶体由20S核心颗粒(core particle, CP)和19S调节颗粒(regulatory particle, RP)组成。20S CP是蛋白酶体的降解部位,包含水解活性位点；19S RP作为多聚泛素化蛋白的受体,负责将蛋白底物去泛素化并将蛋白解折叠,并运送至20S CP。目前20SCP的抑制剂硼替佐米已经在临床上用于治疗多发性骨髓瘤和淋巴瘤,但易产生耐药性,且对实体瘤的治疗效果差。研究显示,19S RP中的一些去泛素化酶参与肿瘤的发生发展并可以作为潜在的肿瘤治疗靶点。
PSMD7是19S蛋白酶体lid中的一个去泛素化酶亚基,对19S RP结构组织的分析表明,在组成lid亚复合体的9个亚基中,PSMD7位于中央核心部位,同时和其他4个亚基相结合,说明PSMD7是19S蛋白酶体lid结构中的重要组成部分,但它在内的具体功能还不完全清楚。为了了解蛋白酶体在纤毛/鞭毛中的分子机制,并寻找肿瘤治疗的有效靶点,本课题从两个方面对PSMD7在胞内可能发挥的功能进行了研究：1.PSMD7是否参与纤毛/鞭毛的解聚?2.它是否可以影响肿瘤的发生发展?
第一部分蛋白酶体亚基PSMD7参与纤毛/鞭毛蛋白kinesin的降解
目的
了解鞭毛解聚过程中蛋白酶体活性的变化,以及PSMD7与纤毛/鞭毛蛋白之间的相互作用,初步研究PSMD7是否参与纤毛/鞭毛的解聚,为进一步研究UPS影响纤毛/鞭毛功能的分子机制打下基础。
方法
1泛素-蛋白酶体系统在鞭毛解聚后的激活状态
用免疫荧光和Western blotting方法检测所提取鞭毛中的泛素结合体系。用IBMX诱导鞭毛解聚后,观察鞭毛中泛素化蛋白的聚集情况；甘油密度梯度离心纯化细胞蛋白酶体,用荧光多肽底物检测蛋白酶体活性的变化；实时荧光定量PCR检测盐藻PSMD7(DsPSMD7)的表达变化。
2PSMD7和纤毛/鞭毛kinesin的结合
酵母双杂交技术分别检测盐藻中DsPSMD7和鞭毛kinesin(KCBP)及293T细胞中PSMD7(HsPSMD7)和纤毛kinesin(Kif3a)的相互作用,并进一步用GST pulldown和免疫共沉淀验证二者之间的相互作用。用蛋白酶体抑制剂分别处理盐藻和293T细胞,观察KCBP和Kif3a在胞内的降解情况。
结果
1泛素-蛋白酶体系统在鞭毛解聚后的激活状态
鞭毛发生解聚后,鞭毛内泛素化蛋白的量增多,胞内蛋白酶体的活性增加了将近2倍,说明泛素-蛋白酶体系统体参与了盐藻鞭毛解聚过程。DsPSMD7在鞭毛解聚过程中的mRNA水平表达量增高,在鞭毛解聚后30min表达量达到最高。
2PSMD7和纤毛／鞭毛kinesin的结合
酵母双杂交显示DsPSMD7和KCBP,HsPSMD7和Kif3a可以相互作用,且GST pulldown和免疫共沉淀进一步验证了二者之间的结合。用蛋白酶体抑制剂分别处理细胞后,KCBP和Kif3a两个kinesin在胞内的聚集增多,说明纤毛／鞭毛kinesin的降解由蛋白酶体参与。
结论
鞭毛解聚后蛋白酶体活性增强,同时PSMD7的表达量增加。纤毛或鞭毛kinesin都可以和PSMD7相互作用,并被蛋白酶体降解,说明PSMD7参与纤毛／鞭毛的解聚。
第二部分蛋白酶体亚基PSMD7在食管鳞癌发生中的作用
目的
鉴于去泛素化酶对肿瘤细胞的生存能力是必需的,我们探讨了PSMD7对蛋白酶体活性的影响,以及在食管鳞癌发生发展中的作用,为深入研究PSMD7在肿瘤细胞中作用的分子机制,以及UPS能否通过影响纤毛/鞭毛的功能来参与肿瘤的发生发展提供实验依据。
方法
1蛋白酶体在正常食管上皮细胞和食管鳞癌细胞中的表达差异
分别提取正常食管上皮细胞Het-1A和食管鳞癌细胞EC9706和Eca109的细胞核、细胞质和中心体,Western blotting检测PSMD7在各亚细胞结构中的表达。免疫荧光观察PSMD7在正常食管上皮细胞和食管鳞癌细胞中的定位。
2慢病毒介导的PSMD7基因干扰对食管鳞癌细胞增殖、周期和凋亡影响的体外研究
检测感染PSMD7RNAi慢病毒的EC9706细胞内泛素化蛋白的聚集情况和蛋白酶体活性的变化,分别用CCK-8实验和流式细胞仪检测细胞的增殖、周期和凋亡的变化。并用Western blotting检测PSMD7基因干扰后对p53表达的影响。同时将PSMD7RNAi慢病毒联合硼替佐米处理食管鳞癌细胞,观察细胞蛋白酶体功能和凋亡的变化。
3慢病毒介导的PSMD7基因干扰对移植瘤生长的影响
建立裸鼠移植瘤模型,观察PSMD7基因干扰后对食管鳞癌细胞EC9706的致瘤能力的影响。同时用硼替佐米对荷瘤裸鼠治疗两周,测量肿瘤大小以绘制生长曲线,并计算抑瘤率。病理组织学观察成瘤细胞的形态变化,免疫组化检测细胞泛素化蛋白的表达,TUNEL法分析不同处理组的裸鼠移植瘤组织细胞凋亡的变化。
结果
1蛋白酶体在正常食管上皮细胞和食管鳞癌细胞中的表达差异
PSMD7在两株食管鳞癌细胞EC9706和Eca109中的表达均高于正常食管上皮细胞Het-IA,在三种细胞的细胞核中的表达没有明显变化,但在食管鳞癌细胞质和中心体中的表达明显高于正常食管上皮细胞。
2慢病毒介导的PSMD7基因干扰对食管鳞癌细胞增殖、周期和凋亡影响的体外研究
PSMD7RNAi可以使EC9706胞内多泛素化蛋白增加,蛋白酶体的活性降低,抑制细胞的增殖,促进细胞的凋亡,还可以影响胞内p53的功能。此外PSMD7基因干扰后还可以增强硼替佐米对蛋白酶体功能的抑制和肿瘤细胞的凋亡。
3慢病毒介导的PSMD7基因干扰对移植瘤生长的影响
PSMD7基因干扰后的EC9706细胞在裸鼠体内生长缓慢,肿瘤体积只有对照的组的1/2。其联合蛋白酶体抑制剂硼替佐米治疗对肿瘤抑制率最高,肿瘤体积是对照组的40%,病理组织学检查发现成瘤细胞的异质性降低。免疫组化结果显示PSMD7基因干扰组、硼替佐米治疗组和联合治疗组细胞中的泛素化蛋白均增多,TUNEL显示治疗组的细胞都有不同程度的凋亡,其中联合治疗组凋亡的比例最大。
结论
PSMD7在食管鳞癌细胞中的高表达,且定位发生改变。PSMD7RNAi可以降低食管鳞癌细胞蛋白酶体的活性,使细胞增殖能力降低,并诱导细胞的凋亡,对肿瘤生长有明显的抑制作用,还可以增强食管鳞癌细胞对硼替佐米的敏感性。抑制PSMD7的表达能降低蛋白酶体的功能来诱导肿瘤细胞的凋亡,提示它可能是食管鳞癌的一个有效治疗靶点。
The ubiquitin proteasome system (UPS) is the major protein degradation system in eukaryotic cells and regulates a variety of cellular functions, including removal of damaged proteins, cell cycle regulation, apoptosis and signal transduction. Cancer cells depend on high proteasome activity to remove harmful immature and irregular proteins generated by rapid proliferation and to strength the anti-apoptosis ability. Studies have shown that the ubiquitin-UNTjunction system are present in flagella of model organism of algae Chlamydomonas reinhardtii and Dunaliella salina, and recently it has also been identified in the ciliome of Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Homo sapiens. So we infer that UPS may be involved in the biological function of cilia/flagella.
Cilia and flagella are UNTserved organelles during evolution with similar structure and origin from basal body protruding from cell surface. Flagella exist in lower eukaryotes whose genome structures are more simple and easy to study. The intraflagellar transport mechanism was first identified in the model organisms Chlamydomonas reinhardtii, and then was UNTfirmed in the cilia of higher organelle. Nearly all kinds of human cells have cilia. Cilia disassembly is closely related to cell-cycle Control, which releases the centrosome to form the mitotic spindle and cilia are regenerated after the completion of cell cycle. Tumor cells usually have abnormal cilia and there are multiple tumor-related signaling pathways in cilia, but the molecular mechanism between them is not clear. A large number of ciliary proteins will be degraded during disassembly. Although the ubiquitin UNTjunction system has been identified in cilia/flagella, whether proteasome is involved in the ciliary/flagellar disassembly and whether UPS could be involved in the tumorigenesis remains unclear.
However, cilia are hard to be observed and disappeared during cell culture in vitro. Flagella are longer and can be induce to disassembly by simple operation which could be used as model organelle to help us to better understand the molecular mechanism between UPS and ciliary disassembly. Dunaliella salina and Chlamydomonas reinhardtii are eukaryotic green algae with the difference that the former has a layer of cell wall but the latter not. The flagellar structure of Dunaliella salina is more similar with mammalian cilia. So the flagella of Dunaliella salina were used as a model organelle in this study to provide evidence for the research of cilia and tumorigenesis.
Based on both structure and function the proteasome can be further divided into two sub-complexes, a central20S proteolytic core particle (CP) that is capped at either end by one or two19S regulatory particles (19S RP). The20S CP is the degradation unit and UNTtains the active sites enquired to hydrolyze proteins into peptides. The19S RP Controls the degradation of ubiquitin-tagged substrates by acting as a receptor for polyubiquitinated proteins and facilitating their ATP-dependent translocation into the catalytic chamber of the20S CP. Inhibitors of20S CP bortezomib is used for treatment of multiple myeloma and lymphoma, but these cancer cells are easily to produce resistance to bortezomib. Besides, bortezomib has poor effect in solid tumor treatment. Several19S RP associated deubiquitinases have been shown to be involved in cancer progression and as such are emerging targets for anti-cancer therapies.
PSMD7is a deubiquitinase subunit in the lid of19S RP proteasome and occupies a central position within this subcomplex showed by structural organizational analysis. PSMD7is centered by other4subunits forming the heterotetrameric core structure, suggesting the essential role of PSMD7in the lid subcomplex of19S RP. However, the cellular function of it remains unknown. In this study, in order to provide an evidence for the molecular mechanism of proteasome in the ciliary/flagellar disassembly and to find therapy targets for cancer, we investigate the possible function of PSMD7:1. whether is it involved in the ciliary/flagellar disassembly?2. whether does it play a role in tumorigenesis?
Part1Proteasome subunit PSMD7is involved in the degradation of the ciliary/flagellar protein kinesin
Objective
The proteasome activity during flagellar disassembly and interactions of PSMD7with ciliary/flagellar proteins were observed to preliminary study whether PSMD7participated in ciliary/flagellar disassembly and provide basis for further research on the molecular mechanism of UPS in ciliary/flagellar function.
Methods
1The activity of ubiquitin-proteasome system after flagellar disassembly
Immunofluorescence and Western blotting methods were used to examine the ubiquitin-UNTjunction system in the isolated flagella. After flagellum disassembly induced by IBMX, ubiquitin protein aggregation was observed, the proteasome was purified by glycerol density gradient centrifugation and its activity was determined using fluorescence peptide substrate, and the mRNA expression of PSMD7of D.salina (DsPSMD7) was analyzed by real-time fluorescent quantitative PCR.
2The binding of PSMD7with ciliary/flagellar kinesin
Interactions of DsPSMD7with KCBP in D.salina cells and HsPSMD7with Kif3a in293T cells were detected by yeast two hybrid technology, which were further UNTfirmed by GST pulldown and co-immunoprecipitation. The degradations of KCBP and Kif3a by proteasome were determined by proteasome inhibitor treatment.
Results
1The activity of ubiquitin-proteasome system after flagellar disassembly
After flagellar disassembly, ubiquitinated proteins were accumulated and the proteasome activity was increased nearly2times, indicating that the ubiquitin-proteasome system is involved in the flagellar disassembly of D.salina. The mRNA level of DsPSMD7was upregulated during flagella disassembly, and reached the highest at30min after disassembly.
2The binding of PSMD7with ciliary/flagellar kinesin
Yeast two hybrid showed that DsPSMD7interacted with KCBP and HsPSMD7interacted with Kif3a, which were UNTfirmed by GST pulldown and co-immunoprecipitation. Two kinesin, KCBP and Kif3a became accumulated in cells after proteasome inhibitor treatment, suggesting that these cilia/flagella kinesin were degraded by proteasome.
UNTclusion
Expression level of PSMD7, which mediates degradation of the ciliary or flagellar kinesin, is elevated during flagellar disassembly suggesting that PSMD7may participate in ciliary or flagellar disassembly.
Part2Functional study of proteasome subunit PSMD7in esophageal squamous cell carcinomas
Objective
1Differential expression of proteasome between normal esophageal epithelium cells and esophageal squamous cancer cells
The nucleus, cytoplasm and centrosome were respectively extracted from normal esophageal epithelium and esophageal squamous cancer cells. The translational expressions of PSMD7were detected in various subcellular structures by Western blotting. The localizations of PSMD7were observed by immunofluorescence.
2Effect of PSMD7gene knockdown mediated by lentiviral mediated RNAi on the cell proliferation, cycle and apoptosis of esophageal squamous cancer cells
The accumulation of ubiquitinated proteins and proteasome activity were examined in EC9706cells infected by PSMD7RNAi lentiviral particles. Cell proliferation was analyzed by CCK-8, cell cycle and apoptosis were tested by flow cytometry instrument. The p53level was inspected after PSMD7gene knockdown using Western blotting. The proteasome function and apoptosis of EC9706cells were investigated after PSMD7RNAi lentiviral particles and bortezomib treatment.
3The inhibition of xenografted tumor cell growth by PSMD7gene knockdown mediated by lentiviral mediated RNAi
Xenograft tumor nude mice models were development to explore the tumorigenic capacity of EC9706cells with PSMD7gene knockdown. At the same time, xenograft tumor nude mice were treated by bortezomib for two weeks.The tumor volume was measured and the tumor growth curves were drawn and tumor inhibition rates were calculated. The pathological morphology of tumor cell was observed, the accumulation of ubiquitinated proteins in the tumor cells were surveyed by immunohistochemical,and tumor cell apoptosis in tumor tissues were analyzed by TUNEL method.
Results
1Differential expression of proteasome between normal esophageal epithelium cells and esophageal squamous cancer cells
The expression of PSMD7in two of esophageal squamous cancer cells, EC9706and Eca109, were higher than that in normal esophageal epithelium. There was no obvious change in the expressed of it in the nucleus of these three cells, but the esophageal squamous cancer cells had significantly higher expression in the cytoplasm and centrosome than normal esophageal epithelial cells.
2Effect of PSMD7gene knockdown mediated by lentiviral mediated RNAi on the cell proliferation, cycle and apoptosis of esophageal squamous cancer cells
The ubiquitinated proteins were accumulated and the proteasome activity was reduced in EC9706cells after the PSMD7gene knockdown by lentiviral mediated RNAi. PSMD7RNAi also inhibited the proliferation, promoted the apoptosis and affected the function of intracellular p53in EC9706cells. Moreover, the proteasomal functional inhibition and apoptosis induced by bortezomib were enhanced by PSMD7RNAi.
3The inhibition of xenografted tumor cell growth by PSMD7gene knockdown mediated by lentiviral mediated RNAi
PSMD7RNAi EC9706cells showed lower growth rate in nude mice and the tumor volume of it is only the1/2of the Control group. It had the highest tumor inhibition rate combined with bortezomib therapy with40%tumor volume of the Control group. Pathological histology examination showed reduced tumor cell heterogeneity and increased accumulation of ubiquitinated proteins after PSMD7RNAi and bortezomib treatment. The treatment groups have more apoptosis cells than the Control, in which the combined treatment group showed the best effect in apoptotic induction.
UNTclusion
PSMD7RNAi can decrease the proliferation, and induce apoptosis of esophageal squamous cancer cells. Also, PSMD7knockdown can effectively inhibit the growth of transplantable tumors in nude mice through inducing apoptosis of tumor cells and raising sensitivity of cancer cells to bortezomib, suggesting that it is likely an effective therapeutic target for esophageal squamous cell carcinoma.
引文
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