肿瘤相关蛋白RalB和STARD7对肝癌细胞生物学影响的实验研究
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摘要
肝癌是一种严重威胁人体健康的恶性肿瘤,其发病率在所有恶性肿瘤中据第3位。肝癌在东南亚地区具有较高的发病率,中国广东、台湾两地是肝癌的高发地区。肝癌的发病机制目前仍不是特别清楚,一般认为与病毒侵袭、化学污染物的接触、环境及饮食习惯、遗传易感性及其他方面的因素有关。目前临床对于肝癌的治疗方法以手术治疗为主,配合放疗和化疗,但是由于肝癌诊断较为困难,发现时多为晚期,因此治疗效果并不十分显著。恶性肿瘤的基因诊断与治疗是近年来研究和开发的热点,同时发现一些相对于恶性肿瘤的诊断及治疗靶标,但是由于肝脏器官体积较大、功能复杂,因此迄今为止,还并没有发现针对于肝癌的诊断及治疗靶标。
RalB是小G蛋白Ral(Ras like)家族的成员之一,Ral家族由RalA和RalB组成。Ral是Ras超家族的一个成员,具有参与介导许多不同细胞外信号活化的潜能。Ral蛋白与Ras具有55%的氨基酸同源性,其作为辅助因子并活化,从而参与肿瘤基因Ras诱导的细胞转化,Ral通过相应的信号通路和不同的效应因子,引起基因转录的改变从而影响细胞形态的改变,进而影响细胞的转化。小G蛋白Ral家族参与肿瘤的发生、侵袭和转移,但对于Ral家族成员RalA和RalB的各自生物学功能并未完全研究清楚。RalB对于维持肿瘤细胞的存活具有特异性的作用,RalB还能够抑制不同肿瘤细胞的程序性死亡,RalA的生物学功能主要在于促进不同的肿瘤细胞锚定非依赖性增殖,推测RalA和RalB可能具有协同参与肿瘤转化、介导肿瘤细胞增殖和存活信号。同时,RalB能够增强肿瘤细胞的活力,据有促进不同肿瘤细胞迁移的生物学作用。
SATRD7是一编码295个氨基酸残基,分子量为34.7kDa的新型蛋白质,其生物学功能至今未明。通过生物信息学分析,发现其全长序列中含有一个类固醇敏感性调节蛋白(StAR)相关脂类转运体结构域(START)。START涉及脂类结合介导的细胞信号转导、脂类转运和调节。START发生突变或错误表达与基因错配、自身免疫性疾病以及肿瘤相关。对于SATRD7的研究,目前限于对其在mRNA水平上的研究。研究发现SATRD7在绒毛膜癌细胞,结肠直肠癌细胞和肝癌细胞中高表达。通过计算机的同源性检测发现推断的蛋白质与磷酸卵磷酯转移蛋白(PCTP)具有同源性,提示SATRD7参与磷脂介导的肿瘤信号活动。
目前虽然对于RalB和SATRD7通过相应的实验研究,对其生物学功能有了一定方面的了解,但是相对RalB和SATRD7在肝癌方面的研究并未见相应的报道,因此,我们设计了一系列的实验,通过RalB和SATRD7对肝癌细胞生物学特性,如肿瘤细胞的增殖、存活、侵袭和转移的影响的研究,对于RalB和SATRD7的功能有进一步的了解,从而为未来将其作为肿瘤生物治疗靶标的可能性提供理论依据。
方法:首先,采用Western-blot技术对RalB和STARD7在不同的肝癌细胞和其他肿瘤细胞表达进行鉴定。同时,采用免疫组织化学技术、免疫细胞化学技术对RalB和STARD7分别进行组织和亚细胞定位。对于RalB和STARD7分别采用肝组织细胞器分离技术和激光共聚焦技术进一步鉴定其在组织和细胞中的定位。
其次,采用RT-PCR技术从293T细胞中扩增出RalB和STARD7目的基因。将目的基因通过转化连接到pMD18-T载体上,提取含有目的基因的质粒,经PCR、双酶切、测序鉴定后,将经双酶切的目的基因与真核载体pcDNA3.1(-)和pDsRed_1-C3连接,经转化得到含有RalB和STARD7目的基因的重组质粒,后经双酶切重组质粒,测序正确后将pcDNA3.1(-)/RalB转染至SMMC7721细胞,将pcDNA3.1(-)/STARD7转染至HepG2细胞,并将转染后的细胞采用G418筛选出稳定表达株,用于进一步的实验。
再次,采用MTT法分别检测稳定转染pcDNA3.1(-)/RalB、转染pcDNA3.1(-)空载体的SMMC7721的细胞,转染pcDNA3.1(-)/STARD7、转染pcDNA3.1(-)空载体的HepG2细胞的生长状态;通过饥饿实验分析高表达RalB对于肿瘤细胞存活状态的影响;通过流式细胞仪分析分析高表达RalB对肿瘤细胞凋亡的影响;通过Transwell方法检测高表达RalB对促进肝癌细胞侵袭转移能力的影响;
最后,通过凝胶迁移实验分析高表达RalB对NF-κB的激活能力,以及从mRNA水平检测转染RalB基因对于细胞周期蛋白B_1、D_1、E_1、E_2以及基质金属蛋白酶MMP2表达变化的影响,从而初步探讨RalB对于肝癌细胞生长及转移的作用机制的影响。
结果:
1.通过Western-blot技术检测了RalB和STARD7在Chang、Bel7402、QGY7703、SMMC7721和HepG2细胞中的表达,结果显示同正常肝细胞Chang比较,RalB在Bel7402和SMMC7721中表达增高,其中在SMMC7721中增高明显,而在QGY7703和HepG2中表达并不明显;STARD7在Bel7402、QGY7703和HepG2中表达增高,在SMMC7721中不表达。同时检测了RalB在其他肿瘤细胞中的表达,发现RalB在不同肿瘤细胞中有不同程度的高表达。
2.通过免疫组织化学实验检测了RalB和STARD7在人肝癌组织中的表达,结果表明同正常肝组织相比,RalB在肝癌组织的细胞膜上表达,同时在细胞核中有散在的表达;STARD7主要在肝癌组织的胞浆中表达。通过免疫细胞化学技术和肝组织细胞器分离技术,显示RalB主要在细胞膜和细胞核中表达。免疫细胞化学技术和激光共聚焦显示STARD7在细胞浆中表达,并在核内有散在的表达。
3.成功构建真核表达载体pcDNA3.1(-)/RalB、pcDNA3.1(-)/STARD7,以及带有红色荧光标记的融合表达真核表达载体pDsRed_1-C3/RalB,pDsRed_1-C3/STARD7。
4.用脂质体转染法将重组质粒pcDNA3.1(-)/RalB转染至肝癌细胞SMMC7721;将重组质粒pcDNA3.1(-)/STARD7转染至肝癌细胞HepG2。采用RT-PCR及Western—Blot法检测,经G418筛选获得的稳定转染细胞,分别从mRNA及蛋白水平鉴定,同对照组比较,结果表明转染细胞中RalB和STARD7的表达量增高,提示成功构建稳定表达RalB和STARD7的细胞株。
5.用同样方法将pDsRed_1-C3/RalB和pDsRed_1-C3/STARD7真核表达质粒转染入293T和肾癌786-0细胞,通过观察发现RalB集中表达于细胞核和细胞膜;STARD7则定位于胞浆中。
6.采用MTT法检测pcDNA3.1(-)/RalB稳定转染的SMMC7721细胞,同转染pcDNA3.1(-)空载体的SMMC7721细胞的生长特性比较;pcDNA3.1(-)/STARD7稳定转染的HepG2细胞,同转染pcDNA3.1(-)空载体的HepG2细胞的生长特性比较,结果表明RalB的过表达能在细胞生长的早期促进肝癌细胞的增殖,而过表达STARD7对于肝癌细胞的生长并无明显的促进作用。
7.Transwell实验显示同转染pcDNA3.1(-)空载体的SMMC7721细胞相比,过表达RalB的SMMC7721细胞的侵袭转移水平有较为明显增高,RalB可增强肝癌细胞的侵袭转移能力。
8.采用无血清培养收集原点、24h、36h、48h的pcDNA3.1(-)/RalB的SMMC7721细胞,经流式细胞仪分析结果发现,同pcDNA3.1(-)空载体的SMMC7721细胞比较发现,过表达RalB可抑制SMMC7721细胞的凋亡;同时,用抗RalB的单克隆抗体,通过Western—Blot法检测无血清和正常血清培养的肝癌细胞中RalB的表达,发现无血清培养的肝癌细胞中RalB的表达明显高于正常血清培养的肝癌细胞,提示RalB可以抑制肝癌细胞的凋亡,维持肝癌细胞的存活。
9.凝胶迁移实验观察到转染RalB基因的SMMC7721细胞核蛋白出现了特异的迁移带,说明RalB能够在肝癌细胞中活化NF—κB,从而产生对于肝癌细胞的生物学作用。
10.通过RT-PCR检测,发现转染RalB基因的SMMC7721细胞中的MMP2同pcDNA3.1(-)空载体比较,表达水平升高。提示RalB基因对肝癌细胞转移特性的影响可能与通过促进MMP2的表达有关。
结论:过表达RalB可促进SMMC7721肝癌细胞的早期增殖、促进肝癌细胞的侵袭转移、抑制肝癌细胞的凋亡、维持肝癌细胞的存活,实验显示RalB可能是通过激活NF—κB,从而调节MMP2的表达,以此促进肝癌细胞的转移。SATRD7基因对于肝癌细胞HepG2无明显的增殖作用,需进一步进行实验研究。
Introduction:Hepatocellular carcinoma is a kind of malignant tumor which threatens the health of human beings.With the third morbidity of all malignant tumors,hepatoma has a high morbidity in Southeast areas of Asia,especially in the Guang dong and Taiwan provinces of China.It is unknown for the pathogenesis of hepatoma,but generally researchers believed that it is associated with virus infection, chemical pollution,environmental factors,dietary factors,genetic predisposition,and so on.The therapy for hepatoma depends on the clinical operation matched with radiotherapy and chemotherapy at present.But it is difficult for the diagnosis of hepatoma,and also to be found in the final period,so therapeutic efficacy is not satisfied.It is extremely hot for the research and development to genetic diagnosis and therapy of malignant tumors,and some targets for diagnosis and therapy had been found recently.Because the complicated functions of liver,no target of diagnosis and therapy treated has been detected until now.
RalB is one member of,small G protein,Ral family which constituted by RalB and RalA.With the potential to mediate the action of many extracellular signals,Ral belongs to the superfamily of Ras,also displaying 55%amino acid sequence identity. As the help-factor to be actived,Ral can participate the cellular transformation induced by oncogenic Ras.Ral may influence cell growth by altering gene transcription throuth different signal cascade way and effect factors,and so result in oncogenic transformation through changes in cell shape.Ral can participate the oncogenic transformation,invasion and metastasis,but it is elusive for the biological functions of RalA and RalB.RalB is required for the survival of tumor cells but not normal cells,and also prevent the programmed cell death of different tumor cells.But the function of RalA is focused on the anchorage independent proliferation.It is supposed that RalA and RalB have the ability to participate oncogenic transformation, and to mediate the proliferation and survival signals of tumor cells.Meanwhile,RalB may increase the survival ability of tumor cells,and promote the migration of different cells.
SATRD7 is a new protein which encodes 295 amino acid residues with a molecular weight of approximately 34.7kDa.It is unknown for the biological function of SATRD7,but there is a steroidogenic acute regulatory protein(StAR)-related lipid transfer(START) domain in its total sequence.START is often present in the transportation and regulation of liquid,and even in the cell signaling mediated by lipid binding.Mutation or misexpression of START proteins is linked to genetic disorders,autoimmune disease and cancer.The reserchment for SATRD7 is just on the mRNA level.It is dedicated that SATRD7 has high expression in choriocarcinoma cells,colorectal adenocarcinoma cell and hepatocellular carcinoma.It has been fount that SATRD7 share some identical area to PCTP,which means SATRD7 play a role in the phospholipid-mediated signal of trophoblastic tumout cellular events.
Although some researches for the function of RalB and SATRD7 have been tested,there is no report for RalB and SATRD7 on Hepatocellular carcinoma.So we design a series of experiments to explore the bionomics of RalB and SATRD7,for example,the influence to the proliferation,survival,invation and metastasis.We can provide scientfic data for RalB and SATRD7 as target of diagnosis and therapy in the future.
Methods:Fristly,the expression of RalB and STARD7 was identified in different tumor cells by Western-blot.And RalB and STARD7 were localized in tissue and cell by immunohistochemistry and immunocytochemical technique. Separation and fractionation of organelles of human liver was used as a method to detect the localization of RalB in tissue,and also laser copolymerization technique is for STARD7 in cells furtherlly.
Secondly,RalB and STARD7 were extracted from 293T cell line.And then the genes were connected to the pMD18-T vector,extracting the plasmids with genes,for PCR,double enzyme digesting and sequencing,to connect with eckaryotic vector pcDNA3.1(-) and pDsRed_1-C3.We got the plasmids transformed with RalB and STARD7 which had been sequencing.And then pcDNA3.1(-)/RalB was transfected into SMMC7721 cells,pcDNA3.1(-)/STARD7 was into HepG2 cell.The transfected cells were bolted by G418 for the stable expressing line.
Thirdly,MTT assay was used for the station of cell growing in pcDNA3.1(-)/RalB,pcDNA3.1(-) of SMMC7721 cell transfected,and even for the pcDNA3.1(-)/STARD7 of HepG2 cell line.The starvation assay was used to detected the influence of high-expression of RalB to the survival station of tumor cell lines.Flow cytometry was for the influence of high-expression of RalB to the apoptosis of tumor cells,and Transwells was for the contribution of high-expression of RalB for the metastasis ability of tumor cells.
Finally,EMSA was used to analyze the role of high-expression of RalB to the transcription factor of NF-κB.And RalB was detected to influence the expression of cyclins and MMP2,so we may approach the mechanism of action of RalB to the growth and metastasis of tumor lines of liver.
Results:
1.The expression of RalB and STARD7 in normal and tumor hepatocytes explored by Western-blot.The result showed that RalB is high-expressed in Bel7402 and SMMC7721,especially in SMMC7721,compared with the normal hepatocyte Chang.STARD7 is high-expressed in Bel7402,QGY7703 and HepG2,but failed to express in SMMC7721.We also detected RalB expressed in other tumor lines.As a result,RalB is high expressed in different levels in tumor cells,compared with the control group.
2.The identification of RalB and STARD7 in human liver cancer tissue detected by immunohistochemistry.The result indicated RalB was expressed at plasma membrane,and also found in nucleus.STARD7 was found endochylema in tumor tissue.RalB was identified in plasma membrane and nucleus by the separation and fractionation of organelles and immunocytochemical technique.STARD7 was also shown in cytoplasm and nucleus scattered by laser copolymerization technique.
3.pcDNA3.1(-)/RalB and pcDNA3.1(-)/STARD7 were constructed successfully, and also for the pDsRed_1-C3/RalB and pDsRed_1-C3/STARD7.
4.Recombinant plasmid of pcDNA3.1(-)/RalB was transfected into SMMC7721 by liposome transfection assay,pcDNA3.1(-)/STARD7 was transfected into HepG2 cell line.The stalbe transfected cell line was analyzed by RT-PCR and Western-blot. The result showed that the expression of RalB and STARD7 transfected is higher than that of control group.
5.The eukaryotic plasmid,pDsRed_1-C3/RalB and pDsRed_1-C3/STARD7,was transfected in the same method,and the result dedicated that RalB was expression in plasma membrane and nucleus,but STARD7 was found in cytoplasm.
6.MTT was used to explore the growth character of SMMC7721 transfected with PcDNA3.1(-)/RalB.The result showed that RalB overexpressed can promote the proliferation in the early period of the cell growth.But STARD7 overexpressed could not promote the growth of HepG2 transfected with STARD7.
7.Compared with the control group,the result of Transwell showed that the ability to invation and metastasis has been promoted in SMMC7721 cell transfected with RalB.
8.After Starvation assay,we incubated SMMC7721 transfected with RalB in the free medium.And then we collect cells in four time point,analyzed by flow cytometry,we fount over-expressed RalB can prevent the tumor cell from apoptosis. Meantime,we measured the expression of RalB in the tumor cell cultured with free serum and normal serum.The result dedicated that the expression of RalB with free serum is higher than that with normal serum.So we conclude that RalB can prevent the malignant hepatocyte from apoptosis and sustain the survival of tumor cell.
9.The special bane has been observed in the nucleic protein of SMMC7721 transfected with RalB,as a result,it is hinted RalB could activate the transcription factor of NF-κB in SMMC7721.The result hinted that NF-κB activated by RalB may generate the effect which can result in the biological change of tumor cells.
10.Compared with the control group,the MMP2 in the cell transfected with RalB has a high expression level.The result may mean the influence on the metastasis of hepatoma carcinoma cell by RalB is related to the expression of MMP2.
Conclusion:Over-expression of RalB may promote the proliferation of hepatoma carcinoma cell,SMMC7721,in the early period,and increases the ability of invation and metastasis in HCCs.RalB may also prevent HCCs from apoptosis and maintain the survival of tumor cell.We dedicated that RalB may result in NF-κB to be actived,as a result to promote the expression of MMP2,and further mediate the effect of metastasis for hepatoma carcinoma cell.But for STARD7,there is no apparent proliferation in HepG2,so further experiment is required.
RalB是小G蛋白Ral(Ras like)家族的成员之一,Ral家族由RalA和RalB组成。Ral是Ras超家族的一个成员,具有参与介导许多不同细胞外信号活化的潜能。Ral蛋白与Ras具有55%的氨基酸同源性,其作为辅助因子并活化,从而参与肿瘤基因Ras诱导的细胞转化,Ral通过相应的信号通路和不同的效应因子,引起基因转录的改变从而影响细胞形态的改变,进而影响细胞的转化。小G蛋白Ral家族参与肿瘤的发生、侵袭和转移,但对于Ral家族成员RalA和RalB的各自生物学功能并未完全研究清楚。RalB对于维持肿瘤细胞的存活具有特异性的作用,RalB还能够抑制不同肿瘤细胞的程序性死亡,RalA的生物学功能主要在于促进不同的肿瘤细胞锚定非依赖性增殖,推测RalA和RalB可能具有协同参与肿瘤转化、介导肿瘤细胞增殖和存活信号。同时,RalB能够增强肿瘤细胞的活力,据有促进不同肿瘤细胞迁移的生物学作用。
SATRD7是一编码295个氨基酸残基,分子量为34.7kDa的新型蛋白质,其生物学功能至今未明。通过生物信息学分析,发现其全长序列中含有一个类固醇敏感性调节蛋白(StAR)相关脂类转运体结构域(START)。START涉及脂类结合介导的细胞信号转导、脂类转运和调节。START发生突变或错误表达与基因错配、自身免疫性疾病以及肿瘤相关。对于SATRD7的研究,目前限于对其在mRNA水平上的研究。研究发现SATRD7在绒毛膜癌细胞,结肠直肠癌细胞和肝癌细胞中高表达。通过计算机的同源性检测发现推断的蛋白质与磷酸卵磷酯转移蛋白(PCTP)具有同源性,提示SATRD7参与磷脂介导的肿瘤信号活动。
目前虽然对于RalB和SATRD7通过相应的实验研究,对其生物学功能有了一定方面的了解,但是相对RalB和SATRD7在肝癌方面的研究并未见相应的报道,因此,我们设计了一系列的实验,通过RalB和SATRD7对肝癌细胞生物学特性,如肿瘤细胞的增殖、存活、侵袭和转移的影响的研究,对于RalB和SATRD7的功能有进一步的了解,从而为未来将其作为肿瘤生物治疗靶标的可能性提供理论依据。
方法:首先,采用Western-blot技术对RalB和STARD7在不同的肝癌细胞和其他肿瘤细胞表达进行鉴定。同时,采用免疫组织化学技术、免疫细胞化学技术对RalB和STARD7分别进行组织和亚细胞定位。对于RalB和STARD7分别采用肝组织细胞器分离技术和激光共聚焦技术进一步鉴定其在组织和细胞中的定位。
其次,采用RT-PCR技术从293T细胞中扩增出RalB和STARD7目的基因。将目的基因通过转化连接到pMD18-T载体上,提取含有目的基因的质粒,经PCR、双酶切、测序鉴定后,将经双酶切的目的基因与真核载体pcDNA3.1(-)和pDsRed_1-C3连接,经转化得到含有RalB和STARD7目的基因的重组质粒,后经双酶切重组质粒,测序正确后将pcDNA3.1(-)/RalB转染至SMMC7721细胞,将pcDNA3.1(-)/STARD7转染至HepG2细胞,并将转染后的细胞采用G418筛选出稳定表达株,用于进一步的实验。
再次,采用MTT法分别检测稳定转染pcDNA3.1(-)/RalB、转染pcDNA3.1(-)空载体的SMMC7721的细胞,转染pcDNA3.1(-)/STARD7、转染pcDNA3.1(-)空载体的HepG2细胞的生长状态;通过饥饿实验分析高表达RalB对于肿瘤细胞存活状态的影响;通过流式细胞仪分析分析高表达RalB对肿瘤细胞凋亡的影响;通过Transwell方法检测高表达RalB对促进肝癌细胞侵袭转移能力的影响;
最后,通过凝胶迁移实验分析高表达RalB对NF-κB的激活能力,以及从mRNA水平检测转染RalB基因对于细胞周期蛋白B_1、D_1、E_1、E_2以及基质金属蛋白酶MMP2表达变化的影响,从而初步探讨RalB对于肝癌细胞生长及转移的作用机制的影响。
结果:
1.通过Western-blot技术检测了RalB和STARD7在Chang、Bel7402、QGY7703、SMMC7721和HepG2细胞中的表达,结果显示同正常肝细胞Chang比较,RalB在Bel7402和SMMC7721中表达增高,其中在SMMC7721中增高明显,而在QGY7703和HepG2中表达并不明显;STARD7在Bel7402、QGY7703和HepG2中表达增高,在SMMC7721中不表达。同时检测了RalB在其他肿瘤细胞中的表达,发现RalB在不同肿瘤细胞中有不同程度的高表达。
2.通过免疫组织化学实验检测了RalB和STARD7在人肝癌组织中的表达,结果表明同正常肝组织相比,RalB在肝癌组织的细胞膜上表达,同时在细胞核中有散在的表达;STARD7主要在肝癌组织的胞浆中表达。通过免疫细胞化学技术和肝组织细胞器分离技术,显示RalB主要在细胞膜和细胞核中表达。免疫细胞化学技术和激光共聚焦显示STARD7在细胞浆中表达,并在核内有散在的表达。
3.成功构建真核表达载体pcDNA3.1(-)/RalB、pcDNA3.1(-)/STARD7,以及带有红色荧光标记的融合表达真核表达载体pDsRed_1-C3/RalB,pDsRed_1-C3/STARD7。
4.用脂质体转染法将重组质粒pcDNA3.1(-)/RalB转染至肝癌细胞SMMC7721;将重组质粒pcDNA3.1(-)/STARD7转染至肝癌细胞HepG2。采用RT-PCR及Western—Blot法检测,经G418筛选获得的稳定转染细胞,分别从mRNA及蛋白水平鉴定,同对照组比较,结果表明转染细胞中RalB和STARD7的表达量增高,提示成功构建稳定表达RalB和STARD7的细胞株。
5.用同样方法将pDsRed_1-C3/RalB和pDsRed_1-C3/STARD7真核表达质粒转染入293T和肾癌786-0细胞,通过观察发现RalB集中表达于细胞核和细胞膜;STARD7则定位于胞浆中。
6.采用MTT法检测pcDNA3.1(-)/RalB稳定转染的SMMC7721细胞,同转染pcDNA3.1(-)空载体的SMMC7721细胞的生长特性比较;pcDNA3.1(-)/STARD7稳定转染的HepG2细胞,同转染pcDNA3.1(-)空载体的HepG2细胞的生长特性比较,结果表明RalB的过表达能在细胞生长的早期促进肝癌细胞的增殖,而过表达STARD7对于肝癌细胞的生长并无明显的促进作用。
7.Transwell实验显示同转染pcDNA3.1(-)空载体的SMMC7721细胞相比,过表达RalB的SMMC7721细胞的侵袭转移水平有较为明显增高,RalB可增强肝癌细胞的侵袭转移能力。
8.采用无血清培养收集原点、24h、36h、48h的pcDNA3.1(-)/RalB的SMMC7721细胞,经流式细胞仪分析结果发现,同pcDNA3.1(-)空载体的SMMC7721细胞比较发现,过表达RalB可抑制SMMC7721细胞的凋亡;同时,用抗RalB的单克隆抗体,通过Western—Blot法检测无血清和正常血清培养的肝癌细胞中RalB的表达,发现无血清培养的肝癌细胞中RalB的表达明显高于正常血清培养的肝癌细胞,提示RalB可以抑制肝癌细胞的凋亡,维持肝癌细胞的存活。
9.凝胶迁移实验观察到转染RalB基因的SMMC7721细胞核蛋白出现了特异的迁移带,说明RalB能够在肝癌细胞中活化NF—κB,从而产生对于肝癌细胞的生物学作用。
10.通过RT-PCR检测,发现转染RalB基因的SMMC7721细胞中的MMP2同pcDNA3.1(-)空载体比较,表达水平升高。提示RalB基因对肝癌细胞转移特性的影响可能与通过促进MMP2的表达有关。
结论:过表达RalB可促进SMMC7721肝癌细胞的早期增殖、促进肝癌细胞的侵袭转移、抑制肝癌细胞的凋亡、维持肝癌细胞的存活,实验显示RalB可能是通过激活NF—κB,从而调节MMP2的表达,以此促进肝癌细胞的转移。SATRD7基因对于肝癌细胞HepG2无明显的增殖作用,需进一步进行实验研究。
Introduction:Hepatocellular carcinoma is a kind of malignant tumor which threatens the health of human beings.With the third morbidity of all malignant tumors,hepatoma has a high morbidity in Southeast areas of Asia,especially in the Guang dong and Taiwan provinces of China.It is unknown for the pathogenesis of hepatoma,but generally researchers believed that it is associated with virus infection, chemical pollution,environmental factors,dietary factors,genetic predisposition,and so on.The therapy for hepatoma depends on the clinical operation matched with radiotherapy and chemotherapy at present.But it is difficult for the diagnosis of hepatoma,and also to be found in the final period,so therapeutic efficacy is not satisfied.It is extremely hot for the research and development to genetic diagnosis and therapy of malignant tumors,and some targets for diagnosis and therapy had been found recently.Because the complicated functions of liver,no target of diagnosis and therapy treated has been detected until now.
RalB is one member of,small G protein,Ral family which constituted by RalB and RalA.With the potential to mediate the action of many extracellular signals,Ral belongs to the superfamily of Ras,also displaying 55%amino acid sequence identity. As the help-factor to be actived,Ral can participate the cellular transformation induced by oncogenic Ras.Ral may influence cell growth by altering gene transcription throuth different signal cascade way and effect factors,and so result in oncogenic transformation through changes in cell shape.Ral can participate the oncogenic transformation,invasion and metastasis,but it is elusive for the biological functions of RalA and RalB.RalB is required for the survival of tumor cells but not normal cells,and also prevent the programmed cell death of different tumor cells.But the function of RalA is focused on the anchorage independent proliferation.It is supposed that RalA and RalB have the ability to participate oncogenic transformation, and to mediate the proliferation and survival signals of tumor cells.Meanwhile,RalB may increase the survival ability of tumor cells,and promote the migration of different cells.
SATRD7 is a new protein which encodes 295 amino acid residues with a molecular weight of approximately 34.7kDa.It is unknown for the biological function of SATRD7,but there is a steroidogenic acute regulatory protein(StAR)-related lipid transfer(START) domain in its total sequence.START is often present in the transportation and regulation of liquid,and even in the cell signaling mediated by lipid binding.Mutation or misexpression of START proteins is linked to genetic disorders,autoimmune disease and cancer.The reserchment for SATRD7 is just on the mRNA level.It is dedicated that SATRD7 has high expression in choriocarcinoma cells,colorectal adenocarcinoma cell and hepatocellular carcinoma.It has been fount that SATRD7 share some identical area to PCTP,which means SATRD7 play a role in the phospholipid-mediated signal of trophoblastic tumout cellular events.
Although some researches for the function of RalB and SATRD7 have been tested,there is no report for RalB and SATRD7 on Hepatocellular carcinoma.So we design a series of experiments to explore the bionomics of RalB and SATRD7,for example,the influence to the proliferation,survival,invation and metastasis.We can provide scientfic data for RalB and SATRD7 as target of diagnosis and therapy in the future.
Methods:Fristly,the expression of RalB and STARD7 was identified in different tumor cells by Western-blot.And RalB and STARD7 were localized in tissue and cell by immunohistochemistry and immunocytochemical technique. Separation and fractionation of organelles of human liver was used as a method to detect the localization of RalB in tissue,and also laser copolymerization technique is for STARD7 in cells furtherlly.
Secondly,RalB and STARD7 were extracted from 293T cell line.And then the genes were connected to the pMD18-T vector,extracting the plasmids with genes,for PCR,double enzyme digesting and sequencing,to connect with eckaryotic vector pcDNA3.1(-) and pDsRed_1-C3.We got the plasmids transformed with RalB and STARD7 which had been sequencing.And then pcDNA3.1(-)/RalB was transfected into SMMC7721 cells,pcDNA3.1(-)/STARD7 was into HepG2 cell.The transfected cells were bolted by G418 for the stable expressing line.
Thirdly,MTT assay was used for the station of cell growing in pcDNA3.1(-)/RalB,pcDNA3.1(-) of SMMC7721 cell transfected,and even for the pcDNA3.1(-)/STARD7 of HepG2 cell line.The starvation assay was used to detected the influence of high-expression of RalB to the survival station of tumor cell lines.Flow cytometry was for the influence of high-expression of RalB to the apoptosis of tumor cells,and Transwells was for the contribution of high-expression of RalB for the metastasis ability of tumor cells.
Finally,EMSA was used to analyze the role of high-expression of RalB to the transcription factor of NF-κB.And RalB was detected to influence the expression of cyclins and MMP2,so we may approach the mechanism of action of RalB to the growth and metastasis of tumor lines of liver.
Results:
1.The expression of RalB and STARD7 in normal and tumor hepatocytes explored by Western-blot.The result showed that RalB is high-expressed in Bel7402 and SMMC7721,especially in SMMC7721,compared with the normal hepatocyte Chang.STARD7 is high-expressed in Bel7402,QGY7703 and HepG2,but failed to express in SMMC7721.We also detected RalB expressed in other tumor lines.As a result,RalB is high expressed in different levels in tumor cells,compared with the control group.
2.The identification of RalB and STARD7 in human liver cancer tissue detected by immunohistochemistry.The result indicated RalB was expressed at plasma membrane,and also found in nucleus.STARD7 was found endochylema in tumor tissue.RalB was identified in plasma membrane and nucleus by the separation and fractionation of organelles and immunocytochemical technique.STARD7 was also shown in cytoplasm and nucleus scattered by laser copolymerization technique.
3.pcDNA3.1(-)/RalB and pcDNA3.1(-)/STARD7 were constructed successfully, and also for the pDsRed_1-C3/RalB and pDsRed_1-C3/STARD7.
4.Recombinant plasmid of pcDNA3.1(-)/RalB was transfected into SMMC7721 by liposome transfection assay,pcDNA3.1(-)/STARD7 was transfected into HepG2 cell line.The stalbe transfected cell line was analyzed by RT-PCR and Western-blot. The result showed that the expression of RalB and STARD7 transfected is higher than that of control group.
5.The eukaryotic plasmid,pDsRed_1-C3/RalB and pDsRed_1-C3/STARD7,was transfected in the same method,and the result dedicated that RalB was expression in plasma membrane and nucleus,but STARD7 was found in cytoplasm.
6.MTT was used to explore the growth character of SMMC7721 transfected with PcDNA3.1(-)/RalB.The result showed that RalB overexpressed can promote the proliferation in the early period of the cell growth.But STARD7 overexpressed could not promote the growth of HepG2 transfected with STARD7.
7.Compared with the control group,the result of Transwell showed that the ability to invation and metastasis has been promoted in SMMC7721 cell transfected with RalB.
8.After Starvation assay,we incubated SMMC7721 transfected with RalB in the free medium.And then we collect cells in four time point,analyzed by flow cytometry,we fount over-expressed RalB can prevent the tumor cell from apoptosis. Meantime,we measured the expression of RalB in the tumor cell cultured with free serum and normal serum.The result dedicated that the expression of RalB with free serum is higher than that with normal serum.So we conclude that RalB can prevent the malignant hepatocyte from apoptosis and sustain the survival of tumor cell.
9.The special bane has been observed in the nucleic protein of SMMC7721 transfected with RalB,as a result,it is hinted RalB could activate the transcription factor of NF-κB in SMMC7721.The result hinted that NF-κB activated by RalB may generate the effect which can result in the biological change of tumor cells.
10.Compared with the control group,the MMP2 in the cell transfected with RalB has a high expression level.The result may mean the influence on the metastasis of hepatoma carcinoma cell by RalB is related to the expression of MMP2.
Conclusion:Over-expression of RalB may promote the proliferation of hepatoma carcinoma cell,SMMC7721,in the early period,and increases the ability of invation and metastasis in HCCs.RalB may also prevent HCCs from apoptosis and maintain the survival of tumor cell.We dedicated that RalB may result in NF-κB to be actived,as a result to promote the expression of MMP2,and further mediate the effect of metastasis for hepatoma carcinoma cell.But for STARD7,there is no apparent proliferation in HepG2,so further experiment is required.
引文
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[1]S .Durand, S Angeletti, S Genti-Raimondi.GTTl/StarD7, a Novel Phosphatidylcholine Transfer Protein-like Highly Expressed in Gestational Trophoblastic Tumour: Cloning and Characterization. Placenta, 2004 ,25: 37-44.
[2] Fabien Alpy, Catherine Tomasetto.Give lipids a START: the StAR-related lipid transfer (START) domain in mammals. Journal of Cell Science, 2005, 118(13):2791-2801.
[3] Ponting C.P, Aravind L. START: a lipid-binding domain in StAR, HD-ZIP and signaling proteins. Trends Biochem. Sci. 1999,24:130-132.
[4]Schultz J., Milpetz F., Bork P. et al. SMART, a simple modular architecture research tool: identification of signaling domains. Proc. Natl. Acad. Sci. USA1998,95:5857-5864.
[5]Wirtz KW. Phospholipid transfer proteins. Annu Rev Biochem. 1991,60:73-99.
[6]Bailey A L, Cullis P R. Membrane fusion with cationic Liposomes:effects of target membrane lipid composition. Biochemistry, 1997, 36: 1628-1634.
[1]S. Durand, S. Angeletti , S. Genti-Raimondi. GTT1/StarD7, a Novel Phosphatidylcholine Transfer Protein-like Highly Expressed in Gestational Trophoblastic Tumour: Cloning and Characterization. Placenta, 2004, 25, 37-44.
[2]Simone Kaiser, Michal Toborek. Liposome mediated high-efficiency transfection of human endothelial cellls. J Vasc Res, 2001; 38(2):381-386.
[3]Mayu Sugiyama, MitusoMatsuura, Yoshito Takeuchi. Possible mechanism of polycation liposome(PCL)-mediated gene transfer.Biochimica Et Biophysica Acta,2004; 1660: 24-30.
[4]Bailey A L, Cullis P R. Membrane fusion with cationic Liposomes:effects of target membrane lipid composition. Biochemistry, 1997; 36: 1628-1634.
[5]Suzanne Jackowski.Cell Cycle Regulation of Membrane Phospholipid Metabolism.The Journal of Biological Chemistry. 1996,271(34): 20219-20222.
[6]Terce'F, Brun, H, Vance, D.E.Requirement of phosphatidylcholine for normal progression through the cell cycle in C3H/10T1/fibroblasts. Journal of Lipid Research. 1994, 35:2130-2142.
[2]YvonaWard,Warner Wang,Elisa Woodhouse,et al.Signal Pathways Which Promote Invasion and Metastasis:Critical and Distinct Contributions of Extracellular Signal-Regulated Kinase and Ral-Specific Guanine Exchange Factor Pathways.Molecullar and Cellullar Biology,2001,21(17):5958-5969.
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[6] Hsieh CL, Swaroop A, Francke U. Chromosomal localization and cDNA sequence of human ralB, a GTP binding protein.Somat Cell Mol Genet,1990,16(4):407-410.
[7] Yuchen Chien, Michael A. White.RAL GTPases are linchpin modulators of human tumour-cell proliferation and survival. EMBO reports, 2003, 4(8):800-806.
[8] Samuel C, Falsetti, De-an Wang, et al.Geranylgeranyltransferase I Inhibitors Target RalB To Inhibit Anchorage-Dependent Growth and Induce Apoptosis and RalA To Inhibit Anchorage-Independent Growth. Molecular and Cellcular Biology, 2007,27: 8003-8014.
[9] Kian-Huat Lim, Kevin O'Hayer, Stacey J. Adam,ea tl.Divergent Roles for RalA and RalB in Malignant Growth of Human Pancreatic Carcinoma Cells. Current Biology, 2006,2385-2394.
[10] Lim KH, Baines AT, Fiordalisi JJ, et.al.Activation of RalA is critical for Ras-induced tumorigenesis of human cells. Cancer Cell.2005, 7(6):533-45.
[11] Michail Shipitsin, Larry A. Feig. RalA but Not RalB Enhances Polarized Delivery of Membrane Proteins to the Basolateral Surface of Epithelial Cells.Molecular and Cellcular Biology, 2004, 24(13): 5746-5756.
[12] Carine Rosse, Anastassia Hatzoglou, Maria-Carla Parrini, et.al. RalB Mobilizes the Exocyst To Drive Cell Migration. Molecular and Celluar Biology, 2006,26(2):727-734.
[13] Fabien Alpy, Catherine Tomasetto.Give lipids a START: the StAR-related lipid transfer (START) domain in mammals. Journal of Cell Science, 2005, 118(13):2791-2801.
[14] Ponting C.P, Aravind L. START: a lipid-binding domain in StAR, HD-ZIP and signaling proteins. Trends Biochem. Sci. 1999,24:130-132.
[15]Schultz J., Milpetz F., Bork P. et al. SMART, a simple modular architecture research tool: identification of signaling domains. Proc. Natl. Acad. Sci.USA1998, 95: 5857-5864.
[16]Wirtz KW. Phospholipid transfer proteins. Annu Rev Biochem. 1991,60:73-99.
[17]Terce'F, Brun, H, Vance, D.E.Requirement of phosphatidylcholine for normal progression through the cell cycle in C3H/10T1/fibroblasts. Journal of Lipid Research. 1994,35:2130-2142.
[18]S. Durand, S. Angeletti , S. Genti-Raimondi. GTT1/StarD7, a Novel Phosphatidylcholine Transfer Protein-like Highly Expressed in Gestational Trophoblastic Tumour: Cloning and Characterization. Placenta, 2004, 25, 37-44.
[1] Wildey GM, Viggeswarapu M, Rim S, Denker JK. Isolation of cDNA clones and tissue expression of rat ral A and ral B GTP-binding proteins. Biochem Biophys Res Commun, 1993 ,15;194(1):552-9.
[2] Hsieh CL, Swaroop A, Francke U. Chromosomal localization and cDNA sequence of human ralB, a GTP binding protein.Somat Cell Mol Genet,1990,16(4):407-410.
[3]Jilkina O, Bhullar RP.Generation of antibodies specific for the RalA and RalB GTP-binding proteins and determination of their concentration and distribution in human platelets. Biochim Biophys Acta. 1996 Nov 8;1314(1-2): 157-66.
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[5]Lim KH, Baines AT, Fiordalisi JJ, et.al.Activation of RalA is critical for Ras-induced tumorigenesis of human cells. Cancer Cell.2005, 7(6):533-45.
[6]Michail Shipitsin, Larry A. Feig. RalA but Not RalB Enhances Polarized Delivery of Membrane Proteins to the Basolateral Surface of Epithelial Cells. Molecular and Cellcular Biology, 2004, 24(13): 5746-5756.
[7] Samuel C, Falsetti, De-an Wang, et al.Geranylgeranyltransferase I Inhibitors Target RalB To Inhibit Anchorage-Dependent Growth and Induce Apoptosis and RalA To Inhibit Anchorage-Independent Growth. Molecular and Cellcular Biology,2007,27: 8003-8014.
[8]Kian-Huat Lim, Kevin O'Hayer, Stacey J. Adam,ea tl.Divergent Roles for RalA and RalB in Malignant Growth of Human Pancreatic Carcinoma Cells. Current Biology, 2006,2385-2394.
[9] Carine Rosse, Anastassia Hatzoglou, Maria-Carla Parrini, et.al. RalB Mobilizes the Exocyst To Drive Cell Migration. Molecular and Celluar Biology, 2006,26(2): 727-734.
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[1]Simone Kaiser, Michal Toborek. Liposome mediated high-efficiency transfection of human endothelial cellls. J Vasc Res, 2001; (2):381-386.
[2]Mayu Sugiyama, MitusoMatsuura, Yoshito Takeuchi. Possiblemechanism of polycation liposome-mediated gene transfer.Biochimica Et Biophysica Acta,2004; 1660: 24-30.
[3]Bailey A L, Cullis P R. Membrane fusion with cationic Liposomes :effects of target membrane lipid composition. Biochemistry, 1997; 36: 1628-1634.
[1]Yuchen Chien, Michael A. White.RAL GTPases are linchpin modulators of human tumour-cell proliferation and survival. EMBO reports, 2003,4 (8):800-806.
[2]Lim KH, Baines AT, Fiordalisi JJ, et.al..Activation of RalA is critical for Ras-induced tumorigenesis of human cells. Cancer Cell.2005 Jun;7(6):533-45.
[3]Dale 0. Henry, Serge A. Moskalenko, Kiran J. Kaur, et al.Ral GTPases contribute to Regulation of Cyclin D1 through Activation of NF-kB. Molecular and Celluar Biology, Nov. 2000,20(21):8084-8092.
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[6] Carine Rosse, Anastassia Hatzoglou, Maria-Carla Parrini, et.al. RalB Mobilizes the Exocyst To Drive Cell Migration. Molecular and Celluar Biology, 2006,26(2):727-734.
[7]Gary Oxford, Charles R. Owens, Brian J. Titus, Tonia L. Foreman, et.al. RalA and RalB: Antagonistic Relatives in Cancer Cell Migration. Cancer Res, 2005,65(16):7111-7120.
[8]Kian-Huat Lim, Kevin O'Hayer, Stacey J. Adam,ea tl.Divergent Roles for RalA and RalB in Malignant Growth of Human Pancreatic Carcinoma Cells. Current Biology, 2006,2385-2394.
[9]YvonaWard, Warner Wang, Elisa Woodhouse,et al. Signal Pathways Which Promote Invasion and Metastasis:Critical and Distinct Contributions of Extracellular Signal-Regulated Kinase and Ral-Specific Guanine Exchange Factor Pathways. Molecullar and Cellullar Biology, 2001,21(17): 5958-5969.
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[1]S .Durand, S Angeletti, S Genti-Raimondi.GTTl/StarD7, a Novel Phosphatidylcholine Transfer Protein-like Highly Expressed in Gestational Trophoblastic Tumour: Cloning and Characterization. Placenta, 2004 ,25: 37-44.
[2] Fabien Alpy, Catherine Tomasetto.Give lipids a START: the StAR-related lipid transfer (START) domain in mammals. Journal of Cell Science, 2005, 118(13):2791-2801.
[3] Ponting C.P, Aravind L. START: a lipid-binding domain in StAR, HD-ZIP and signaling proteins. Trends Biochem. Sci. 1999,24:130-132.
[4]Schultz J., Milpetz F., Bork P. et al. SMART, a simple modular architecture research tool: identification of signaling domains. Proc. Natl. Acad. Sci. USA1998,95:5857-5864.
[5]Wirtz KW. Phospholipid transfer proteins. Annu Rev Biochem. 1991,60:73-99.
[6]Bailey A L, Cullis P R. Membrane fusion with cationic Liposomes:effects of target membrane lipid composition. Biochemistry, 1997, 36: 1628-1634.
[1]S. Durand, S. Angeletti , S. Genti-Raimondi. GTT1/StarD7, a Novel Phosphatidylcholine Transfer Protein-like Highly Expressed in Gestational Trophoblastic Tumour: Cloning and Characterization. Placenta, 2004, 25, 37-44.
[2]Simone Kaiser, Michal Toborek. Liposome mediated high-efficiency transfection of human endothelial cellls. J Vasc Res, 2001; 38(2):381-386.
[3]Mayu Sugiyama, MitusoMatsuura, Yoshito Takeuchi. Possible mechanism of polycation liposome(PCL)-mediated gene transfer.Biochimica Et Biophysica Acta,2004; 1660: 24-30.
[4]Bailey A L, Cullis P R. Membrane fusion with cationic Liposomes:effects of target membrane lipid composition. Biochemistry, 1997; 36: 1628-1634.
[5]Suzanne Jackowski.Cell Cycle Regulation of Membrane Phospholipid Metabolism.The Journal of Biological Chemistry. 1996,271(34): 20219-20222.
[6]Terce'F, Brun, H, Vance, D.E.Requirement of phosphatidylcholine for normal progression through the cell cycle in C3H/10T1/fibroblasts. Journal of Lipid Research. 1994, 35:2130-2142.