Primary Study Concerned The Biologcal Influence On HCC Performed By Tumor Related RalB And STARD7

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 pDsRed₁-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 pDsRed₁-C3/RalB and pDsRed₁-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,pDsRed₁-C3/RalB and pDsRed₁-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.