真核翻译延伸因子1A1通过调控cyclin D2表达促进肝癌细胞增殖
|
摘要
目的 :研究真核翻译延伸因子1A1(eukaryotic translation elongation factor1A1,eEF1A1)表达对原发性肝癌细胞周期和增殖的影响,并探讨其分子机制。方法 :免疫组织化学法检测101例肝癌及癌旁组织中eEF1A1的表达水平,并进一步分析肝癌组织中eEF1A1表达与肝癌患者临床病理参数的相关性。采用实时荧光定量PCR和蛋白质印迹法检测肝癌细胞Huh7、SMMC7721、MHCC97H、Hep3B和正常肝细胞HL-7702中eEF1A1 mRNA和蛋白的表达水平。在肝癌Huh7和SMMC7721细胞中分别转染特异性针对eEF1A1基因的shRNA-1/2(即eEF1A1-shRNA-1/2)和过表达质粒pcDNA3.1-eEF1A1,并采用实时荧光定量PCR和蛋白质印迹法验证eEF1A1表达的变化,然后采用EdU法和FCM法分别检测肝癌细胞增殖和细胞周期的变化。另外,蛋白质印迹法检测转染eEF1A1 shRNA-1/2的肝癌Huh7细胞和转染pcDNA3.1-eEF1A1的SMMC7721细胞中cyclin D2以及信号转导与转录激活子1(signal transducer and activator of transcription 1,STAT1)通路蛋白的表达变化。结果 :肝癌组织中eEF1A1表达水平明显高于癌旁组织(P <0.001),肝癌细胞中eEF1A1表达水平明显高于正常肝细胞(P <0.001),并且eEF1A1高表达与肝癌大小及TNM分期密切相关(P <0.01,P <0.05)。eEF1A1-shRNA-1/2转染后,肝癌Huh7细胞中eEF1A1 mRNA及蛋白的表达水平明显降低(P值均<0.01),G_1期细胞所占比例明显升高(P <0.01),肝癌细胞的增殖活性明显降低(P <0.01);pcDNA3.1-eEF1A1转染后肝癌SMMC7721细胞中eEF1A1 mRNA及蛋白的表达水平明显升高(P值均<0.01),G_1期细胞所占比例明显降低(P <0.05),肝癌细胞的增殖活性明显增高(P <0.05)。随着eEF1A1基因表达下调,Huh7细胞中cyclin D2、STAT1总蛋白和核蛋白水平均明显下降(P值均<0.05);而过表达eEF1A1后,SMMC7721细胞中cyclin D2、STAT1总蛋白和核蛋白水平均明显升高(P值均<0.05);过表达eEF1A1基因的同时加入STAT1抑制剂作用后,cyclin D2、STAT1总蛋白和核蛋白水平均无明显变化(P值均> 0.05)。结论 :eEF1A1通过STAT1通路调控cyclin D2表达,从而促进肝癌细胞周期进展和细胞增殖。
Objective: To study the effects of eukaryotic translation elongation factor 1A1(eEF1A1) on the cell cycle and proliferation of hepatocellular carcinoma(HCC) cells, and to explore the molecular mechanism.Methods: The expression of eEF1A1 protein in 101 HCC tissues and their adjacent tissues were detected by immunohistochemistry, and the correlations between the expression of eEF1A1 protein and the clinicopathologic parameters of HCC patients was further analyzed. The expressions of eEF1A1 mRNA and protein in HCC cells(including Huh7, SMMC7721, MHCC97H and Hep3B) and the normal liver cells HL-7702 were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The specific shRNA-1/2 targeting eEF1A1 gene(eEF1A1-shRNA-1/2) and the overexpression plasmid pcDNA3.1-eEF1A1 were respectively transfected into Huh7 and SMMC7721 cells, and the alteration of eEF1A1 expression was verified by real-time fluorescent quantitative PCR and Western blotting, respectively. Then the proliferation and cell cycle of HCC cells were detected by EdU and FCM assays, respectively. In addition, the expressions of cyclin D2 and the signal transducer and activator of transcription 1(STAT1)-related pathway proteins in Huh7 cells transfected with eEF1A1-shRNA-1/2 and in SMMC7721 cells transfected with pcDNA3.1-eEF1A1 were detected by Western blotting.Results: The expression level of eEF1A1 protein in HCC tissues was markedly higher than that in the adjacent tissues(P < 0.001). The expression levels of eEF1A1 mRNA and protein in four HCC cell lines were significantly higher than those in normal live cells(all P < 0.001). The high expression of eEF1A1 was closely related to tumor size and TNM stage in HCC patients(P < 0.01, P < 0.05). After transfection of eEF1A1-shRNA-1/2 into Huh7 cells, the expression levels of eEF1A1 mRNA and protein were significantly decreased(both P < 0.01), the proportion of G_1 phase-cells increased significantly(P < 0.01), and the proliferation activity of Huh7 cells was significantly reduced(P < 0.01). After transfection of pcDNA3.1-eEF1A1 into SMMC7721 cells, the expression levels of eEF1A1 mRNA and protein were significantly increased(both P < 0.01), the proportion of G_1 phase-cells was significantly reduced(P < 0.05), and the proliferation activity of SMMC7721 cells increased significantly(P < 0.05). In addition, the expression levels of cyclin D2, total and nuclear STAT1 proteins were decreased(all P < 0.05) in Huh7 cells with the downregulation of eEF1A1 expression. After eEF1A1 overexpression, the expression levels of cyclin D2, total and nuclear STAT1 proteins were increased(all P < 0.05) in SMMC7721 cells. However, the expression levels of cyclin D2, total and nuclear STAT1 proteins were unchanged(all P > 0.05) in SMMC7721 cells after eEF1A1 overexpression and treatment with STAT1 inhibitor fludarabine.Conclusion: eEF1A1 regulates the expression of cyclin D2 through STAT1 pathway, thereby promoting the cell cycle progression and proliferation of primary HCC cells.
Objective: To study the effects of eukaryotic translation elongation factor 1A1(eEF1A1) on the cell cycle and proliferation of hepatocellular carcinoma(HCC) cells, and to explore the molecular mechanism.Methods: The expression of eEF1A1 protein in 101 HCC tissues and their adjacent tissues were detected by immunohistochemistry, and the correlations between the expression of eEF1A1 protein and the clinicopathologic parameters of HCC patients was further analyzed. The expressions of eEF1A1 mRNA and protein in HCC cells(including Huh7, SMMC7721, MHCC97H and Hep3B) and the normal liver cells HL-7702 were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The specific shRNA-1/2 targeting eEF1A1 gene(eEF1A1-shRNA-1/2) and the overexpression plasmid pcDNA3.1-eEF1A1 were respectively transfected into Huh7 and SMMC7721 cells, and the alteration of eEF1A1 expression was verified by real-time fluorescent quantitative PCR and Western blotting, respectively. Then the proliferation and cell cycle of HCC cells were detected by EdU and FCM assays, respectively. In addition, the expressions of cyclin D2 and the signal transducer and activator of transcription 1(STAT1)-related pathway proteins in Huh7 cells transfected with eEF1A1-shRNA-1/2 and in SMMC7721 cells transfected with pcDNA3.1-eEF1A1 were detected by Western blotting.Results: The expression level of eEF1A1 protein in HCC tissues was markedly higher than that in the adjacent tissues(P < 0.001). The expression levels of eEF1A1 mRNA and protein in four HCC cell lines were significantly higher than those in normal live cells(all P < 0.001). The high expression of eEF1A1 was closely related to tumor size and TNM stage in HCC patients(P < 0.01, P < 0.05). After transfection of eEF1A1-shRNA-1/2 into Huh7 cells, the expression levels of eEF1A1 mRNA and protein were significantly decreased(both P < 0.01), the proportion of G_1 phase-cells increased significantly(P < 0.01), and the proliferation activity of Huh7 cells was significantly reduced(P < 0.01). After transfection of pcDNA3.1-eEF1A1 into SMMC7721 cells, the expression levels of eEF1A1 mRNA and protein were significantly increased(both P < 0.01), the proportion of G_1 phase-cells was significantly reduced(P < 0.05), and the proliferation activity of SMMC7721 cells increased significantly(P < 0.05). In addition, the expression levels of cyclin D2, total and nuclear STAT1 proteins were decreased(all P < 0.05) in Huh7 cells with the downregulation of eEF1A1 expression. After eEF1A1 overexpression, the expression levels of cyclin D2, total and nuclear STAT1 proteins were increased(all P < 0.05) in SMMC7721 cells. However, the expression levels of cyclin D2, total and nuclear STAT1 proteins were unchanged(all P > 0.05) in SMMC7721 cells after eEF1A1 overexpression and treatment with STAT1 inhibitor fludarabine.Conclusion: eEF1A1 regulates the expression of cyclin D2 through STAT1 pathway, thereby promoting the cell cycle progression and proliferation of primary HCC cells.
引文
[1]TORRE LA,BRAY F,SIEGEL RL,et al.Global cancer statistics,2012[J].CA Cancer J Clin,2015,65(2):87-108.
[2]LI J,HUANG L,LIU CF,et al.Risk factors and surgical outcomes for spontaneous rupture of BCLC stages A and B hepatocellular carcinoma:a case-control study[J].World J Gastroenterol,2014,20(27):9121-9127.
[3]FINN RS.Emerging targeted strategies in advanced hepatocellular carcinoma[J].Semin Liver Dis,2013,33(Suppl 1):S11-S19.
[4]BERTOLI C,SKOTHEIM JM,DE BRUIN RA.Control of cell cycle transcription during G1 and S phases[J].Nat Rev Mol Cell Biol,2013,14(8):518-528.
[5]MUSGROVE EA,CALDON CE,BARRACLOUGH J,et al.Cyclin D as a therapeutic target in cancer[J].Nat Rev Cancer,2011,11(8):558-572.
[6]HUAN GF,ZHAO H,DU Z,et al.MiR-615 inhibits prostate cancer cell proliferation and invasion by directly targeting Cyclin D2[J/OL].Oncol Res,2018(2018-02-22)[2018-04-28].https://www.ingentaconnect.com/content/cog/or/pre-prints/contentorm-a-1980_huang;jsessionid=nqdv5lcltmbv.x-ic-live-02.doi:10.3727/096504018X15190399381143.
[7]ZENG Q,TAO X,HUANG F,et al.Overexpression of miR-155 promotes the proliferation and invasion of oral squamous carcinoma cells by regulating BCL6/cyclin D2[J].Int J Mol Med,2016,37(5):1274-1280.
[8]KAPP LD,LORSCH JR.The molecular mechanics of eukaryotic translation[J].Annu Rev Biochem,2004,73:657-704.
[9]SCAGGIANTE B,DAPAS B,GRASSI G,et al.Interaction of G-rich GT oligonucleotides with nuclearassociated eEF1A is correlated with their antiproliferative effect in haematopoietic human cancer cell lines[J].FEBS J,2006,273(7):1350-1361.
[10]KOBAYASHI Y,YONEHARA S.Novel cell death by downregulation of eEF1A1 expression in tetraploids[J].Cell Death Differ,2009,16(1):139-150.
[11]SCAGGIANTE B,DAPAS B,BONIN S,et al.Dissecting the expression of EEF1A1/2 genes in human prostate cancer cells:the potential of EEF1A2 as a hallmark for prostate transformation and progression[J].Brit J Cancer,2012,106(1):166-173.
[12]ZHU G,YAN W,HE HC,et al.Inhibition of proliferation,invasion,and migration of prostate cancer cells by downregulating elongation factor-1alpha expression[J].Mol Med,2009,15(11/12):363-370.
[13]LIU X,CHEN L,GE J,et al.The ubiquitin-like protein FAT10 stabilizes eEF1A1 expression to promote tumor proliferation in a complex manner[J].Cancer Res,2016,76(16):4897-4907.
[14]晏琛,刘秀霞,戈进,等.RNA干扰真核翻译延长因子1A1抑制肝癌Hep3B细胞的增殖[J].肿瘤,2014,34(11):1016-1022.
[15]HUANG J,ZHENG C,SHAO J,et al.Overexpression of eEF1A1 regulates G1-phase progression to promote HCCproliferation through the STAT1-cyclin D1 pathway[J].Biochem Biophys Res Commun,2017,494(3/4):542-549.
[16]OLIVERI RS,WETTERSLEV J,GLUUD C.Hepatocellular carcinoma[J].Lancet,2012,380(9840):470.
[17]LUO C,XIONG H,CHEN L,et al.GRP78promotes hepatocellular carcinoma proliferation by increasing FAT10expression through the NF-kappaB pathway[J].Exp Cell Res,2018,365(1):1-11.
[18]GRASSI G,SCAGGIANTE B,FARRA R,et al.The expression levels of the translational factors eEF1A1/2 correlate with cell growth but not apoptosis in hepatocellular carcinoma cell lines with different differentiation grade[J].Biochimie,2007,89(12):1544-1552.
[19]AL-MAGHREBI M,ANIM JT,OLALU AA.Up-regulation of eukaryotic elongation factor-1 subunits in breast carcinoma[J].Anticancer Res,2005,25(3c):2573-2577.
[20]THORNTON S,ANAND N,PURCELL D,et al.Not just for housekeeping:protein initiation and elongation factors in cell growth and tumorigenesis[J].J Mol Med(Berl),2003,81(9):536-548.
[21]GANGWANI L,MIKRUT M,GALCHEVA-GARGOVA Z,et al.Interaction of ZPR1 with translation elongation factor-1alpha in proliferating cells[J].J Cell Biol,1998,143(6):1471-1484.
[22]ZHONG D,ZHANG JF,YANG SA,et al.The SAM domain of the RhoGAP DLC1 binds EF1A1 to regulate cell migration[J].J Cell Sci,2009,122(3):414-424.
[23]LI YL,WANG J,ZHANG CY,et al.MiR-146a-5p inhibits cell proliferation and cell cycle progression in NSCLC cell lines by targeting CCND1 and CCND2[J].Oncotarget,2016,7(37):59287-59298.
[2]LI J,HUANG L,LIU CF,et al.Risk factors and surgical outcomes for spontaneous rupture of BCLC stages A and B hepatocellular carcinoma:a case-control study[J].World J Gastroenterol,2014,20(27):9121-9127.
[3]FINN RS.Emerging targeted strategies in advanced hepatocellular carcinoma[J].Semin Liver Dis,2013,33(Suppl 1):S11-S19.
[4]BERTOLI C,SKOTHEIM JM,DE BRUIN RA.Control of cell cycle transcription during G1 and S phases[J].Nat Rev Mol Cell Biol,2013,14(8):518-528.
[5]MUSGROVE EA,CALDON CE,BARRACLOUGH J,et al.Cyclin D as a therapeutic target in cancer[J].Nat Rev Cancer,2011,11(8):558-572.
[6]HUAN GF,ZHAO H,DU Z,et al.MiR-615 inhibits prostate cancer cell proliferation and invasion by directly targeting Cyclin D2[J/OL].Oncol Res,2018(2018-02-22)[2018-04-28].https://www.ingentaconnect.com/content/cog/or/pre-prints/contentorm-a-1980_huang;jsessionid=nqdv5lcltmbv.x-ic-live-02.doi:10.3727/096504018X15190399381143.
[7]ZENG Q,TAO X,HUANG F,et al.Overexpression of miR-155 promotes the proliferation and invasion of oral squamous carcinoma cells by regulating BCL6/cyclin D2[J].Int J Mol Med,2016,37(5):1274-1280.
[8]KAPP LD,LORSCH JR.The molecular mechanics of eukaryotic translation[J].Annu Rev Biochem,2004,73:657-704.
[9]SCAGGIANTE B,DAPAS B,GRASSI G,et al.Interaction of G-rich GT oligonucleotides with nuclearassociated eEF1A is correlated with their antiproliferative effect in haematopoietic human cancer cell lines[J].FEBS J,2006,273(7):1350-1361.
[10]KOBAYASHI Y,YONEHARA S.Novel cell death by downregulation of eEF1A1 expression in tetraploids[J].Cell Death Differ,2009,16(1):139-150.
[11]SCAGGIANTE B,DAPAS B,BONIN S,et al.Dissecting the expression of EEF1A1/2 genes in human prostate cancer cells:the potential of EEF1A2 as a hallmark for prostate transformation and progression[J].Brit J Cancer,2012,106(1):166-173.
[12]ZHU G,YAN W,HE HC,et al.Inhibition of proliferation,invasion,and migration of prostate cancer cells by downregulating elongation factor-1alpha expression[J].Mol Med,2009,15(11/12):363-370.
[13]LIU X,CHEN L,GE J,et al.The ubiquitin-like protein FAT10 stabilizes eEF1A1 expression to promote tumor proliferation in a complex manner[J].Cancer Res,2016,76(16):4897-4907.
[14]晏琛,刘秀霞,戈进,等.RNA干扰真核翻译延长因子1A1抑制肝癌Hep3B细胞的增殖[J].肿瘤,2014,34(11):1016-1022.
[15]HUANG J,ZHENG C,SHAO J,et al.Overexpression of eEF1A1 regulates G1-phase progression to promote HCCproliferation through the STAT1-cyclin D1 pathway[J].Biochem Biophys Res Commun,2017,494(3/4):542-549.
[16]OLIVERI RS,WETTERSLEV J,GLUUD C.Hepatocellular carcinoma[J].Lancet,2012,380(9840):470.
[17]LUO C,XIONG H,CHEN L,et al.GRP78promotes hepatocellular carcinoma proliferation by increasing FAT10expression through the NF-kappaB pathway[J].Exp Cell Res,2018,365(1):1-11.
[18]GRASSI G,SCAGGIANTE B,FARRA R,et al.The expression levels of the translational factors eEF1A1/2 correlate with cell growth but not apoptosis in hepatocellular carcinoma cell lines with different differentiation grade[J].Biochimie,2007,89(12):1544-1552.
[19]AL-MAGHREBI M,ANIM JT,OLALU AA.Up-regulation of eukaryotic elongation factor-1 subunits in breast carcinoma[J].Anticancer Res,2005,25(3c):2573-2577.
[20]THORNTON S,ANAND N,PURCELL D,et al.Not just for housekeeping:protein initiation and elongation factors in cell growth and tumorigenesis[J].J Mol Med(Berl),2003,81(9):536-548.
[21]GANGWANI L,MIKRUT M,GALCHEVA-GARGOVA Z,et al.Interaction of ZPR1 with translation elongation factor-1alpha in proliferating cells[J].J Cell Biol,1998,143(6):1471-1484.
[22]ZHONG D,ZHANG JF,YANG SA,et al.The SAM domain of the RhoGAP DLC1 binds EF1A1 to regulate cell migration[J].J Cell Sci,2009,122(3):414-424.
[23]LI YL,WANG J,ZHANG CY,et al.MiR-146a-5p inhibits cell proliferation and cell cycle progression in NSCLC cell lines by targeting CCND1 and CCND2[J].Oncotarget,2016,7(37):59287-59298.