siRNA干扰肝癌相关抗原Cdc25C表达对Hepa1-6肝癌细胞增殖和迁移的影响及机制研究
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摘要
目的:探讨小干扰RNA(siRNA)抑制肝癌相关抗原细胞分裂周期蛋白25(Cdc25C)表达对小鼠肝癌细胞Hepa1-6增殖和迁移的作用及其机制。方法:将细胞分为空白对照组(未转染)、阴性对照组(转染阴性siRNA)及3个实验组(转染siRNA-Cdc25C-1组、转染siRNA-Cdc25C-2组、转染siRNA-Cdc25C-3组)。转染8 h后,通过荧光显微镜观察转染效率,并采用Western blotting法检测各组Cdc25C蛋白的表达,筛选出最有效的siRNA序列;用荧光定量PCR(qPCR)法检测内质网应激反应(ERS)相关蛋白葡萄糖调节蛋白78(GRP78)、X-盒结合蛋白(XBP-1)、C/EBP同源蛋白(CHOP)的表达,CCK-8法和Transwell实验检测Hepa1-6细胞增殖和迁移能力。结果:成功构建Cdc25C低表达的Hepa1-6细胞株,转染siRNA-Cdc25C-1组的转染效率最高且Cdc25C蛋白表达水平最低。与阴性对照组比较,转染siRNA-Cdc25C-1组Hepa1-6细胞的迁移率明显降低,细胞增殖抑制率及GRP78、XBP-1、CHOP基因相对表达量明显升高(P<0.05)。空白对照组与阴性对照组比较差异均无统计学意义(均P>0.05)。结论:Cdc25C基因低表达能抑制肝癌细胞增殖和迁移,其机制可能与ERS有关。
Objective:To investigate the effects of silencing Cdc25 C gene on proliferation and migration of Hepa1-6 cell line.Methods:Hepa1-6 cells were transfected with either mock,negative control siRNA,Cdc25 C-siRNA-1,Cdc25 cC-siRNA-2 or Cdc25 C-siRNA-3.After 8 hours of transfection,fluorescent staining and western blotting were used to select the siRNA with the highest transfection efficiency.The expressions of endoplasmic reticulum stress(ERS) related glucose regulation protein-78(GRP78),X-box binding protein-1(XBP-1),and C/EBP homologous protein(CHOP) were determined by quantitative fluorescence PCR(qPCR).The proliferation and migration of Hepa1-6 cells were evaluated by CCK-8 method and Transwell assay,respectively.Results:Hepa1-6 cell line with low expression of Cdc25 C was successfully constructed.Cdc25 C-siRNA-1 with the highest transfection efficiency was selected for subsequent tests.Comparing the negative control group to the Cdc25 C-siRNA-1 group,there was a significant reduction in cell number and migration rate of Hepa1-6 cells with Cdc25 C siRNA treatment,and a concomitant increase in the expression of GRP78,XBP-1 and CHOP mRNA(P<0.05).No significant difference was found between the mock and negative control siRNA transfections(P>0.05).Conclusion:Low expression of Cdc25 C inhibited the proliferation and migration of Hepa1-6 cells,and the mechanism might be related to the ERS.
Objective:To investigate the effects of silencing Cdc25 C gene on proliferation and migration of Hepa1-6 cell line.Methods:Hepa1-6 cells were transfected with either mock,negative control siRNA,Cdc25 C-siRNA-1,Cdc25 cC-siRNA-2 or Cdc25 C-siRNA-3.After 8 hours of transfection,fluorescent staining and western blotting were used to select the siRNA with the highest transfection efficiency.The expressions of endoplasmic reticulum stress(ERS) related glucose regulation protein-78(GRP78),X-box binding protein-1(XBP-1),and C/EBP homologous protein(CHOP) were determined by quantitative fluorescence PCR(qPCR).The proliferation and migration of Hepa1-6 cells were evaluated by CCK-8 method and Transwell assay,respectively.Results:Hepa1-6 cell line with low expression of Cdc25 C was successfully constructed.Cdc25 C-siRNA-1 with the highest transfection efficiency was selected for subsequent tests.Comparing the negative control group to the Cdc25 C-siRNA-1 group,there was a significant reduction in cell number and migration rate of Hepa1-6 cells with Cdc25 C siRNA treatment,and a concomitant increase in the expression of GRP78,XBP-1 and CHOP mRNA(P<0.05).No significant difference was found between the mock and negative control siRNA transfections(P>0.05).Conclusion:Low expression of Cdc25 C inhibited the proliferation and migration of Hepa1-6 cells,and the mechanism might be related to the ERS.
引文
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[2] RAZA A,SOOD G K.Hepatocellular carcinoma review:Current treatment and evidence-based medicine[J].World J Gastroenterol,2014,20(15):4115-4127.
[3] TAKIZAWA C G,MORGAN D O.Control of mitosis by changes in the subcellular location of cyclin-B1-Cdk1 and Cdc25C[J].Curr Opin Cell Biol,2000,12(6):658-665.
[4] 莫发荣.细胞周期蛋白Cdc25C的研究[J].生命的化学,2010,30(6):889-892.
[5] CHEN M S,HUROV J,WHITE L S,et al.Absence of apparent phenotype in mice lacking Cdc25C protein phosphatase[J].Am Soc Microbiol,2001,21(12) :3853-3861.
[6] OZEN M,ITTMANN M.Increased expression and activity of CDC25C phosphatase and an alternatively spliced variant in prostate cancer[J].Clin Cancer Res,2005,11(13):4701-4706.
[7] WANG Z H,TROPE C G,FLORENES V A,et al.Over expression of CDC25B,CDC25C and phospho-CDC25C (Ser216) in vulvar squamous cell carcinomas are associated with malignant features and aggressive cancer phenotypes[J].BMC Cancer,2010(10):233.
[8] 李多杰,吴礼高,朱超莽,等.Cdc25C在放疗敏感的食管鳞状细胞癌中高表达[J].中国组织化学与细胞化学杂志,2017,26(2):147-151.
[9] 张琪惠,李春梅,黄天明,等.CDC25C抗体在肝病患者中的血清学检测[J].世界华人消化杂志,2015,23(36):5848-5853.
[10] 陈承晓,张琪惠,李春梅,等.人肝癌相关抗原Cdc25C特异性多肽抗体的制备及其应用[J].山东医药,2016,56(13):1-3.
[11] TABAS I,RON D.Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress[J].Nat Cell Biol,2011(13):184-190.
[12] AS SELAH T,BIECHE I,MANSOURI A,et al.In vivo hepatic endoplasmic reticulum stress in patients with chronic hepatitis C[J].J Pathol,2010,221(3):264-274.
[13] WEI Y,WANG D,TOPCZEWSKI F,et al.Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells[J].Am J Physiol Endocrinol and Me Tab,2006,291(2):E275-E281.
[14] NAGESH P K,HATAMI E,CHOWDHURY P,et al.Tannic acid induces endoplasmic reticulum stress-mediated apoptosis in prostate cancer[J].Cancers,2018,10(3):68.
[15] YADAV R K,CHAE S W,KIM H R,et al.Endoplasmic reticulum stress and cancer[J].J Cancer Prev,2014,19(2):75-88.
[16] LO Y H,LIN I L,LIN C F,et al.Novel acyclic enediynes inhibit cyclin A and Cdc25C expression and induce apoptosis phenomenon to show potent antitumor proliferation[J].Bioorganic & Medicinal Chemistry,2007,15(13):4528-4536.