胃癌组织MYH-9蛋白表达及MYH-9 siRNA下调SGC-7901细胞MYH-9表达对细胞侵袭转移的影响
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摘要
目的非肌性肌球蛋白重链9基因(non-muscular myosin heavy chain 9,MYH-9)在多种恶性肿瘤中高表达,且预后差。检测胃癌组织MYH-9的表达及利用RNA干扰(RNA interference,RNAi)技术特异性,抑制胃癌SGC-7901细胞中MYH-9的表达对细胞侵袭和转移的影响,探讨在胃癌治疗中将MYH-9作为基因治疗靶点的价值。方法选取2009-06-01-2010-01-01南昌大学第一附属医院126例胃癌及癌旁组织。利用RNAi技术将MYH-9siRNA转染到胃癌SGC-7901细胞中,蛋白质印迹法检测MYH-9蛋白的表达,RT-PCR法检测MYH-9mRNA的表达;运用细胞迁移和侵袭实验检测MYH-9siRNA下调胃癌SGC-7901细胞中MYH-9的表达对细胞侵袭转移的影响。结果 MYH-9在胃癌组织中的表达显著高于癌旁组织,χ2=77.83,P<0.001。胃癌MYH-9蛋白表达与肿瘤Lauren分型(χ2=5.296,P=0.020)、肿瘤分化程度(χ2=14.39,P=0.002)、肿瘤浸润深度(χ2=5.296,P=0.026)、肿瘤淋巴结转移数(χ2=11.15,P=0.003)、脉管癌栓(χ2=5.255,P=0.022)、肿瘤远处转移状态(χ2=6.683,P=0.009)和TNM分期(χ2=10.73,P=0.002)密切相关。MYH-9蛋白表达(HR=2.825,95%CI=1.104~3.706,P=0.041)、胃癌远处转移状态(HR=1.749,95%CI=0.983~3.503,P=0.035)和胃癌淋巴结转移数(HR=3.235,95%CI=1.579~5.358,P<0.001)可作为预测患者不良预后的独立指标。与空白对照组及阴性对照组比较,特异性siRNA可以高效抑制SGC-7901细胞MYH-9基因的表达,在mRNA水平其表达抑制率分别为87.5%(t=48.22,P<0.001)和86.9%(t=57.30,P<0.001);在蛋白质水平其表达抑制率分别为52.9%(t=11.01,P<0.001)和60.1%(t=15.53,P<0.001)。在侵袭实验中,实验组细胞数为(49.5±13.8)个,显著低于阴性对照组(112±4.34)个(t=15.53,P<0.001)和空白对照组(116±5.57)个(t=28.12,P<0.001)。在迁移实验中,实验组细胞数为(81.5±7.26)个,显著低于阴性对照组(185±7.33)个(t=47.29,P<0.001)和空白对照组(189±3.46)个,t=46.40,P<0.001。结论 MYH-9高表达患者预后较差,MYH-9可能成为胃癌基因治疗的新靶点。
OBJECTIVE MYH-9has a high expression in different tumors,and has a poor survival.This study is to detect the expression of MYH-9in the tissue of gastric carcinoma.RNA interference(RNAi)was employed to inhibit the expression of MYH-9in SGC-7901 cells and to evaluate the effects of MYH-9as a target for gene therapy in gastric carcinoma.METHODS MYH-9protein expression was examined by immunohistochemistry in 126 gastric adenocarcinoma and adjacent normal tissues.SGC-7901 cells were transfected with MYH-9siRNA.MYH-9protein and mRNA levels were detected using Western Blotting and RT-PCR.The migration and invasion assay were used to evaluate the effect of siRNA-mediated inhibition of MYH-9on invasion and metastasis in gastric carcinoma SGC-7901 cells.RESULTS MYH-9protein was overexpressed in GC compared with the adjacent normal gastric epithelium(χ2=77.83,P<0.001).Elevated MYH-9expression was strongly correlated with Lauren classification(2χ=5.296,P=0.020),tumor differentiation(2χ=14.39,P=0.002),depth of wall invasion(2χ=5.296,P=0.026),mean no.of MLNs(2χ=11.15,P=0.003),vascular tumor embolus(2χ=5.255,P=0.022),distant metastasis staus(2χ=6.683,P=0.009)and clinical stage(2χ=10.73,P=0.002).Cox regression model demonstrated that MYH-9expression level(HR=2.825,95%CI=1.104-3.706,P=0.041),distant metastasis status(HR=1.749,95%CI=0.983-3.503,P=0.035)and mean no.of MLNs(HR=3.235,95%CI=1.579-5.358,P<0.001)were the independent prognostic factors of gastric cancer.The specific siRNA can efficiently block the expression of MYH-9both at mRNA and protein levels.Compared with the blank control group and negative control group,the expression inhibition rates were 87.5%(t=48.22,P<0.001)and 86.9%(t=57.30,P<0.001)at mRNA level,respectively.The expression inhibition rates were 52.9%(t=11.01,P<0.001)and 60.1%(t=15.53,P<0.001)at potein level,respectively.The result of in vitro invasion assay showed that Lenti-MYH9-siRNA cells had significantly reduced invasiveness as compared with negative control cells(t=15.53,P<0.001)and blank control cells(t=28.12,P<0.001).The result of in vitro migration assay showed that Lenti-MYH9-siRNA cells had significantly reduced invasiveness as compared with negative control cells(t=47.29,P<0.001)and blank control cells(t=46.40,P<0.001).CONCLUSIONS MYH-9might be a good target for gastric cancer treatment.
OBJECTIVE MYH-9has a high expression in different tumors,and has a poor survival.This study is to detect the expression of MYH-9in the tissue of gastric carcinoma.RNA interference(RNAi)was employed to inhibit the expression of MYH-9in SGC-7901 cells and to evaluate the effects of MYH-9as a target for gene therapy in gastric carcinoma.METHODS MYH-9protein expression was examined by immunohistochemistry in 126 gastric adenocarcinoma and adjacent normal tissues.SGC-7901 cells were transfected with MYH-9siRNA.MYH-9protein and mRNA levels were detected using Western Blotting and RT-PCR.The migration and invasion assay were used to evaluate the effect of siRNA-mediated inhibition of MYH-9on invasion and metastasis in gastric carcinoma SGC-7901 cells.RESULTS MYH-9protein was overexpressed in GC compared with the adjacent normal gastric epithelium(χ2=77.83,P<0.001).Elevated MYH-9expression was strongly correlated with Lauren classification(2χ=5.296,P=0.020),tumor differentiation(2χ=14.39,P=0.002),depth of wall invasion(2χ=5.296,P=0.026),mean no.of MLNs(2χ=11.15,P=0.003),vascular tumor embolus(2χ=5.255,P=0.022),distant metastasis staus(2χ=6.683,P=0.009)and clinical stage(2χ=10.73,P=0.002).Cox regression model demonstrated that MYH-9expression level(HR=2.825,95%CI=1.104-3.706,P=0.041),distant metastasis status(HR=1.749,95%CI=0.983-3.503,P=0.035)and mean no.of MLNs(HR=3.235,95%CI=1.579-5.358,P<0.001)were the independent prognostic factors of gastric cancer.The specific siRNA can efficiently block the expression of MYH-9both at mRNA and protein levels.Compared with the blank control group and negative control group,the expression inhibition rates were 87.5%(t=48.22,P<0.001)and 86.9%(t=57.30,P<0.001)at mRNA level,respectively.The expression inhibition rates were 52.9%(t=11.01,P<0.001)and 60.1%(t=15.53,P<0.001)at potein level,respectively.The result of in vitro invasion assay showed that Lenti-MYH9-siRNA cells had significantly reduced invasiveness as compared with negative control cells(t=15.53,P<0.001)and blank control cells(t=28.12,P<0.001).The result of in vitro migration assay showed that Lenti-MYH9-siRNA cells had significantly reduced invasiveness as compared with negative control cells(t=47.29,P<0.001)and blank control cells(t=46.40,P<0.001).CONCLUSIONS MYH-9might be a good target for gastric cancer treatment.
引文
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[7]徐宝国,杨永栋,于霞,等.胃癌组织C-Met和MMP-11表达与肝转移相关性研究[J].中华肿瘤防治杂志,2014,21(9):678-683.
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[11]Liu D,Zhang L,Shen Z,et al.Clinicopathological significance of MYH-9overexpression in human gastric cancer[J].Int J Mol Sci,2012,13(11):15291-15304.
[12]Du M,Wang G,Ismail TM,et al.S100Pdissociates myosin IIA filaments and focal adhesion sites to reduce cell adhesion and enhance cell migration[J].J Biol Chem,2012,287(19):15330-15344.
[13]Arora S,Saha S,Roy S,et al.Role of Nonmuscle MyosinⅡin Migration of Wharton’s Jelly-Derived Mesenchymal Stem Cells[J].Stem Cells Dev,2015,24(17):2065-2077.
[14]Komatsu S,Ikebe M.ZIPK is critical for the motility and contractility of VSMCs through the regulation of nonmuscle myosinⅡisoforms[J].Am J Physiol Heart Circ Physiol,2014,306(9):H1275-H1286.
[15]Conti CM,Saleh AD,Brinster LR,et al.Conditional deletion of nonmuscle myosinⅡ-A in mouse tongue epithelium results in squamous cell carcinoma[J].Sci Rep,2015,5:14068.
[16]Schramek D,Sendoel A,Segal JP,et al.Direct in vivo RNAi screen unveils myosinⅡa as a tumor suppressor of squamous cell carcinomas[J].Science,2014,343(6168):309-313.
[17]Chen Z,Huang Y,Shen X,et al.Short hairpin RNA targeting autotaxin reduces human gastric carcinoma AGS cell proliferative,migratory and invasive capabilities in vitro and causes tumor regression in vivo[J].Oncol Rep,2013,29(3):1087-1093.
[18]Huang Y,Arora P,Mcculloch CA,et al.The collagen receptor DDR1regulates cell spreading and motility by associating with myosinⅡA[J].J Cell Sci,2009,122(Pt 10):1637-1646.
[19]Elliott PR,Irvine AF,Jung HS,et al.Asymmetric mode of Ca2+-S100A4interaction with nonmuscle myosinⅡA generates nanomolar affinity required for filament remodeling[J].Structure,2012,20(4):654-666.
[20]侯继院,刘兴安,单国用,等.S100A4调节MMP-13表达对甲状腺癌细胞侵袭能力的影响[J].中华肿瘤防治杂志,2015,22(7):514-518.
[21]Chai J,Jamal MM.S100A4in esophageal cancer:is this the one to blame?[J].World J Gastroenterol,2012,18(30):3931-3935.
[22]邓春阳,冯建勋,张海英,等.羊水干细胞诱导肾移植免疫耐受及对氧化应激的影响[J].中国组织工程研究,2015,(45):7342-7349.
[23]贠志敏,李素波,张雪,等.酶解法清除猪皮a-Gal抗原的研究[J].军事医学,2015,39(12):938-940.
[24]储成艳,吴云红,朱亮.巨噬细胞在移植排斥中的作用及其机制的研究进展[J].细胞与分子免疫学杂志,2015,31(12):1721-1723,1727.
[2]勾文峰,杨雪峰,赵爽,等.胃癌组织及细胞株细胞骨架蛋白Fascin-1表达临床意义分析[J].中华肿瘤防治杂志,2014,21(18):1400-1404.
[3]Dulyaninova NG,House RP,Betapudi V,et al.Myosin-ⅡA heavychain phosphorylation regulates the motility of MDA-MB-231carcinoma cells[J].Mol Biol Cell,2007,18(8):3144-3155.
[4]Zetterberg E,Carlsson AM,Najm J,et al.Thrombin generation in two families with MYH9-related platelet disorder[J].Platelets,2016,27(3):264-267.
[5]Thomas DG,Yenepalli A,Denais CM,et al.Non-muscle myosin IIB is critical for nuclear translocation during 3Dinvasion[J].J Cell Biol,2015,210(4):583-594.
[6]He H,Wang D,Yao H,et al.Transcriptional factors p300and MRTF-A synergistically enhance the expression of migration-related genes in MCF-7breast cancer cells[J].Biochem Biophys Res Commun,2015,467(4):813-820.
[7]徐宝国,杨永栋,于霞,等.胃癌组织C-Met和MMP-11表达与肝转移相关性研究[J].中华肿瘤防治杂志,2014,21(9):678-683.
[8]Sellers JR.Myosins:a diverse superfamily[J].Biochim Biophys Acta,2000,1496(1):3-22.
[9]Katono K,Sato Y,Jiang SX,et al.Prognostic significance of MYH9expression in resected non-small cell lung cancer[J/CD].PLoS One,2015,10(3):e121460.
[10]Xia ZK,Yuan YC,Yin N,et al.Nonmuscle myosinⅡA is associated with poor prognosis of esophageal squamous cancer[J].Dis Esophagus,2012,25(5):427-436.
[11]Liu D,Zhang L,Shen Z,et al.Clinicopathological significance of MYH-9overexpression in human gastric cancer[J].Int J Mol Sci,2012,13(11):15291-15304.
[12]Du M,Wang G,Ismail TM,et al.S100Pdissociates myosin IIA filaments and focal adhesion sites to reduce cell adhesion and enhance cell migration[J].J Biol Chem,2012,287(19):15330-15344.
[13]Arora S,Saha S,Roy S,et al.Role of Nonmuscle MyosinⅡin Migration of Wharton’s Jelly-Derived Mesenchymal Stem Cells[J].Stem Cells Dev,2015,24(17):2065-2077.
[14]Komatsu S,Ikebe M.ZIPK is critical for the motility and contractility of VSMCs through the regulation of nonmuscle myosinⅡisoforms[J].Am J Physiol Heart Circ Physiol,2014,306(9):H1275-H1286.
[15]Conti CM,Saleh AD,Brinster LR,et al.Conditional deletion of nonmuscle myosinⅡ-A in mouse tongue epithelium results in squamous cell carcinoma[J].Sci Rep,2015,5:14068.
[16]Schramek D,Sendoel A,Segal JP,et al.Direct in vivo RNAi screen unveils myosinⅡa as a tumor suppressor of squamous cell carcinomas[J].Science,2014,343(6168):309-313.
[17]Chen Z,Huang Y,Shen X,et al.Short hairpin RNA targeting autotaxin reduces human gastric carcinoma AGS cell proliferative,migratory and invasive capabilities in vitro and causes tumor regression in vivo[J].Oncol Rep,2013,29(3):1087-1093.
[18]Huang Y,Arora P,Mcculloch CA,et al.The collagen receptor DDR1regulates cell spreading and motility by associating with myosinⅡA[J].J Cell Sci,2009,122(Pt 10):1637-1646.
[19]Elliott PR,Irvine AF,Jung HS,et al.Asymmetric mode of Ca2+-S100A4interaction with nonmuscle myosinⅡA generates nanomolar affinity required for filament remodeling[J].Structure,2012,20(4):654-666.
[20]侯继院,刘兴安,单国用,等.S100A4调节MMP-13表达对甲状腺癌细胞侵袭能力的影响[J].中华肿瘤防治杂志,2015,22(7):514-518.
[21]Chai J,Jamal MM.S100A4in esophageal cancer:is this the one to blame?[J].World J Gastroenterol,2012,18(30):3931-3935.
[22]邓春阳,冯建勋,张海英,等.羊水干细胞诱导肾移植免疫耐受及对氧化应激的影响[J].中国组织工程研究,2015,(45):7342-7349.
[23]贠志敏,李素波,张雪,等.酶解法清除猪皮a-Gal抗原的研究[J].军事医学,2015,39(12):938-940.
[24]储成艳,吴云红,朱亮.巨噬细胞在移植排斥中的作用及其机制的研究进展[J].细胞与分子免疫学杂志,2015,31(12):1721-1723,1727.