肌动蛋白2基因(ACTG2)对肝癌肿瘤细胞侵袭转移促进作用的实验研究
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摘要
目的:明确平滑肌肠道肌动蛋白ACTG2在肝癌细胞(HCC)侵袭转移过程中的作用,为治疗HCC转移提供有希望的治疗靶标。方法:通过病毒逆转录载体沉默ACTG2即制备shACTG2,通过Western Blot、免疫荧光、PCR实验验证ACTG2被成功干扰,接着以体外划痕实验、Transwell实验,考察Control组、Scramble组及shACTG2组肝癌细胞侵袭与转移与ACTG2表达的相关性;qRT-PCR用来扩增ACTG2mRNA,采用Western Blot验证ACTG2-OE高表达模型成功建立后,通过体内裸鼠实验性转移模型,在尾静脉注射HepG2细胞、ACTG2-OE、ACTG2shRNA质粒转染的SMMC-7721细胞,考察裸鼠体内肝癌转移至肺的节结数。结果:shACTG2组无论在划痕实验还是Transwell实验都显示出较低侵袭转移的倾向,并且与HepG2对照组相比具有显著性差异,体内实验结果也显示肺结节转移数shACTG2组最少,与对照组及ACTG2-OE高表达组比较均具有显著性差异。结论:ACTG2在HCC细胞侵袭转移中具有促进作用。
Objective:To identify the role of ACTG2 in the metastatic process of hepatocellular carcinoma(HCC) cells,we did the following experiments hoping to provide therapeutic targets for treating HCC metastases.Methods:ACTG2 was silenced by building a lentivirus silence retroviral vector(shACTG2),and the ACTG2 interference were then validated by using Western blot,immunofluorescence and PCR tests.Wound healing assay and transwell experiments in vitro were used to investigate the correlation of metastasis and ACTG2 through the Control group,Scramble group and shACTG2 groups;QRT-PCR were used to amplify ACTG2 mRNA and Western blot were used to test whether an ACTG2 over expression model(ACTG2-OE) was successfully established.After lateral tail vein injection of HepG2 cells,ACTG2-OE HepG2 cells or ACTG2 shRNA plasmid transfected SMMC-7721 cells,the number of knots of liver cancer transferred to lungs in nude mice were counted.Results:In both wound healing assay and transwell experiments,cells in the shACTG2 group showed lower tendency of invasion and metastasis compared with those of the HepG2 control group(P<0.01).In vivo experiment results also showed that lung nodular metastases in the shACTG2 group were the lowest,and were significantly different from those in the control group and the ACTG2-OE group.Conclusion:ACTG2 plays a role in the migration of HCC cells.
Objective:To identify the role of ACTG2 in the metastatic process of hepatocellular carcinoma(HCC) cells,we did the following experiments hoping to provide therapeutic targets for treating HCC metastases.Methods:ACTG2 was silenced by building a lentivirus silence retroviral vector(shACTG2),and the ACTG2 interference were then validated by using Western blot,immunofluorescence and PCR tests.Wound healing assay and transwell experiments in vitro were used to investigate the correlation of metastasis and ACTG2 through the Control group,Scramble group and shACTG2 groups;QRT-PCR were used to amplify ACTG2 mRNA and Western blot were used to test whether an ACTG2 over expression model(ACTG2-OE) was successfully established.After lateral tail vein injection of HepG2 cells,ACTG2-OE HepG2 cells or ACTG2 shRNA plasmid transfected SMMC-7721 cells,the number of knots of liver cancer transferred to lungs in nude mice were counted.Results:In both wound healing assay and transwell experiments,cells in the shACTG2 group showed lower tendency of invasion and metastasis compared with those of the HepG2 control group(P<0.01).In vivo experiment results also showed that lung nodular metastases in the shACTG2 group were the lowest,and were significantly different from those in the control group and the ACTG2-OE group.Conclusion:ACTG2 plays a role in the migration of HCC cells.
引文
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[2]Jiang G,Zhang L,Zhu Q,et al.CD146 promotes metastasis and predicts poor prognosis of hepatocellular carcinoma[J].J Exp Clin Cancer Res,2016,35:38.
[3]Edfeldt K,Hellman P,Westin G,et al.A plausible role for actin gamma smooth muscle 2(ACTG2)in small intestinal neuroendocrine tumorigenesis[J].BMCEndocr Disord,2016,16:19.
[4]Choi YK,Cho SG,Woo SM,et al.Herbal extract SH003 suppresses tumor growth and metastasis of MDA-MB-231 breast cancer cells by inhibiting STAT3-IL-6 signaling[J].Mediators Inflamm,2014,2014:492173.
[5]Ono K,Wang X,Han J.Resistance to tumor necrosis factor-induced cell death mediated by PMCA4 deficiency[J].Mol Cell Biol,2001,21(24):8276-88.
[6]Miao Y,Zheng W,Li N,et al.MicroRNA-130b targets PTEN to mediate drug resistance and proliferation of breast cancer cells via the PI3K/Akt signaling pathway[J].Sci Rep,2017,7:41942.
[7]Tang B,Tang F,Wang Z,et al.Overexpression of CTNND1 in hepatocellular carcinoma promotes carcinous characters through activation of Wnt/beta-catenin signaling[J].J Exp Clin Cancer Res,2016,35(1):82.
[8]Benzoubir N,Mussini C,Lejamtel C,et al.Gammasmooth muscle actin expression is associated with epithelial-mesenchymal transition and stem-like properties in hepatocellular carcinoma[J].PLoS One,2015,10(6):e0130559.
[9]Mas VR,Maluf DG,Archer KJ,et al.Genes involved in viral carcinogenesis and tumor initiation in hepatitis C virus-induced hepatocellular carcinoma[J].Mol Med,2009,15(3-4):85-94.
[10]Wurmbach E,Chen YB,Khitrov G,et al.Genomewide molecular profiles of HCV-induced dysplasia and hepatocellular carcinoma[J].Hepatology,2007,45(4):938-47.
[11]Wu Yu,Liu Zhao Guo,Shi Mei Qin,et al.Identification of ACTG2 functions as a promoter gene in hepatocellular carcinoma cells migration and tumor metastasis[J].Biochem Biophys Res Commun,2017,491(2):537-44.