慢病毒介导的IGF-1R基因沉默对肝细胞癌的增殖和迁移的影响
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摘要
目的探讨慢病毒介导的胰岛素样生长因子1受体(IGF-1R)基因沉默对肝细胞癌的增殖和迁移的影响。方法设计并筛选高效特异性胰岛素样生长因子1受体(IGF-1R)基因的小干扰RNA,构建稳定遗传的慢病毒载体质粒,观察其转染沉默IGF-1R基因的肝癌细胞株Huh7细胞增殖、迁移以及侵袭能力,进一步观察其对PI3K/Akt及Bax/Bcl-2通路的影响。结果采用倒置相差荧光显微镜观察转染效果,镜下可见较多细胞特异性表达GFP绿色荧光,转染率超过80%。scrambled阴性对照组以及空白对照组IGF-1R mRNA及蛋白的相对表达量显著高于LV-IGF-1R-RNAi转染组(P<0.01)。成功转染后,培养细胞72 h、96 h,LV-IGF-1R-RNAi转染组增值率显著低于scrambled阴性对照组以及空白对照组(P<0.05或P<0.01)。侵袭实验和迁移实验均证实细胞明显受到抑制(P<0.01)。蛋白免疫印迹法显示,LV-IGF-1R-RNAi转染组p-PI3K、p-Akt及Bcl-2相对表达量显著低于scrambled阴性对照组以及空白对照组(P<0.05);而Bax显著高于scrambled阴性对照组以及空白对照组(P<0.05)。结论RNAi技术沉默IGF-1R基因表达能显著抑制肝癌细胞株Huh7细胞的增殖、侵袭作用,而这一作用可能与抑制PI3K/Akt通路蛋白磷酸化及调节Bax/Bcl-2通路蛋白表达有关。
Objective To investigate the effects of lentivirus mediated insulin-like growth factor 1 receptor( IGF-1R) gene silencing on proliferation and migration of hepatocellular carcinoma. Methods High specificity of insulin-like growth factor 1 receptor( IGF-1R) small interfering RNA gene lentiviral vector plasmid,genetic stability were designed and screened,Huh7 cells proliferation of the transfected IGF-1R gene silencing,migration and invasion ability were observed,its effect on PI3K/Akt and Bax/Bcl-2 pathway were further observed. Results The effect of transfection was observed by inverted microscope,GFP expression of green fluorescence microscope showed more cell specificity,the transfection rate of more than 80%. Scrambled negative control group and blank control group IGF-1R mRNA and protein expression was significantly higher than that of LV-IGF-1R-RNAi group( P < 0. 01). After transfection,cells of 72 h,96 h,LV-IGF-1R-RNAi group the value of scrambled was significantly lower than the negative control group and blank control group( P < 0. 05 or P < 0. 01). Invasion assay and migration assay showed that cell was significantly inhibited( P < 0. 01). Western blotting showed that LV-IGF-1R-RNAi transfection group p-PI3 K,p-Akt and Bcl-2 expression was significantly lower than that of scrambled negative control group and blank control group( P < 0.05); while the Bax was significantly higher than that of scrambled negative control group and blank control group( P < 0. 05). Conclusion RNAi silencing IGF-1R expression can significantly inhibit Huh7 cell proliferation of hepatocellular carcinoma cell lines,invasion,and this effect may be related to the inhibition of PI3K/Akt pathway protein phosphorylation and regulation of Bax/Bcl-2 pathway protein expression.
Objective To investigate the effects of lentivirus mediated insulin-like growth factor 1 receptor( IGF-1R) gene silencing on proliferation and migration of hepatocellular carcinoma. Methods High specificity of insulin-like growth factor 1 receptor( IGF-1R) small interfering RNA gene lentiviral vector plasmid,genetic stability were designed and screened,Huh7 cells proliferation of the transfected IGF-1R gene silencing,migration and invasion ability were observed,its effect on PI3K/Akt and Bax/Bcl-2 pathway were further observed. Results The effect of transfection was observed by inverted microscope,GFP expression of green fluorescence microscope showed more cell specificity,the transfection rate of more than 80%. Scrambled negative control group and blank control group IGF-1R mRNA and protein expression was significantly higher than that of LV-IGF-1R-RNAi group( P < 0. 01). After transfection,cells of 72 h,96 h,LV-IGF-1R-RNAi group the value of scrambled was significantly lower than the negative control group and blank control group( P < 0. 05 or P < 0. 01). Invasion assay and migration assay showed that cell was significantly inhibited( P < 0. 01). Western blotting showed that LV-IGF-1R-RNAi transfection group p-PI3 K,p-Akt and Bcl-2 expression was significantly lower than that of scrambled negative control group and blank control group( P < 0.05); while the Bax was significantly higher than that of scrambled negative control group and blank control group( P < 0. 05). Conclusion RNAi silencing IGF-1R expression can significantly inhibit Huh7 cell proliferation of hepatocellular carcinoma cell lines,invasion,and this effect may be related to the inhibition of PI3K/Akt pathway protein phosphorylation and regulation of Bax/Bcl-2 pathway protein expression.
引文
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[12]Abdulamir AS,Hafidh RR,Abubakar F.Different inflammatory mechanisms in lungs of severe and mild asthma:crosstalk of NF-kappa-B,TGFbeta1,Bax,Bcl-2,IL-4 and Ig E[J].Scand J Clin Lab Invest,2016,69(4):487-495.
[2]Habib R,Akhtar J,Taqi M,et al.Lentiviral vector-mediated survivin ShRNA delivery in gastric cancer cell lines significantly inhibits cell proliferation and tumor growth[J].Oncol Rep,2016,34(2):859-867.
[3]万璟,李小毛,舒珊荣,等.慢病毒介导的靶向沉默胰岛素样生长因子1型受体siRNA对人子宫内膜癌细胞迁移和侵袭能力的影响[J].中国病理生理杂志,2012,28(8):1352-157.
[4]别彩群,黄秋燕,颜英,等.转染沉默IGF1R基因的肝癌细胞株增殖、迁移及侵袭能力观察[J].山东医药,2016,56(11):1-4.
[5]罗武,张春强.RNA干扰技术在急性脊髓损伤早期治疗中的应用[J].临床和实验医学杂志,2012,11(6):471-473,475.
[6]Mc Ginnis KM.RNAi for functional genomics in plants[J].Brief Funct Genomics,2015,9(2):111-117.
[7]Singh P,Alex JM,Bast F.Insulin receptor(IR)and insulin-like growth factor receptor1(IGF-1R)signaling systems:novel treatment strategies for cancer[J].Med Oncol,2015,31(1):805.
[8]Yan XD,Yao M,Wang L,et al.Over-expression of insulin-like growth factor-I receptor as a pertinent biomarker for hepatocytes malignant transformation[J].World J Gastroenterol,2016,19(36):6084-6092.
[9]Ding Y,Wang B,Chen X,et al.Staurosporine suppresses survival of Hep G2 cancer cells through Omi/Htr A2-mediated inhibition of PI3K/Akt signaling pathway[J].Tumour Biol,2017,39(3):1010428317694317.
[10]Matsushita K,Gotoh K,Eguchi H,et al.Agenesis of the left hepatic lobe undergoing laparoscopic hepatectomy for hepatocellular carcinoma:a case report[J].Surg Case Rep,2017,3(1):50.
[11]Rouette A,Parent S,Girouard J,et al.Cisplatin increases B-celllymphoma-2 expression via activation of protein kinase C and Akt2 in endometrial cancer cells[J].Int J Cancer,2012,130(8):1755-1767.
[12]Abdulamir AS,Hafidh RR,Abubakar F.Different inflammatory mechanisms in lungs of severe and mild asthma:crosstalk of NF-kappa-B,TGFbeta1,Bax,Bcl-2,IL-4 and Ig E[J].Scand J Clin Lab Invest,2016,69(4):487-495.