基于RNAi技术研究参芪扶正注射液联合IFN-α对肝癌细胞增殖能力的影响
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摘要
目的:研究参芪扶正注射液与干扰素α(IFN-α)联用时对肝癌细胞增殖能力的影响,并通过RNA干扰技术明确其对IFN-α协同增效的作用机制。方法:运用细胞增殖实验(MTT)技术检测IFN-α注射液(6000 IU)单独或与参芪扶正注射液(0.5 g/L)联合时对SMMC-7721细胞增殖能力的影响;用实时荧光定量聚合酶链反应(Real-time PCR)技术和蛋白质印迹法(Western-blot)分别检测IFN-α单独或与参芪扶正注射液联用时,实验组、阴性对照组(NaCl)和空白对照组中STAT1的RNA转录和蛋白质表达情况,确定参芪扶正注射液对STAT1转录和表达的影响;运用Western blot检验STAT1基因经RNA干扰后SMMC-7721的STAT1蛋白质表达,确定干扰效果;MTT实验检测IFN-α单独或联合参芪扶正注射液作用于STAT1基因"敲低"细胞株时,对细胞增殖能力的影响。结果:与单独使用IFN-α及参芪扶正注射液相比,小剂量参芪扶正注射液与IFN-α联用可以增强IFN-α对人肝癌细胞SMMC-7721增殖能力的抑制作用,并上调STAT1 mRNA和蛋白质的表达;SMMC-7721的STAT1基因"敲低"细胞株中STAT1蛋白表达明显降低;针对SMMC-7721的STAT1基因"敲低"细胞株,参芪扶正注射液协同IFN-α与单独IFN-α及阴性对照组相比,对细胞增殖能力的影响无显著性差异。结论:参芪扶正注射液通过上调人肝癌细胞SMMC-7721中STAT1的表达,是提升干扰素α对肝癌细胞增殖能力抑制作用的主要机制之一。
Objective: To study the effect of Shenqi Fuzheng Injection Combined with interferon alpha(IFN-alpha) on the proliferation of hepatocellular carcinoma cells, and to clarify the mechanism of its synergistic effect on IFN-alpha by RNA interference. Methods: By using cell proliferation assay(MTT) detection of interferon alpha(IFN-alpha) injection(6000 IU) alone or with Shenqifuzheng Injection(0.5 g/L) effects on the proliferation ability of SMMC-7721 cells combined; using real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) and Western blot(Western-blot) were detected respectively. IFN-alone or with Shenqi Fuzheng Injection Combined with, the experimental group, negative control group(NaCl) and STAT1 control group the expression of RNA mRNA and protein, to determine the effect of Shenqi Fuzheng Injection on the expression and transcription of STAT1; expression of STAT1 protein by Western blot test of STAT1 gene by RNA interference SMMC-7721 sure, the jamming effect is stable; IFN-alpha MTT assay alone or combined With Shenqifuzheng Injection on STAT1 gene knockdown cell lines, the The effect of cell proliferation.Results: Compared with the use of IFN-alpha and Shenqi Fuzheng injection alone, can inhibit the enhancement of IFN-alpha on the proliferation of human hepatocellular carcinoma SMMC-7721 cells combined with small dose of Shenqifuzheng injection and IFN-alpha, and STAT1 mRNA up-regulated expression and protein; STAT1 gene SMMC-7721 was knocked decreased expression of STAT1 protein and low cell lines; "SMMC-7721 for STAT1 gene knockdown cell lines, Shenqifuzheng injection compared with single IFN-collaborative IFN-alpha alpha and negative control group, no significant effect on cell proliferation. Conclusion: Shenqifuzheng injection through the expression of STAT1 in human hepatocellular carcinoma cells SMMC-7721, is one of the main mechanisms effectively enhance the inhibitory effect of interferon alpha on the proliferation of hepatoma cells.
Objective: To study the effect of Shenqi Fuzheng Injection Combined with interferon alpha(IFN-alpha) on the proliferation of hepatocellular carcinoma cells, and to clarify the mechanism of its synergistic effect on IFN-alpha by RNA interference. Methods: By using cell proliferation assay(MTT) detection of interferon alpha(IFN-alpha) injection(6000 IU) alone or with Shenqifuzheng Injection(0.5 g/L) effects on the proliferation ability of SMMC-7721 cells combined; using real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) and Western blot(Western-blot) were detected respectively. IFN-alone or with Shenqi Fuzheng Injection Combined with, the experimental group, negative control group(NaCl) and STAT1 control group the expression of RNA mRNA and protein, to determine the effect of Shenqi Fuzheng Injection on the expression and transcription of STAT1; expression of STAT1 protein by Western blot test of STAT1 gene by RNA interference SMMC-7721 sure, the jamming effect is stable; IFN-alpha MTT assay alone or combined With Shenqifuzheng Injection on STAT1 gene knockdown cell lines, the The effect of cell proliferation.Results: Compared with the use of IFN-alpha and Shenqi Fuzheng injection alone, can inhibit the enhancement of IFN-alpha on the proliferation of human hepatocellular carcinoma SMMC-7721 cells combined with small dose of Shenqifuzheng injection and IFN-alpha, and STAT1 mRNA up-regulated expression and protein; STAT1 gene SMMC-7721 was knocked decreased expression of STAT1 protein and low cell lines; "SMMC-7721 for STAT1 gene knockdown cell lines, Shenqifuzheng injection compared with single IFN-collaborative IFN-alpha alpha and negative control group, no significant effect on cell proliferation. Conclusion: Shenqifuzheng injection through the expression of STAT1 in human hepatocellular carcinoma cells SMMC-7721, is one of the main mechanisms effectively enhance the inhibitory effect of interferon alpha on the proliferation of hepatoma cells.
引文
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[12]Fu S,Wei J,Wang G,et al.The key role of PML in IFN-αinduced cellular senescence of human mesenchymal stromal cells[J].Inter J Oncol,2015,46(1):351-359.
[13]樊志伟.参芪扶正注射液联合干扰素-α治疗中晚期原发性肝癌的临床及实验研究[D].北京中医药大学,2012.
[14]Hamano Y,Zeisberg M,Sugimoto H,et al.Physiological levels of tumstatin,a fragment of collagen IVα3 chain,are generated by MMP-9 proteolysis and suppress angiogenesis viaαVβ3 integrin[J].Cancer Cell,2003,3(6):589.
[15]Harris B,Roeder RG.Structural relationships of low molecular weight viral RNAs synthesized by RNA polymeraseⅢin nuclei from adenovirus 2-infected cells[J].J Biol Chem,1978,253(12):4120-4127.
[16]张岩,田素礼.RNA干扰技术在肝细胞癌治疗方面的研究现状及进展[J].中国现代普通外科进展,2014,17(5):370-374.
[17]丁治国,何蓓,陈晓珩,等.STAT1干扰慢病毒载体的构建及其基因沉默效应测定[J].中国现代普通外科进展,2015,18(11):841-844.
[18]丁治国,何蓓,陈晓珩,等.针对人STAT1基因的RNA干扰有效靶点的设计与筛选[J].中国现代普通外科进展,2015,18(12):925-928.
[19]丁治国,何蓓,陈晓珩,等.STAT1过表达慢病毒载体的构建及其在SMMC-7721细胞中的表达[J].中国现代普通外科进展,2013,16(12):931-934.
[20]吕彬,李盼,马向慧,等.参芪扶正注射液药效物质基础的研究及抗肿瘤作用靶点的预测[J].中国药理学通报,2015(B11):202-202.
[2]Pugnale P,Pazienza V,Guilloux K,et al.Hepatitis delta virus inhibits alpha interferon signaling[J].Hepatology,2009,49(2):398.
[3]Li T,Dong ZR,Guo ZY,et al.Aspirin enhances IFN-α-induced growth inhibition and apoptosis of hepatocellular carcinoma via JAK1/STAT1 pathway[J].Cancer Gene Therapy,2013,20(6):366.
[4]汪唐顺,陈晓珩,丁治国,等.参芪扶正注射液联合干扰素-α治疗中晚期原发性肝癌[J].中国实验方剂学杂志,2014,20(18):174-177.
[5]刘国栋,肖桂林.参芪扶正注射液联合化疗治疗原发性肝癌的临床疗效观察[J].现代生物医学进展,2010,10(7):1348-1350.
[6]占国清,李儒贵,谭华炳,等.参芪扶正注射液对中晚期肝癌肝动脉化疗栓塞术后患者生存质量的影响[J].湖北医药学院学报,2013(4):325-328.
[7]丁治国,李乃卿,陶德胜,等.参芪扶正注射液对小鼠肝转移癌组织基因表达谱的影响[J].中国中西医结合杂志,2008,28(2):135-138.
[8]高翔.慢病毒介导的STAT1基因过表达和STAT1基因沉默在肝癌MHCC97L细胞中的应用[D].北京中医药大学,2014.
[9]陈晓珩.参芪扶正注射液通过STAT1协同干扰素治疗肝细胞癌的机制研究[D].北京中医药大学,2017.
[10]Cha L,Berry CM,Nolan D,et al.Interferon-alpha,immune activation and immune dysfunction in treated HIV infection[J].Clin Transl Immunol,2014,3(2):e10.
[11]Wang G,Sun Y,He Y,et al.miR-26a Promoted by Interferon-Alpha Inhibits Hepatocellular Carcinoma Proliferation and Migration by Blocking EZH2[J].Genet Test Mol Biomarkers,2015,19(1):30-36.
[12]Fu S,Wei J,Wang G,et al.The key role of PML in IFN-αinduced cellular senescence of human mesenchymal stromal cells[J].Inter J Oncol,2015,46(1):351-359.
[13]樊志伟.参芪扶正注射液联合干扰素-α治疗中晚期原发性肝癌的临床及实验研究[D].北京中医药大学,2012.
[14]Hamano Y,Zeisberg M,Sugimoto H,et al.Physiological levels of tumstatin,a fragment of collagen IVα3 chain,are generated by MMP-9 proteolysis and suppress angiogenesis viaαVβ3 integrin[J].Cancer Cell,2003,3(6):589.
[15]Harris B,Roeder RG.Structural relationships of low molecular weight viral RNAs synthesized by RNA polymeraseⅢin nuclei from adenovirus 2-infected cells[J].J Biol Chem,1978,253(12):4120-4127.
[16]张岩,田素礼.RNA干扰技术在肝细胞癌治疗方面的研究现状及进展[J].中国现代普通外科进展,2014,17(5):370-374.
[17]丁治国,何蓓,陈晓珩,等.STAT1干扰慢病毒载体的构建及其基因沉默效应测定[J].中国现代普通外科进展,2015,18(11):841-844.
[18]丁治国,何蓓,陈晓珩,等.针对人STAT1基因的RNA干扰有效靶点的设计与筛选[J].中国现代普通外科进展,2015,18(12):925-928.
[19]丁治国,何蓓,陈晓珩,等.STAT1过表达慢病毒载体的构建及其在SMMC-7721细胞中的表达[J].中国现代普通外科进展,2013,16(12):931-934.
[20]吕彬,李盼,马向慧,等.参芪扶正注射液药效物质基础的研究及抗肿瘤作用靶点的预测[J].中国药理学通报,2015(B11):202-202.