靶向siRNA干扰hIAP-2联合姜黄素抑制肾癌GRC-1细胞增殖的实验研究
|
摘要
目的应用hIAP-2特异性siRNA干扰肾癌GRC-1细胞hIAP-2基因的表达,探讨siRNA-hIAP-2联合姜黄素对肾癌GRC-1细胞增殖和凋亡的影响。方法体外化学合成针对hIAP-2的siRNA,通过阳离子脂质体将siRNA-hIAP-2转染肾癌GRC-1细胞,实时荧光定量PCR检测hIAP-2 m RNA表达水平,免疫印迹检测其蛋白表达水平,MTT法及流式细胞术分别测定siRNA-hIAP-2联合姜黄素处理后对肾癌GRC-1细胞增殖及凋亡的影响。结果靶向hIAP-2的siRNA可以有效抑制肾癌GRC-1细胞的hIAP-2的表达,siRNA-hIAP-2和姜黄素均可抑制GRC-1细胞增殖并诱导其一定程度的凋亡,当联合应用靶向hIAP-2的siRNA和姜黄素时,上述作用显著增强(P<0.05)。结论体外化学合成的siRNA-hIAP-2可以有效抑制肾癌GRC-1细胞的hIAP-2的表达,并能显著增强姜黄素诱导肾癌细胞GRC-1凋亡的敏感度,提示hIAP-2可作为姜黄素治疗肾癌的有效增敏靶点。
Objective To investigate the therapeutic effect of targeting siRNA-hIAP-2 combined curcumin against GRC-1 cell line. Methods Renal cancer cell GRC-1 was cultured and transfected with siRNA-hIAP-2. Then real time PCR and Western blotting were performed to detect the changes of m RNA and protein of hIAP-2. Finally, MTT assay and flow cytometry were used to analyse the proliferation and apoptosis of targeting hIAP-2 combined curcumin against GRC-1 cell line. Result Si RNA-hIAP-2 could down-regulate the expression of hIAP-2 m RNA and protein. Both curcumin and siRNA-hIAP-2 could induce apoptosis and inhibit proliferation in GRC-1 cell line. Moreover, a synergic effect against proliferation and survival of combined siRNA-hIAP-2 with curcumin in GRC-1 cell line was noted. Conclusion Downregulation of the expression of hIAP-2 could enhance the chemosensitivity of GRC-1 to curcumin, suggesting that hIAP-2 could be a potential therapeutic target to sensitize renal cell carcinoma to curcumin.
Objective To investigate the therapeutic effect of targeting siRNA-hIAP-2 combined curcumin against GRC-1 cell line. Methods Renal cancer cell GRC-1 was cultured and transfected with siRNA-hIAP-2. Then real time PCR and Western blotting were performed to detect the changes of m RNA and protein of hIAP-2. Finally, MTT assay and flow cytometry were used to analyse the proliferation and apoptosis of targeting hIAP-2 combined curcumin against GRC-1 cell line. Result Si RNA-hIAP-2 could down-regulate the expression of hIAP-2 m RNA and protein. Both curcumin and siRNA-hIAP-2 could induce apoptosis and inhibit proliferation in GRC-1 cell line. Moreover, a synergic effect against proliferation and survival of combined siRNA-hIAP-2 with curcumin in GRC-1 cell line was noted. Conclusion Downregulation of the expression of hIAP-2 could enhance the chemosensitivity of GRC-1 to curcumin, suggesting that hIAP-2 could be a potential therapeutic target to sensitize renal cell carcinoma to curcumin.
引文
[1]Tamm I,Wang Y,Sausville E,et al.IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas(CD95),Bax,caspases,and anticancer drugs[J].Cancer Res,1998,58(23):5315-5320.
[2]Wang Y,Luo YH.The Inhibitory Effect of h IAP-2-si RNA on Hep G2 cells[J].Anti-tumor Pharmacy,2011,1(5):426-430.[王杨,罗映晖.h IAP-2-si RNA对hep G2肝癌细胞抑制作用[J].肿瘤药学,2011,1(5):426-430.]
[3]Guo XD,Xu JH.Research progress on antitumor activity of curcumin and its derivatives[J].Strait Pharmaceut J,2011,23(6):15-18.[郭晓丹,许建华.姜黄素及其衍生物抗肿瘤作用的研究进展[J].海峡药学,2011,23(6):15-18.]doi:10.3969/j.issn.1006-3765.2011.06.005.
[4]Ma HM,Xiong L,Liao W.Effects of curcumin on human renal cell carcinoma 786-O cells in vitro[J].Lishizhen Med Materia Med Res,2012,23(12):2995-2996.[马华谋,熊亮,廖伟,等.姜黄素对人肾透明细胞癌细胞株786-O作用的实验研究[J].时珍国医国药,2012,23(12):2995-2996.]doi:10.3969/j.issn.1008-0805.2012.12.020.
[5]Liu HY,Wang HY,Ye S,et al.Research progress of curcumin pharmacological action and mechanism[J].China J Mod Med,2012,22(6):48-51.[刘红艳,王海燕,叶松,等.姜黄素药理作用及其机制研究进展[J].中国现代医药杂志,2012,22(6):48-51.]
[6]Liu SH,Wang XH.Research progress of inhibitor of apoptosis protein-2[J].Anhui Med Pharmaceut J,2009,13(1):11-12.[刘善浩,汪兴洪.细胞凋亡抑制蛋白-2研究进展[J].安徽医药,2009,13(1):11-12.]doi:10.3969/j.issn.1009-6469.2009.01.005.
[7]Guo C.RNAi-mediated silencing of the h IAP-2 gene inhibits the proliferation and invasion of SMMC-7721 cells[D].Changsha:Central South University,2014.[郭超.si RNA沉默h IAP-2基因表达对肝癌细胞SMMC-7721增殖与侵袭能力的影响[D].长沙:中南大学,2014.]
[8]Debata PR,Begum S,Mata A,et al.Curcumin potentiates the ability of sunitinib to eliminate the VHL-lacking renal cancer cells 786-O:rapid inhibition of Rb phosphorylation as a preamble to cyclin D1 inhibition[J].Anticancer Agents Med Chem,2013,13(10):1508-1513.
[9]Jin Z,Cai Y,Zhang Y,et al.The effect of curcumin on PI3-K/Akt and MEK/ERK pathway in colon cancer[J].J Mod Oncol,2015,23(12):1629-1631.[金子,蔡颖,张晔,等.姜黄素对人结肠癌细胞PI3-K/Akt和MEK/ERK信号通路的影响[J].现代肿瘤医学,2015,23(12):1629-1631.]doi:10.3969/j.issn.1672-4992.2015.12.01.
[10]Liu TY,Tan ZJ,Jiang L,et al.Curcumin induces apoptosis in gallbladder carcinoma cell line GBC-SD cells[J].Cancer Cell Int,2013,13(1):64.doi:10.1186/1475-2867-13-64.
[11]Liao H,Wang Z,Deng Z,et al.Curcumin inhibits lung cancer invasion and metastasis by attenuating GLUT1/MT1-MMP/MMP2 pathway[J].Int J Clin Exp Med,2015,8(6):8948-8957.
[12]Lu WD,Qin Y,Yang C,et al.Effect of curcumin on human colon cancer multidrug resistance in vitro and in vivo[J].Clinics(Sao Paulo),2013,68(5):694-701.doi:10.6061/clinics/2013(05)18.
[13]Chen P,Li J,Jiang HG,et al.Curcumin reverses cisplatin resistance in cisplatin-resistant lung caner cells by inhibiting FA/BRCA pathway[J].Tumour Biol,2015,36(5):3591-3599.doi:10.1007/s13277-014-2996-4.
[14]Zhao X,Chen Q,Li Y,et al.Doxorubicin and curcumin codelivery by lipid nanoparticles for enhanced treatment of diethylnitrosamine-induced hepatocellular carcinoma in mice[J].Eur J Pharm Biopharm,2015,93:27-36.doi:10.1016/j.ejpb.2015.03.003.
[15]Liu ZX,Li RM,Wang XX.Effect of Chk1 Gene Silencing on Curcumin-induced Apoptosis Susceptibility of Human Gastric Cancer SGC7901 Cells[J].Cancer Res Prev Treat,2013,40(8):748-751.[刘志祥,李瑞明,王晓勋.沉默Chk1基因对姜黄素诱导胃癌细胞SGC7901凋亡敏感度的影响[J].肿瘤防治研究,2013,40(8):748-751.]doi:10.3971/j.issn.1000-8578.2013.08.006.
[16]Wu ZJ.The Effect of Lentivius-Mediated Stable RNAi Targeting bcr/abl Gene on Biological Characters and Curcumin Antitumor Action in K562 Cell Line[D].Fuzhou:Fujian Medical University,2009.[吴枝娟.慢病毒介导bcr/abl基因长期沉默对K562细胞生物学特性与姜黄素抗肿瘤作用影响及其相关机制研究[D].福州:福建医科大学,2009.]
[17]Li XY,Shao SL,Zhang WW,et al.Plasmid mediated RNAi silencing of MDR1 gene enhanced the sensitivity of leukemia mdr cell HT9 to curcumin[J].Jiangsu Agricul Sci,2014,42(1):22-25.[李旭艳,邵淑丽,张伟伟,等.质粒介导的RNAi沉默mdr1基因增强白血病耐药细胞HT9对姜黄素的敏感性[J].江苏农业科学,2014,42(1):22-25.]
[2]Wang Y,Luo YH.The Inhibitory Effect of h IAP-2-si RNA on Hep G2 cells[J].Anti-tumor Pharmacy,2011,1(5):426-430.[王杨,罗映晖.h IAP-2-si RNA对hep G2肝癌细胞抑制作用[J].肿瘤药学,2011,1(5):426-430.]
[3]Guo XD,Xu JH.Research progress on antitumor activity of curcumin and its derivatives[J].Strait Pharmaceut J,2011,23(6):15-18.[郭晓丹,许建华.姜黄素及其衍生物抗肿瘤作用的研究进展[J].海峡药学,2011,23(6):15-18.]doi:10.3969/j.issn.1006-3765.2011.06.005.
[4]Ma HM,Xiong L,Liao W.Effects of curcumin on human renal cell carcinoma 786-O cells in vitro[J].Lishizhen Med Materia Med Res,2012,23(12):2995-2996.[马华谋,熊亮,廖伟,等.姜黄素对人肾透明细胞癌细胞株786-O作用的实验研究[J].时珍国医国药,2012,23(12):2995-2996.]doi:10.3969/j.issn.1008-0805.2012.12.020.
[5]Liu HY,Wang HY,Ye S,et al.Research progress of curcumin pharmacological action and mechanism[J].China J Mod Med,2012,22(6):48-51.[刘红艳,王海燕,叶松,等.姜黄素药理作用及其机制研究进展[J].中国现代医药杂志,2012,22(6):48-51.]
[6]Liu SH,Wang XH.Research progress of inhibitor of apoptosis protein-2[J].Anhui Med Pharmaceut J,2009,13(1):11-12.[刘善浩,汪兴洪.细胞凋亡抑制蛋白-2研究进展[J].安徽医药,2009,13(1):11-12.]doi:10.3969/j.issn.1009-6469.2009.01.005.
[7]Guo C.RNAi-mediated silencing of the h IAP-2 gene inhibits the proliferation and invasion of SMMC-7721 cells[D].Changsha:Central South University,2014.[郭超.si RNA沉默h IAP-2基因表达对肝癌细胞SMMC-7721增殖与侵袭能力的影响[D].长沙:中南大学,2014.]
[8]Debata PR,Begum S,Mata A,et al.Curcumin potentiates the ability of sunitinib to eliminate the VHL-lacking renal cancer cells 786-O:rapid inhibition of Rb phosphorylation as a preamble to cyclin D1 inhibition[J].Anticancer Agents Med Chem,2013,13(10):1508-1513.
[9]Jin Z,Cai Y,Zhang Y,et al.The effect of curcumin on PI3-K/Akt and MEK/ERK pathway in colon cancer[J].J Mod Oncol,2015,23(12):1629-1631.[金子,蔡颖,张晔,等.姜黄素对人结肠癌细胞PI3-K/Akt和MEK/ERK信号通路的影响[J].现代肿瘤医学,2015,23(12):1629-1631.]doi:10.3969/j.issn.1672-4992.2015.12.01.
[10]Liu TY,Tan ZJ,Jiang L,et al.Curcumin induces apoptosis in gallbladder carcinoma cell line GBC-SD cells[J].Cancer Cell Int,2013,13(1):64.doi:10.1186/1475-2867-13-64.
[11]Liao H,Wang Z,Deng Z,et al.Curcumin inhibits lung cancer invasion and metastasis by attenuating GLUT1/MT1-MMP/MMP2 pathway[J].Int J Clin Exp Med,2015,8(6):8948-8957.
[12]Lu WD,Qin Y,Yang C,et al.Effect of curcumin on human colon cancer multidrug resistance in vitro and in vivo[J].Clinics(Sao Paulo),2013,68(5):694-701.doi:10.6061/clinics/2013(05)18.
[13]Chen P,Li J,Jiang HG,et al.Curcumin reverses cisplatin resistance in cisplatin-resistant lung caner cells by inhibiting FA/BRCA pathway[J].Tumour Biol,2015,36(5):3591-3599.doi:10.1007/s13277-014-2996-4.
[14]Zhao X,Chen Q,Li Y,et al.Doxorubicin and curcumin codelivery by lipid nanoparticles for enhanced treatment of diethylnitrosamine-induced hepatocellular carcinoma in mice[J].Eur J Pharm Biopharm,2015,93:27-36.doi:10.1016/j.ejpb.2015.03.003.
[15]Liu ZX,Li RM,Wang XX.Effect of Chk1 Gene Silencing on Curcumin-induced Apoptosis Susceptibility of Human Gastric Cancer SGC7901 Cells[J].Cancer Res Prev Treat,2013,40(8):748-751.[刘志祥,李瑞明,王晓勋.沉默Chk1基因对姜黄素诱导胃癌细胞SGC7901凋亡敏感度的影响[J].肿瘤防治研究,2013,40(8):748-751.]doi:10.3971/j.issn.1000-8578.2013.08.006.
[16]Wu ZJ.The Effect of Lentivius-Mediated Stable RNAi Targeting bcr/abl Gene on Biological Characters and Curcumin Antitumor Action in K562 Cell Line[D].Fuzhou:Fujian Medical University,2009.[吴枝娟.慢病毒介导bcr/abl基因长期沉默对K562细胞生物学特性与姜黄素抗肿瘤作用影响及其相关机制研究[D].福州:福建医科大学,2009.]
[17]Li XY,Shao SL,Zhang WW,et al.Plasmid mediated RNAi silencing of MDR1 gene enhanced the sensitivity of leukemia mdr cell HT9 to curcumin[J].Jiangsu Agricul Sci,2014,42(1):22-25.[李旭艳,邵淑丽,张伟伟,等.质粒介导的RNAi沉默mdr1基因增强白血病耐药细胞HT9对姜黄素的敏感性[J].江苏农业科学,2014,42(1):22-25.]