龙葵碱联合KLF16基因对胶质瘤细胞增殖、凋亡的影响及其机制研究
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摘要
目的探讨龙葵碱联合Krüppel样转录因子16(KLF16)基因对人胶质瘤U87细胞增殖和凋亡的影响及作用机制。方法体外培养人胶质瘤U87细胞,将特异性KLF16 siRNA和阴性对照siRNA Control转染U87细胞,分别命名为si-KLF16组和si-NC组,应用实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹(Western blot)检测转染后细胞中KLF16的表达情况。使用不同浓度(2.5、5、10、20、30μg/μL)的龙葵碱分别作用si-NC组和si-KLF16组细胞48 h,噻唑蓝(MTT)法测定细胞增殖抑制率并筛选半数抑制浓度(IC50),以IC50为龙葵碱后续实验浓度,根据是否添加龙葵碱处理实验分为4组,si-NC组、si-KLF16组、si-NC+Sol组、si-KLF16+Sol组,分别使用MTT和流式细胞仪测定各组细胞增殖和凋亡能力,Western blot分析各组细胞中Ki-67、PCNA、Bax和Bcl-2蛋白表达情况。结果胶质瘤U87细胞转染后,si-KLF16组细胞中KLF16 mRNA和蛋白表达显著低于si-NC组(P<0.05)。筛选出浓度为20μg/μL的龙葵碱作为后续实验浓度。与si-NC组比,si-KLF16组、si-NC+Sol组和si-KLF16+Sol组细胞增殖能力均受到明显抑制(P<0.05),凋亡率升高(P<0.05),Ki-67、PCNA和Bcl-2蛋白表达下调(P<0.05),Bax蛋白表达上调(P<0.05)。与si-KLF16组和si-NC+Sol组比,si-KLF16+Sol组细胞增殖抑制作用和凋亡促进作用更显著(P<0.05)。结论龙葵碱联合KLF16基因能够协同抑制胶质瘤细胞增殖,诱导细胞凋亡,其作用机制可能与调控Ki-67、PCNA、Bax和Bcl-2蛋白表达有关。
Objective To investigate the effects of solanine combined with Krüppel-like transcription factor 16(KLF16)gene on proliferation and apoptosis of human glioma U87 cells. Methods Human glioma U87 cells were cultured in vitro,and specific KLF16 siRNA and negative control siRNA Control were transfected into U87 cells,named si-KLF16 group and si-NC group,respectively. The expression of KLF16 in transfected cells was detected by real-time quantitative PCR(qRT-PCR)and Western blot. The cells of si-NC group and si-KLF16 group were treated with different concentrations(2.5,5,10,20,30 μg/μL)of solanine for 48 h. The inhibition rate of cell proliferation was determined by MTT assay and half of the cells were screened. Inhibitory concentration(IC50),IC50 is the subsequent experimental concentration of Solanum. Divided into four groups according to whether or not to add the solanine treatment experiment,siNC group,si-KLF16 group,si-NC+Sol group,si-KLF16+Sol group. The proliferation and apoptosis of each group were determined by MTT and flow cytometry. The expressions of Ki-67,PCNA,Bax and Bcl-2 in each group were analyzed by Western blot. Results After transfection of glioma U87 cells,the expression of KLF16 mRNA and protein in si-KLF16 group was significantly lower than that in si-NC group(P<0.05).Solanine at a concentration of 20 μg/μL was selected as the subsequent experimental concentration. Compared with the si-NC group,the cell proliferation ability of the si-KLF16 group,the si-NC+Sol group and the siKLF16+Sol group were significantly inhibited(P<0.05),and the apoptotic rate was increased(P<0.05). Ki-67,PCNA and Bcl-2 protein expression was down-regulated(P<0.05),Bax protein expression was up-regulated(P<0.05). Compared with the si-KLF16 group and the si-NC + Sol group,the cell proliferation inhibition and apoptosis promotion effects of the si-KLF16+Sol group were more significant(P<0.05). Conclusion Solanum nigrum combined with KLF16 gene can synergistically inhibit glioma cell proliferation and induce apoptosis,which may be related to the regulation of Ki-67,PCNA,Bax and Bcl-2 protein expression.
Objective To investigate the effects of solanine combined with Krüppel-like transcription factor 16(KLF16)gene on proliferation and apoptosis of human glioma U87 cells. Methods Human glioma U87 cells were cultured in vitro,and specific KLF16 siRNA and negative control siRNA Control were transfected into U87 cells,named si-KLF16 group and si-NC group,respectively. The expression of KLF16 in transfected cells was detected by real-time quantitative PCR(qRT-PCR)and Western blot. The cells of si-NC group and si-KLF16 group were treated with different concentrations(2.5,5,10,20,30 μg/μL)of solanine for 48 h. The inhibition rate of cell proliferation was determined by MTT assay and half of the cells were screened. Inhibitory concentration(IC50),IC50 is the subsequent experimental concentration of Solanum. Divided into four groups according to whether or not to add the solanine treatment experiment,siNC group,si-KLF16 group,si-NC+Sol group,si-KLF16+Sol group. The proliferation and apoptosis of each group were determined by MTT and flow cytometry. The expressions of Ki-67,PCNA,Bax and Bcl-2 in each group were analyzed by Western blot. Results After transfection of glioma U87 cells,the expression of KLF16 mRNA and protein in si-KLF16 group was significantly lower than that in si-NC group(P<0.05).Solanine at a concentration of 20 μg/μL was selected as the subsequent experimental concentration. Compared with the si-NC group,the cell proliferation ability of the si-KLF16 group,the si-NC+Sol group and the siKLF16+Sol group were significantly inhibited(P<0.05),and the apoptotic rate was increased(P<0.05). Ki-67,PCNA and Bcl-2 protein expression was down-regulated(P<0.05),Bax protein expression was up-regulated(P<0.05). Compared with the si-KLF16 group and the si-NC + Sol group,the cell proliferation inhibition and apoptosis promotion effects of the si-KLF16+Sol group were more significant(P<0.05). Conclusion Solanum nigrum combined with KLF16 gene can synergistically inhibit glioma cell proliferation and induce apoptosis,which may be related to the regulation of Ki-67,PCNA,Bax and Bcl-2 protein expression.
引文
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[15]Fernandezzapico ME,Lomberk GA,Tsuji S,et al.Afunctional family-wide screening of SP/KLF proteins identifies a subset of suppressors of KRAS-Mediated cell growth[J].Biochem J,2011,435(2):529-537.
[16]Ma P,Sun CQ,Wang YF,et al.KLF16promotes proliferation in gastric cancer cells via regulating p21 and CDK4[J].Am J Transl Res,2017,9(6):3027-3036.
[17]Yi YJ,Jia XH,Wang JY,et al.Solanine induced apoptosis and increased chemosensitivity to adriamycin in T-cell acute lymphoblastic leukemia cells[J].Oncol Lett,2018,15(5):7383-7388.
[18]Wang L,Sun QQ,Zhang SJ,et al.Inhibitory effect ofα-solanine on esophageal carcinoma in vitro[J].Exp Ther Med,2016,12(3):1525-1530.
[19]郭玲,盛华均,刘茜,等.龙葵碱对U251细胞增殖及凋亡的影响[J].中草药,2017,48(10):2081-2086.
[20]Lopes JM,Hannisdal E,Bjerkehagen B,et al.Synovial sarcoma.evaluation of prognosis with emphasis on the study of DNA ploidy and proliferation(PCNA and Ki-67)markers[J].Anal Cell Pathol,2016,16(1):45-62.
[21]张新华,王庆亮,赵坤.miR-214对人舌鳞状细胞癌增殖能力影响的体外实验研究[J].中华生物医学工程杂志,2014,20(4):268-271.
[22]Renault TT,Dejean LM,Manon S.A brewing understanding of the regulation of Bax function by Bcl-xLand Bcl-2[J].Mech Ageing Dev,2017,161(Pt B):201-210.
[2]Qin L,Li A,Qu J,et al.Normalization of ADC does not improve correlation with overall survival in patients with high-grade glioma(HGG)[J].J Neuro Oncol,2018,137(2):313-319.
[3]归小龙.龙葵碱抗肿瘤作用机制研究进展[J].中国中医药信息杂志,2016,36(10):1075-1077.
[4]Meng XQ,Zhang W,Zhang F,et al.Solanine-induced reactive oxygen species inhibit the growth of human hepatocellular carcinoma HepG2 cells[J].Oncol Lett,2016,11(3):2145-2151.
[5]周怡,李影,孔鸿儒,等.龙葵碱对胰腺癌细胞裸鼠移植瘤的抑制作用及机制研究[J].肝胆胰外科杂志,2016,28(3):209-212.
[6]Mohsenikia M,Alizadeh AM,Khodayari H,et al.Therapeutic effects of dendrosomal solanine on a metastatic breast tumor[J].Life Sci,2016,148:260-267.
[7]Daftary GS,Lomberk GA,Buttar NS,et al.Detailed structural-functional analysis of the krüppel-like factor16(KLF16)transcription factor reveals novel mechanisms for silencing Sp/KLF sites involved in metabolism and endocrinology[J].J Biol Chem,2011,287(10):7010-7125.
[8]黄志宏,骆文志,蔡兴东.KLF16在肺腺癌中表达的临床意义及机制[J].中国病理生理杂志,2013,29(11):1978-1983.
[9]Gada RP,Tabbaa ZM,Lomberk GA,et al.Uterine transcription factor KLF16 regulates endometrial physiology and metabolism via cytochrome p450 enzyme inhibition[J].Fertil Steril,2011,96(3):S145-S145.
[10]Wang J,Galvao J,Beach K M,et al.Novel roles and mechanism for krüppel-like factor 16(KLF16)regulation of neurite outgrowth and ephrin receptor A5(EphA5)expression in retinal ganglion cells[J].J Biol Chem,2016,291(41):21422.
[11]Sun Q,Xu R,Xu H,et al.Extracranial metastases of high-grade glioma:the clinical characteristics and mechanism.[J].World J Surg Oncol,2017,15(1):181-186.
[12]Auffinger B,Spencer D,Pytel P,et al.The role of glioma stem cells in chemotherapy resistance and glioblastoma multiforme recurrence[J].Expert Rev Neurother,2016,15(7):741-752.
[13]Uhm JH,Porter AB.Treatment of glioma in the 21st century:an exciting decade of postsurgical treatment advances in the molecular era[J].Mayo Clin Proc,2017,92(6):995-1004.
[14]Chen X,Li S,Ke Y,et al.KLF16suppresses human glioma cell proliferation and tumourigenicity by targeting TFAM[J].Artif Cells Nanomed Biotechnol,2018,29:1-8.
[15]Fernandezzapico ME,Lomberk GA,Tsuji S,et al.Afunctional family-wide screening of SP/KLF proteins identifies a subset of suppressors of KRAS-Mediated cell growth[J].Biochem J,2011,435(2):529-537.
[16]Ma P,Sun CQ,Wang YF,et al.KLF16promotes proliferation in gastric cancer cells via regulating p21 and CDK4[J].Am J Transl Res,2017,9(6):3027-3036.
[17]Yi YJ,Jia XH,Wang JY,et al.Solanine induced apoptosis and increased chemosensitivity to adriamycin in T-cell acute lymphoblastic leukemia cells[J].Oncol Lett,2018,15(5):7383-7388.
[18]Wang L,Sun QQ,Zhang SJ,et al.Inhibitory effect ofα-solanine on esophageal carcinoma in vitro[J].Exp Ther Med,2016,12(3):1525-1530.
[19]郭玲,盛华均,刘茜,等.龙葵碱对U251细胞增殖及凋亡的影响[J].中草药,2017,48(10):2081-2086.
[20]Lopes JM,Hannisdal E,Bjerkehagen B,et al.Synovial sarcoma.evaluation of prognosis with emphasis on the study of DNA ploidy and proliferation(PCNA and Ki-67)markers[J].Anal Cell Pathol,2016,16(1):45-62.
[21]张新华,王庆亮,赵坤.miR-214对人舌鳞状细胞癌增殖能力影响的体外实验研究[J].中华生物医学工程杂志,2014,20(4):268-271.
[22]Renault TT,Dejean LM,Manon S.A brewing understanding of the regulation of Bax function by Bcl-xLand Bcl-2[J].Mech Ageing Dev,2017,161(Pt B):201-210.