华蟾素通过miR-106a-5p/STAT3信号通路调控肺癌细胞增殖和凋亡的机制研究
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摘要
目的研究华蟾素对肺癌A549细胞增殖和凋亡的影响,并探讨其作用机制。方法以0. 15μg/ml华蟾素处理肺癌A549细胞,qRT-PCR和Western blot检测miR-106a-5p和STAT3表达量,MTT和流式细胞仪检测细胞增殖和凋亡。转染miR-106a-5p模拟剂(mimic)和STAT3 siRNA至A549细胞中,检测细胞增殖和凋亡。靶基因预测软件预测miR-106a-5p和STAT3靶向结合位点,双荧光素酶报告基因实验检测二者靶向关系。转染miR-106a-5p抑制剂(inhibitor)和pc DNA-STAT3至A549细胞中,并用0. 15μg/ml华蟾素处理,检测细胞增殖和凋亡。结果华蟾素处理的A549细胞中,miR-106a-5p上调表达,STAT3下调表达,细胞增殖抑制率和凋亡率显著升高。过表达miR-106a-5p和抑制STAT3的表达,均可提高A549细胞增殖抑制率和凋亡率。双荧光素酶报告基因实验证实miR-106a-5p和STAT3的靶向结合关系。抑制miR-106a-5p或过表达STAT3,可逆转华蟾素对A549细胞的增殖的抑制和凋亡的促进作用。结论华蟾素可抑制A549细胞的增殖,促进其凋亡,其机制可能与调控miR-106a-5p/STAT3信号通路有关,上述结论可为华蟾素治疗肺癌的分子机制提供新依据。
Objective To study the effect of cinobufotalin on the proliferation and apoptosis of A549 cells and explore its mechanism.Methods A549 cells were treated with cinobufotalin at 0. 15 μg/ml. Expressions of miR-106 a-5 p and STAT3 were detected by qRT-PCR and western blot,and cell proliferation and apoptosis were detected by MTT and flow cytometry. Cell proliferation and apoptosis were detected after transfection with miR-106 a-5 p mimic and STAT3 siRNA into A549 cells. The target binding sites of miR-106 a-5 p and STAT3 were predicted by target gene prediction software and the targeting relationship was detected by dual luciferase reporter gene experiment. MiR-106 a-5 p inhibitor and pcDNA-STAT3 were transfected into A549 cells and treated with 0. 15 μg/ml cinobufotalin to detect cell proliferation and apoptosis.Results In A549 cells treated with cinobufotalin,the expression of miR-106 a-5 p was up-regulated,the expression of STAT3 was down-regulated,and the cell proliferation inhibition rate and apoptosis rate were significantly increased. Both over-expression of miR-106 a-5 p and inhibition of STAT3 expression could improve the proliferation inhibition rate and apoptosis rate of A549 cells. Dual luciferase reporter gene experiment confirmed the targeting binding relationship between miR-106 a-5 p and STAT3. Inhibition of miR-106 a-5 p or over-expression of STAT3 could reverse the inhibitory effect of cinobufotalin on proliferation and apoptosis of A549 cells. Conclusion Cinobufotalin can inhibit the proliferation of A549 cells and promote its apoptosis. The mechanism may be related to the regulation of miR-106 a-5 p/STAT3 signaling pathway,which may provide a new basis for the molecular mechanism of cinobufotalin in the treatment of lung cancer.
Objective To study the effect of cinobufotalin on the proliferation and apoptosis of A549 cells and explore its mechanism.Methods A549 cells were treated with cinobufotalin at 0. 15 μg/ml. Expressions of miR-106 a-5 p and STAT3 were detected by qRT-PCR and western blot,and cell proliferation and apoptosis were detected by MTT and flow cytometry. Cell proliferation and apoptosis were detected after transfection with miR-106 a-5 p mimic and STAT3 siRNA into A549 cells. The target binding sites of miR-106 a-5 p and STAT3 were predicted by target gene prediction software and the targeting relationship was detected by dual luciferase reporter gene experiment. MiR-106 a-5 p inhibitor and pcDNA-STAT3 were transfected into A549 cells and treated with 0. 15 μg/ml cinobufotalin to detect cell proliferation and apoptosis.Results In A549 cells treated with cinobufotalin,the expression of miR-106 a-5 p was up-regulated,the expression of STAT3 was down-regulated,and the cell proliferation inhibition rate and apoptosis rate were significantly increased. Both over-expression of miR-106 a-5 p and inhibition of STAT3 expression could improve the proliferation inhibition rate and apoptosis rate of A549 cells. Dual luciferase reporter gene experiment confirmed the targeting binding relationship between miR-106 a-5 p and STAT3. Inhibition of miR-106 a-5 p or over-expression of STAT3 could reverse the inhibitory effect of cinobufotalin on proliferation and apoptosis of A549 cells. Conclusion Cinobufotalin can inhibit the proliferation of A549 cells and promote its apoptosis. The mechanism may be related to the regulation of miR-106 a-5 p/STAT3 signaling pathway,which may provide a new basis for the molecular mechanism of cinobufotalin in the treatment of lung cancer.
引文
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[23]刘慧琳,魏敏,王国霞.miR-98-5p通过降低STAT3水平促进A549细胞凋亡并抑制其侵袭和迁移[J].细胞与分子免疫学杂志,2018,34(6):522-527.
[24]纪国文,罗志兵,段晶晶,等.三乙酰白藜芦醇对非小细胞肺癌A549细胞增殖及STAT3凋亡通路的影响[J].国际呼吸杂志,2017,37(11):806-810.
[2]Sahu M,Mallick B.Deciphering synergistic regulatory networks of microRNAs in hESCs and fibroblasts[J].Int J Biol Macromol,2018,113:1279-1286.
[3]Shi JG,Shao HJ,Jiang FE,et al.Role of radiation therapy in lung cancer management-a review[J].Eur Rev Med Pharmacol Sci,2016,20(15):3217-3222.
[4]Naylor EC,Desani JK,Chung PK.Targeted Therapy and Immunotherapy for Lung Cancer[J].Surg Oncol Clin N Am,2016,25(3):601-609.
[5]Siegel RL,Miller KD,Jemal A.Cancer statistics,2015[J].CA Cancer J,2015,65(1):5-29.
[6]Tseng CY,Lin CH,Wu LY,et al.Potential Combinational AntiCancer Therapy in Non-Small Cell Lung Cancer with Traditional Chinese Medicine Sun-Bai-Pi Extract and Cisplatin[J].PLo S ONE,2016,11(5):e0155469.
[7]王双双,李柏,翟笑枫.华蟾素注射液抗肿瘤应用及其机制研究进展[J].山东中医药大学学报,2008,32(5):436-438.
[8]涂超,殷俊,贺洁宇.华蟾素注射液联合化疗药物治疗中晚期非小细胞肺癌的Meta分析[J].肿瘤药学,2012,2(1):67-72.
[9]Chen W,Zheng R,Baade PD,et al.Cancer Statistics in China,2015[J].CA Cancer J,2016,66(2):115-132.
[10]He XR,Han SY,Li PP.Recent highlights of Chinese medicine for advanced lung cancer[J].Chin J Integr Med,2017,23(5):323-330.
[11]温洋,白东玉,吴彦岭,等.华蟾素注射液联合氨酚羟考酮治疗中重度癌性内脏痛的疗效观察[J].临床和实验医学杂志,2018,17(7):731-733.
[12]Kai S,Lu JH,Hui PP,et al.Pre-clinical evaluation of cinobufotalin as a potential anti-lung cancer agent[J].Biochem Biophys Res Commun,2014,452(3):768-774.
[13]王卓敏,李宏斌,李迎春,等.华蟾素配合立体定向放射治疗中晚期食管癌28例[J].中国药业,2010,19(17):61-62.
[14]徐咏梅,刘声.华蟾素胶囊联合化疗对中晚期胃癌的疗效观察[J].世界中医药,2016,11(7):1212-1214.
[15]马丽娜,宋兵,金花,等.华蟾素对MDA-MB-231细胞内钙离子和细胞膜电位的影响[J].中国肿瘤临床,2011,38(18):1127-1130.
[16]陈彬.华蟾素联合GP方案治疗中晚期非小细胞肺癌的临床疗效和抗肿瘤机理[J].实用癌症杂志,2016,31(2):224-227.
[17]Bartel DP.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[18]Zhi F,Zhou G,Shao N,et al.miR-106a-5p Inhibits the Proliferation and Migration of Astrocytoma Cells and Promotes Apoptosis by Targeting FASTK[J].PLo S ONE,2013,8(8):e72390.
[19]He QY,Wang GC,Zhang H,et al.MiR-106a-5p Suppresses the Proliferation,Migration,and Invasion of Osteosarcoma Cells by Targeting HMGA2[J].DNA Cell Biol,2016,35(9):506-520.
[20]夏丽琴,冯忠明,陈旭,等.网络筛选乳腺癌相关miRNAs及miR-106a-5p对乳腺癌细胞侵袭迁移能力的影响[J].第三军医大学学报,2017,39(2):130-136.
[21]Pan YJ,Wei LL,Wu XJ,et al.MiR-106a-5p inhibits the cell migration and invasion of renal cell carcinoma through targeting PAK5[J].Cell Death Dis,2017,8(10):e3155.
[22]Yuan ZL,Guan YJ,Wang L,et al.Central role of the threonine residue within the p+1 loop of receptor tyrosine kinase in STAT3 constitutive phosphorylation in metastatic cancer cells[J].Mol Cell Biol,2004,24(21):9390-9400.
[23]刘慧琳,魏敏,王国霞.miR-98-5p通过降低STAT3水平促进A549细胞凋亡并抑制其侵袭和迁移[J].细胞与分子免疫学杂志,2018,34(6):522-527.
[24]纪国文,罗志兵,段晶晶,等.三乙酰白藜芦醇对非小细胞肺癌A549细胞增殖及STAT3凋亡通路的影响[J].国际呼吸杂志,2017,37(11):806-810.