葛根素通过miR-490/E3泛素连接酶抑制非小细胞肺癌细胞生长、侵袭和迁移  

Puerarin Inhibits the Proliferation,Invasion,and Migration of Non-small Cell Lung Cancer Cells through Regulating miR-490/Denticleless E3 Ubiquitin Protein Ligase

作　　者：张钰欣 张臻臻 赵利刚 赵琳琳 ZHANG Yuxin;ZHANG Zhenzhen;ZHAO Ligang;ZHAO Linlin(College of Pharmacy,North China University of Science and Technology,Tangshan,Hebei 063000,China)

机构地区：[1]华北理工大学药学院,河北唐山063000

出　　处：《中国医学科学院学报》2022年第1期91-101,共11页Acta Academiae Medicinae Sinicae

基　　金：河北省中医药管理局项目(2016194)。

摘　　要：目的研究葛根素抑制非小细胞肺癌细胞生长、侵袭和迁移的机制。方法培养A549细胞,采用不同浓度的葛根素处理细胞。CCK-8法检测细胞增殖抑制率(IR);qRT-PCR法检测细胞中miR-490和E3泛素连接酶(DTL)mRNA的表达情况;构建miR-490过表达对照组、miR-490过表达模拟物组、miR-490沉默阴性对照组、miR-490沉默组,采用双荧光素酶报告分析miR-490和DTL的靶向关系。将20μmol/L葛根素处理过的细胞分为对照组、葛根素组、葛根素+miR-490阴性对照组、葛根素+miR-490沉默模拟物组、葛根素+miR-490沉默模拟物+DTL阴性对照组、葛根素+miR-490沉默模拟物+DTL沉默质粒组。Transwell法检测细胞迁移和侵袭。Western blot检测上皮细胞-间充质转化进程标志因子E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)和波形蛋白(Vimentin)的表达。结果随着葛根素浓度升高,细胞抑制率逐步升高,差异有统计学意义(F=105.375,P<0.001);且miR-490表达逐步升高(F=32.919,P<0.001),DTL表达逐步降低(F=116.120,P<0.001)。双荧光素酶报告分析显示,与miR-490过表达对照组相比,miR-490过表达模拟物组荧光素酶活性显著降低(t=7.762,P=0.016),miR-490 mRNA表达显著升高(t=13.319,P<0.001),DTL mRNA表达显著降低(t=7.415,P=0.002);与miR-490沉默阴性对照组相比,miR-490沉默组细胞中miR-490表达显著降低(t=9.523,P=0.001),DTL mRNA的表达显著升高(t=11.305,P<0.001)。Western blot检测结果显示,与miR-490过表达对照组相比,miR-490过表达模拟物组的DTL蛋白表达显著降低(t=7.953,P=0.001);与miR-490沉默阴性对照组相比,miR-490沉默组细胞中的DTL蛋白表达显著升高(t=10.552,P<0.001)。经葛根素处理后,与对照组相比,葛根素组细胞miR-490表达显著升高(t=10.255,P=0.001),DTL mRNA表达显著降低(t=6.682,P=0.003);与葛根素+miR-490阴性对照组相比,葛根素+miR-490沉默模拟物组细胞中miR-490表达显著降低(t=10.995,P<0.001),DTL mRNA表达显著升�Objective To explore the mechanism of puerarin inhibiting the proliferation,invasion,and migration of non-small cell lung cancer cells.Methods A549 cells were cultured and treated with different concentrations of puerarin.The inhibition rate(IR)on cell proliferation was detected by CCK-8,and qRT-PCR was performed to detect the mRNA levels of miR-490 and denticleless E3 ubiquitin protein ligase(DTL).Double luciferase reporter assay was employed to identify the targets of miR-490 and DTL based on the establishment of NC mimic group,miR-490 mimic group,NC inhibitor group,and miR-490 inhibitor group.The cells treated by 20μmol/L puerarin were classified into six groups:DMSO,puerarin,puerarin+NC inhibitor,puerarin+miR-490 inhibitor,puerarin+miR-490 inhibitor+Si-NC,and puerarin+miR-490 inhibitor+Si-DTL.Transwell was used to detect cell migration and invasion.Western blotting was performed to detect the protein levels of epithelial-mesenchymal transition-related markers E-cadherin,N-cadherin,and Vimentin.Results With the increase in puerarin concentration,the IR gradually elevated(F=105.375,P<0.001),miR-490 expression gradually increased(F=32.919,P<0.001),and DTL expression gradually decreased(F=116.120,P<0.001).Compared with NC mimic group,miR-490 mimic group had decreased luciferase activity(t=7.762,P=0.016),raised miR-490 mRNA level(t=13.319,P<0.001),and declined DTL mRNA level(t=7.415,P=0.002).Compared with those in NC inhibitor group,miR-490 demonstrated decreased mRNA level(t=9.523,P=0.001)and DTL presented increased mRNA level(t=11.305,P<0.001)in miR-490 inhibitor group.Western blotting showed that the protein level of DTL was higher in NC mimic group(t=7.953,P=0.001)than in miR-490 mimic group and higher in miR-490 inhibitor group than in NC inhibitor group(t=10.552,P<0.001).Compared with DMSO group,puerarin group showed up-regulated mRNA level of miR-490(t=10.255,P=0.001)while down-regulated mRNA level of DTL(t=6.682,P=0.003).Compared with those in puerarin+NC inhibitor group,the mRNA level of miR-490 declined

关 键 词：葛根素 miR-490 E3泛素连接酶 非小细胞肺癌 生长 迁移 

分 类 号：R734.2[医药卫生—肿瘤]