摘要
目的探讨山慈菇提取物(CAE)对肝癌细胞Huh7增殖、凋亡的影响及其作用机制。方法体外培养Huh7细胞,使用不同浓度(10、20、30 mg/mL)CAE处理24 h;采用MTT法检测细胞存活率,流式细胞术检测细胞凋亡率,实时荧光定量PCR法检测miR-329-3p及含跨膜Bax抑制剂的母体6(TMBIM6)mRNA表达,Western blotting法检测TMBIM6及Cyclin D1、p21、Bcl-2、Bax蛋白表达。双荧光素酶报告实验验证miR-329-3p与TMBIM6的靶向关系。将细胞分为CAE+anti-miR-329-3p组、CAE+anti-miR-NC组,分别转染miR-329-3p特异性寡核苷酸抑制剂(anti-miR-329-3p)及其阴性对照(anti-miR-NC),并加入含有30 mg/mL的CAE处理24 h。参照上法检测细胞存活率、细胞凋亡率、miR-329-3p表达、TMBIM6及凋亡相关蛋白表达。结果随着加入CAE浓度的升高,细胞存活率、TMBIM6 mRNA及蛋白、Cyclin D1、Bcl-2蛋白表达均逐渐降低,细胞凋亡率、miR-329-3p表达、p21及Bax蛋白表达均逐渐升高(P均<0.05)。双荧光素酶报告实验结果显示,miR-329-3p能够靶向结合TMBIM6。与CAE+anti-miR-NC组比较,CAE+anti-miR-329-3p组细胞存活率及TMBIM6、Cyclin D1、Bcl-2蛋白表达升高,细胞凋亡率、miR-329-3p表达及p21、Bax蛋白表达均降低(P均<0.01)。结论CAE可抑制肝癌细胞Huh7增殖、促进细胞凋亡,其机制可能与激活miR-329-3p表达并负向调控其靶基因TMBIM6有关。
Objective To investigate the effect of cremastra appendiculata extracts(CAE)on the proliferation and apoptosis of liver cancer cells and its mechanism.Methods Human liver cancer Huh7 cells were cultured in vitro and treated with CAE at different concentrations(10,20,and 30 mg/mL)for 24 h.MTT was used to detect cell survival rate,flow cytometry was used to detect apoptosis rate,and qRT-PCR was used to detect the expression of miR-329-3p and TMBIM6.Western blotting was used to detect the expression levels of TMBIM6,Cyclin D1,p21,Bcl-2,and Bax.The dual luciferase reporting experiment verified the targeting relationship between miR-329-3p and TMBIM6.Cells were divided into the CAE+anti-miR-329-3p group and CAE+anti-miR-NC group,which were transfected with miR-329-3p specific oligonucleotide inhibitor(anti-miR-329-3p)and the negative control(anti-miR-NC),followed by 30 mg/mL CAE for 24 h.The cell survival rate,apoptosis rate,miR-329-3p expression,TMBIM6 and apoptosis-related protein expression were detected by using the same methods as above.Results With the increased concentrations of CAE,the cell survival rate,TMBIM6 mRNA and protein,Cyclin D1,and Bcl-2 protein levels decreased gradually,and the apoptotic rate and expression levels of miR-329-3p,p21,and Bax increased gradually.Double luciferase reporting experiments confirmed that miR-329-3p was able to target TMBIM6.Compared with the CAE+anti-miR-NC group,the cell survival rate and TMBIM6,Cyclin D1,Bcl-2 protein expression increased,the apoptosis rate,miR-329-3p expression and p21 and Bax protein expression decreased in the CAE+anti-miR-329-3p group(all P<0.01).Conclusion CAE inhibits the proliferation and promotes apoptosis of liver cancer cells by activating the miR-329-3p and negatively regulating its target gene TMBIM6.
作者
方健
王辰男
孟庆刚
FANG Jian;WANG Chennan;MENG Qinggang(Beijing University of Traditional Chinese Medicine,Beijing 100029,China)
出处
《山东医药》
CAS
2020年第11期11-15,共5页
Shandong Medical Journal
