摘要
目的探讨渥曼青霉素对人黑色素瘤C8161细胞迁移和侵袭的影响及对PI3K/AKT/NF-κB通路的调节作用。方法将体外培养C8161细胞分别用不同浓度(2.5、5.0、10.0、15.0、20.0μmol/L)的渥曼青霉素或阳性药物(200μmol/L的5-氟尿嘧啶)处理,对照组细胞不做处理。然后使用倒置显微镜观察C8161细胞形态,活细胞计数(CCK-8)法测定细胞活力,EdU法测定细胞增殖率,划痕法测定细胞迁移情况,Transwell小室检测细胞侵袭情况,RT-qPCR测定EMT相关因子[E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)和波形蛋白(Vimentin)]mRNA表达,蛋白免疫印迹法(WB)测定PI3K/AKT/NF-κB通路相关蛋白(AKT、p-AKT、NF-κB p65和p-NF-κB p65)和EMT相关蛋白(E-cadherin、N-cadherin和Vimentin)表达情况。结果与对照组比较,不同浓度渥曼青霉素和阳性药物处理抑制细胞的生长、增殖率、迁移率、侵袭细胞数、N-cadherin、Vimentin、p-AKT/AKT和p-NF-κB p65/NF-κB p65表达水平,增加了E-cadherin mRNA和蛋白表达水平,且渥曼青霉素处理组呈浓度依赖性;低浓度实验组对细胞的活力没有显著影响(P>0.05),阳性药物显著降低了细胞的活力(P<0.05)。结论渥曼青霉素可显著抑制C8161细胞的迁移、侵袭和EMT,该抑瘤作用可能是通过抑制PI3K/AKT/NF-κB信号通路的活化实现的。
Objective To investigate effects of wortmannin on migration and invasion of human melanoma C8161 cells and its regulation on the PI3K/AKT/NF-κB pathway.Methods C8161 cells were cultured in vitro and exposed to different concentratiosn of wortmannin(2.5,5.0,10.0,15.0 and 20.0μmol/L),or 200μmol/L of 5-fluorouracil as a positive drug,respectively.Cells in control group were not treated.Then,cell morphology was observed under an inverted microscope.Cell Viability,proliferation,migration,invasion and EMT-related factors(E-cadherin,N-cadherin and Vimentin)mRNA expression were measured by cell counting kit-8(CCK-8),EdU,wound healing,transwell and RT-qPCR analyses,respectively.Related protein levels in the PI3K/AKT/NF-κB(AKT,p-AKT,NF-κB p65 and p-NF-κB p65)and EMT(E-cadherin,N-cadherin and Vimentin)were determined by using Western blot(WB).Results Compared with control group,different concentrations of wortmannin and positive control treatment inhibited cell growth,proliferation rate,migration rate,invasive cell numbers,N-cadherin,Vimentin,p-AKT/AKT and p-NF-κB p65/NF expression,while promoted E-cadherin mRNA and protein expression levels.Treatment effect of experimental group was concentration-dependent.Low concentration experimental group had no significant effect on cell viability(P>0.05),and positive control significantly reduced cell viability(P<0.05).Conclusion Wortmannin significantly inhibited growth,proliferation,migration,invasion and EMT of C8161 cells,which may be achieved by inhibiting activation of PI3K/AKT/NF-κB signaling pathway.
作者
杜旭
袁源
简华宏
李晓凤
李建军
Du Xu;Yuan Yuan;Jian Huahong(Department of Dermatological Venereal Diseases,Chongqing Qianjiang Central Hospital,Chongqing,409099,China.)
出处
《四川医学》
CAS
2022年第12期1185-1191,共7页
Sichuan Medical Journal
