胃肠安颗粒通过RUFY3抑制人胃癌MKN45细胞侵袭转移  

Inhibitory Effect of Weichang’an Granule on Invasion and Metastasis of MKN45 Cells in Human Gastric Cancer by RUFY3

作　　者：秦梦梦 李佳[1,2] 李朝燕 赵爱光[1] QIN Meng-meng;LI Jia;LI Zhao-yan;ZHAO Ai-guang(Longhua Hospital,Shanghai University of Traditional Chinese Medicine(TCM),Shanghai 200032,China;Zhengzhou Hospital of TCM,Zhengzhou 450002,China)

机构地区：[1]上海中医药大学附属龙华医院,上海200032 [2]郑州市中医院,郑州450002

出　　处：《中国实验方剂学杂志》2021年第8期66-73,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家重点研发计划“中医药现代化研究”重点专项(2017YFC1700605);国家“重大新药创制”科技重大专项(2017ZX09304-001);国家中医药管理局国家中医临床研究基地业务建设科研专项(JDZX2015068)。

摘　　要：目的:研究健脾复方胃肠安颗粒抑制人胃癌MKN45细胞侵袭转移的分子机制。方法:体外培养人胃癌MKN45细胞,采用细胞增殖与活性检测(CCK-8)检测不同质量浓度的胃肠安颗粒(600,900,1 200,1 500 mg·L^(-1))与人胃癌MKN45细胞共孵育24,48,72 h,对细胞增殖活力的影响;采用蛋白免疫印迹法(Western blot)检测RUN和FYVE结构域蛋3(RUFY3)在人正常胃黏膜细胞和不同胃癌细胞系中的表达;Western blot检测不同质量浓度的胃肠安颗粒干预(600,900,1 200 mg·L^(-1))对人胃癌MKN45细胞中RUFY3蛋白表达的影响;利用慢病毒转染技术,构建稳定沉默RUFY3的人胃癌细胞MKN45细胞;采用Transwell迁移和侵袭实验,检测胃肠安颗粒和沉默RUFY3干预后人胃癌MKN45细胞迁移和侵袭能力的变化;采用Western blot检测各组细胞中上皮间质转化(EMT)相关蛋白E-钙黏蛋白(E-cadherin),N-钙黏蛋白(N-cadherin),波形蛋白(Vimentin),锌指转录因子(SNAIL1,SNAIL2)的表达。结果:与正常胃黏膜组比较,RUFY3在人胃癌细胞中表达明显升高(P<0.05)。与慢病毒阴性组比较,沉默RUFY3组MKN45细胞中RUFY3的蛋白表达显著降低(P<0.01);胃肠安颗粒可抑制人胃癌MKN45细胞中RUFY3的表达(P<0.05,P<0.01),且呈浓度依赖。与空白组比较,胃肠安组和沉默RUFY3均能抑制人胃癌MKN45细胞的侵袭和转移能力(P<0.01)。与空白组比较,胃肠安颗粒干预和沉默RUFY3后,人胃癌MKN45细胞中上皮标志基因E-cadherin蛋白表达显著增加(P<0.01),间质标志基因N-cadherin,Vimentin蛋白表达降低,但差异无统计学意义;锌指转录因子SNAIL1,SNAIL2的蛋白表达显著降低(P<0.01)。结论:健脾复方胃肠安颗粒可能通过RUFY3调控EMT抑制人胃癌MKN45细胞的侵袭和转移。Objective: To explore the molecular mechanism of spleen-strengthening traditional Chinese medicine(TCM)Weichang’an granule in inhibiting the invasion and metastasis of human gastric cancer MKN45 cells. Method: MKN45 cells were cultured in vitro and incubated with different concentrations(600,900,1 200,1 500 mg·L^(-1))of Weichang’an granule for 24,48,72 h. Cell counting kit-8(CCK-8)assay was used to detect its effect on the cell proliferation. Western blot was used to detect the expression of RUN and FYVE domain containing 3(RUFY3)in normal gastric mucosa cells and different gastric cancer cell lines. The expression of RUFY3 in the gastric cancer cells after Weichang’an granule intervention(600, 900,1 200 mg·L^(-1))was detected by Western blot. Lentivirus transfection technique was used to achieve the stable and silenced expression of RUFY3 in gastric cancer MKN45 cells. Transwell assay was used to evaluate the influence of Weichang’an granule and silenced RUFY3 on the metastasis and invasion ability of MKN45. E-cadherin,N-cadherin,Vimentin,Zinc-finger transcription factor(SNAIL1 and SNAIL2)protein expression levels were detected by Western blot. Result: RUFY3 expression in human gastric cancer cells was significantly higher than that in normal gastric mucosa. The protein expression of RUFY3 in MKN45 cells of silenced RUFY3 group was significantly lower than that in Lentivirus negative group(P<0.01). Weichang’an granule inhibited the expression of RUFY3 in human MKN45 gastric cancer cells(P<0.05,P<0.01)in a concentration-dependent manner. As compared with the blank group, both Weichang’an granule and silenced RUFY3 inhibited the metastasis and invasion ability of MKN45(P<0.01). After Weichang’an granule and silenced RUFY3 treatment,the protein expression of epithelial marker gene E-cadherin was up-regulated(P<0.01),the protein expression of Vimentin and N-cadherin decreased,but with no statistical difference,while SNAIL1 and SNAIL2 were both significantly down-regulated(P<0.01). Conclusion: By targ

关 键 词：胃肠安颗粒 健脾中药 胃癌 上皮间质转化(EMT) 侵袭转移 RUN和FYVE结构域蛋3 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]