芪玉三龙方抑制A549肺癌细胞对miRNA21“成瘾”的机制  

Mechanism of Qiyu Sanlong Prescription in Inhibiting "Addiction" of Lung Cancer A549 Cells to miRNA21

作　　者：张星星[1] 高雅婷 王小乐[1,2] 杨勤军 刘桐[1,2] 童佳兵 李泽庚 ZHANG Xing-xing;GAO Ya-ting;WANG Xiao-le;YANG Qin-jun;LIU Tong;TONG Jia-bing;LI Ze-geng(First Affiliated Hospital of Anhui University of Tradition Chinese Medcine(TCM),Key Laboratory of TCM Prevention an d Treatment of Pulmonary Diseases of Anhui Provincial Education Department,Institute of Research Institute of Respiratory Disease of TCM,Hefei 230031,China;Anhui University of Chinese Medicine,Hefei 230012,China)

机构地区：[1]安徽中医药大学第一附属医院,安徽省教育厅中医药防治肺系重大疾病重点实验室,安徽省中医药科学院中医呼吸病防治研究所,合肥230031 [2]安徽中医药大学,合肥230012

出　　处：《中国实验方剂学杂志》2021年第11期35-41,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81874431);国家自然科学基金青年科学基金项目(81804039);安徽省自然科学基金青年项目(1808085QH256)。

摘　　要：目的:探讨芪玉三龙方(QYSL)抑制A549肺癌细胞对微小RNA21(miRNA21)"成瘾"的分子机制。方法:选择人A549肺癌细胞,常规传代培养,分别设置为空白组,空白血清组,含药血清组,干扰小RNA(siRNA)组。制备QYSL含药血清,以前期研究筛选的最佳药物浓度和作用时间进行干预。蛋白免疫印迹法(Western blot)检测及实时荧光定量聚合酶链式反应(Real-time PCR)miRNA21及其靶向信号通路第10号染色体缺失的磷酸酶及张力蛋白同源物(PTEN)/磷脂酰肌醇3-激酶(PI3K)相关分子的蛋白及mRNA的表达。结果:与空白血清组比较,含药血清组和siRNA组的miRNA21 mRNA表达显著降低,PTEN mRNA的表达显著升高,差异具有统计学意义(P<0.01)。与空白血清组比较,含药血清组和siRNA组的PI3K,雷帕霉素靶蛋白(mTOR)mRNA表达显著降低,差异具有统计学意义(P<0.01),但含药血清组蛋白激酶B(Akt)mRNA的表达无明显差异。与空白血清组比较,含药血清组和siRNA组的PTEN蛋白表达明显升高,差异具有统计学意义(P<0.05);含药血清组磷酸化蛋白激酶B(p-Akt),磷酸化雷帕霉素靶蛋白(p-mTOR)蛋白表达明显降低(P<0.05),总蛋白Akt,mTOR表达无明显降低,PI3K蛋白的表达降低,但差异无统计学意义。结论:QYSL可抑制miRNA21的转录,使PTEN表达有所上升,下调PI3K/Akt/mTOR信号通路关键分子表达,从而温和抑制肺癌细胞对致癌基因的"成瘾"现象,阻断肺癌细胞增殖。Objective:To investigate the molecular mechanism of Qiyu Sanlong prescription(QYSL in inhibiting the "addiction" of lung cancer A549 cells to miRNA21. Method: The human lung cancer A549 cells were routinely passaged and divided into the blank group,blank serum group,QYSL-containing serum group,and siRNA group. The prepared QYSL-containing serum was used for intervention,with the optimal concentration and action time determined in previous studies. The protein and mRNA expression levels of miRNA21 and related molecules in its target phosphatase and tensin homolog deleted on chromosome 10(PTEN)/phosphatidylinositol 3-kinase(PI3K)signaling pathway were detected by real-time polymerase chain reaction(Real-time PCR)and Western blot assay. Result:The comparison with the blank serum group revealed that the mRNA expression levels of miRNA21 in the QYSL-containing serum group and the siRNA group were decreased,while the PTEN mRNA expression in the QYSL-containing serum group was increased,showing significant differences(P<0.01). Compared with the blank serum,the QYSL-containing serum and siRNA significantly down-regulated PI3K and mammalian target of rapamycin(mTOR)mRNA expression(P<0.01),whereas the QYSL-containing serum did not change the mRNA expression of protein kinase B(Akt). The protein expression levels of PTEN in the QYSL-containing serum group and the siRNA group were obviously elevated in contrast to that in the blank serum group(P<0.05). Meanwhile,the protein expression levels of phosphorylated Akt(p-Akt)and phosphorylated mTOR(p-mTOR)evidently declined in the QYSL-containing serum group(P<0.05),but there was no significant reduction in total Akt and mTOR protein expression. The PI3K protein expression was slightly down-regulated,with no statistical significance. Conclusion: QYSL inhibits the transcription of miRNA21,increases the expression of PTEN,and reduces the expression of key molecules in PI3K/Akt/mTOR signaling pathway,thus mildly inhibiting the "addiction" of lung cancer cells to oncogenes and blockin

关 键 词：芪玉三龙方 A549肺癌细胞 微小RNA21(miRNA21) 第10号染色体缺失的磷酸酶及张力蛋白同源物(PTEN)/磷脂酰肌醇3-激酶(PI3K)信号通路 

分 类 号：R2-0[医药卫生—中医学] R22