摘要
The present study aimed to determine whether actinomycin X2 (AX2) intercepted the mTOR/PTEN/PI3K/Akt signaling pathway to inhibit human prostate cancer cells (PC-3) in vitro. The effects of AX2 on mTOR, PTEN, PI3K, and Akt at the protein level and mRNA were determined by western blotting and real-time reverse transcription-PCR (RT-PCR), respectively. Concurrently, the effects of AX2 on expression levels of MiRNA144 and MiRNA126 in PC-3 were measured by real-time RT-PCR. The association of MiRNA144 with 3′- UTR of mTOR was identified using the Dual-Luciferase Reporter Gene System. The direct effect of MIRNA144 on the mTOR/PTEN/PI3K/Akt pathway was determined by real-time RT-PCR and western blotting. Apoptosis of PC-3 cells induced by AX2 was determined by MTT and flow cytometry. The results indicated that mTOR/PTEN/PI3K/ Akt were decreased and PTEN was increased by AX (1, 10 µmol/l) at protein and mRNA levels in a dose-dependent manner. MiRNA144 was decreased, whereas MiRNA126 was increased by AX2. MiRNA144 associated with 3′-UTR of mTOR was corroborated. Overexpression of MiRNA144 decreased mTOR, but did not affect PTEN, PI3K, or Akt. The proliferation rates of AX2 on PC-3 cells were decreased. This suggests that AX2 induces apoptosis of PC-3 cells by meddling in the mTOR/PTEN/PI3K/Akt signaling pathway, but compounded by MiRNA144, and AX2 and MiRNA144 AQ2 intercepts it in different ways but crosses on mTOR.
研究背景
mTOR/PTEN/PI3K/Akt信号通路在前列腺癌治疗过程中发挥着至关重要作用。mTOR的两个变型TORC1和TORC2行驶不同功能，其中，TORC1作为重要的Akt信号通路的下调控影响因子，TORC2却作为敏感性生长因子。AX2作为一类类似于放线菌素D的抗生素，可以已知肺结核等其他细菌引起的疾病。本实验室前期研究，MTT 检测到AX2能已知肿瘤细胞增殖，其机制尚不清楚。本文旨在研究mTOR/PTEN/PI3K/Akt信号通路在AX2参与时miRNA治疗前列腺癌的新思路。
方法流程
研究结果
1、mTOR-PTEN-PI3K-Akt信号通路基因表达水平受AX2影响差异不同。通过qPCR和WB检测，发现10 umol/l AX2处理PC-3细胞时，mTOR、PI3K及Akt表达水平明显降低，相反，PTEN表达水平明显增高（图1）。
图1 mTOR-PTEN-PI3K-Akt信号通路基因表达情况
2、不同浓度AX2处理PC-3细胞，miRNA126、miRNA144表达情况（图2）。
图2 miRNA126、144基因表达水平
3、双荧光素酶报告基因检测mTOR基因与miRNA144相互作用（图3）。
图3 双荧光素酶报告基因检测
4、miRNA144 mimic处理PC-3细胞，mTOR mRNA表达显著降低，PTEN、PI3K及AKT无明显变化（图4）。
图4 qPCR检测
5、流式检测AX2处理是细胞凋亡情况（图5）。
图5 流式检测细胞凋亡
伯信合作技术
双荧光素酶报告基因、siRNA、WB及FCM。
参考文献
Liu J, Xie S, Wu Y, et al. Apoptosis of human prostate cancer cells induced by marine actinomycin X2 through the mTOR pathway compounded by MiRNA144.[J]. Anti-cancer drugs, 2016, 27(3).
原文链接
https://www.ncbi.nlm.nih.gov/pubmed/26645890
