芪玉三龙汤调控miRNA21介导的PTEN/PI3K信号通路抑制非小细胞肺癌的机制研究

NSCLC has a high incidence and high mortality. As a oncogene，miRNA21 specifically identify 3’-Untranslated region of target gene and adjust its expression through transcription level ，which has became research hotspot in the development of tumor regulating mechanism. PI3K/Akt/mTOR pathway activation can promote lung cancer proliferation. PTEN can make PIP3 dephosphorylate to PIP2 that negatively regulate the pathway. Our previous research have found that Fuzheng Kangai experience prescription Qiyusanlong decoction.（QYSL）could reduce miRNA21 level and deregulate the expression of key proteins in above pathway. So，we propose that QYSL can inhibit lung cancer proliferation via blocking PI3K/Akt/mTOR signal pathway through inhibiting miRNA21 expression and promoting PTEN activation. We will carry out further in-depth study to adopt transplantation lung tumors model in vivo and lung cancer cells model in vitro respectively. siRNA， RT-qPCR and Western Blot technology will be used to explore molecular mechanism of inhibiting tumor about QYSL，which will provide a scientific basis in popularizing and application for clinical experience prescription.

非小细胞肺癌（NSCLC）发病多，死亡率高，致癌基因miRNA21特异性识别靶基因3’-端非编码区，通过转录后水平调节靶基因表达，在肿瘤发生发展中的调控机制成为研究热点。磷脂酰肌醇-3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白（PI3K/Akt/mTOR）通路激活可促进肺癌增殖，第10号染色体缺失的磷酸酶及张力蛋白同源物（PTEN）可使通路中PIP3去磷酸化为PIP2而负向调控该通路活化。我们前期研究发现扶正抗癌验方芪玉三龙汤可下调肺癌组织miRNA21水平，降低上述通路关键蛋白表达，故提出该方可通过抑制miRNA21表达促进PTEN活化从而阻断PI3K/Akt/mTOR通路传导抑制肺癌增殖的假说。拟进一步深入研究，分别采用肺癌移植瘤体内模型和肺癌细胞株体外模型，运用siRNA、RT-qPCR、Western Blot等技术探讨芪玉三龙汤抑癌增殖的分子机制，为临床验方的推广运用提供科学依据。

非小细胞肺癌（NSCLC）发病多，死亡率高，致癌基因miRNA21特异性识别靶基因3’-端非编码区，通过转录后水平调节靶基因表达，在肿瘤发生发展中的调控机制成为研究热点。磷脂酰肌醇-3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白（PI3K/Akt/mTOR）通路激活可促进肺癌增殖，第10号染色体缺失的磷酸酶及张力蛋白同源物（PTEN）可使通路中PIP3去磷酸化为PIP2而负向调控该通路活化。我们前期研究发现国医大师韩明向教授扶正抗癌验方芪玉三龙汤（QYSL）可下调肺癌组织miRNA21水平，降低上述通路关键蛋白表达，故提出该方可通过抑制miRNA21表达促进PTEN活化从而阻断PI3K/Akt/mTOR通路传导抑制肺癌增殖的假说。本项目采用肺癌移植瘤体内模型和肺癌细胞株体外模型，运用siRNA、RT-qPCR、Western Blot等技术探讨芪玉三龙汤抑癌增殖的分子机制。采用A549肺癌、LLC肺癌细胞两种细胞株为研究对象，分别设置为A549/LLC肺癌组、空白血清组、siRNA组、含药血清组，分别运用CCK-8法和MTT法筛选作用条件，结合现代分子学检测手段。结果发现，QYSL含药血清呈浓度依赖性抑制肺癌细胞活性。与肺癌细胞组比较，含药血清组可以降低PI3K、p-AKT、p-mTOR蛋白及基因转录水平，但对总蛋白AKT、mTOR表达无明显差异，并可以下调该信号轴下游关键分子eIF4E、p70s6k表达。体内采用肺癌移植瘤模型，检测各组miRNA21/PI3K/Akt信号轴关键分子表达，结果发现，QYSL可抑制miRNA21表达，上调PTEN水平，抑制信号轴关键分子表达。所以得出以下结论：QYSL可抑制miRNA21的转录，使PTEN表达有所上升，下调PI3K/Akt/mTOR信号轴关键分子活性，从而温和抑制肺癌细胞增殖。该结果及结论验证了前期假说，证明了假说的科学性和实效性。下一步借助网络药理学和RNA-Seq研究，将继续从基因干扰、外源基因表达等方面深度挖掘该方治疗肺癌的分子机制，开发中药新药，推广临床运用和成果转化，发扬国医大师临床验方。