前癃通胶囊介导miR-216a-5p/TPT1/mTORC1通路调控良性前列腺增生的实验研究  

Experimental study of Qianlongtong Capsule on mediating miR-216a-5p/TPT1/mTORC1 pathway to regulate benign prostate hyperplasia

作　　者：黄鸿宇 郭子莘 朱文雄[3] 袁轶峰[3] 贺菊乔[3] 刘涛[3] 谭梅鑫 杨金玉 曹雨昙 张熙 HUANG Hongyu;GUO Zishen;ZHU Wenxiong;YUAN Yifeng;HE Juqiao;LIU Tao;TAN Meixin;YANG Jinyu;CAO Yutan;ZHANG Xi(Hunan Brain Hospital(Hunan Second People's Hospital),Changsha,Hunan 410021,China;School of Integrated Chinese and Western Medicine,Hunan University of Chinese Medicine,Changsha,Hunan 410208,China;The FirstHospital of Hunan University of Chinese Medicine,Changsha,Hunan 410007,China)

机构地区：[1]湖南省脑科医院(湖南省第二人民医院),湖南长沙410021 [2]湖南中医药大学中西医结合学院,湖南长沙410208 [3]湖南中医药大学第一附属医院,湖南长沙410007

出　　处：《湖南中医药大学学报》2024年第3期374-382,共9页Journal of Hunan University of Chinese Medicine

基　　金：湖南省自然科学基金项目(2021JJ30396);湖南省卫生健康委科研计划项目(202103051239);湖南省中医药科研计划项目(2021108)。

摘　　要：目的通过细胞实验探讨前癃通胶囊(qian long tong capsule,QLTC)能否通过调控miR-216a-5p/肿瘤蛋白翻译控制1/哺乳动物雷帕霉素靶蛋白复合物1(miR-216a-5p/tumor protein translationally controlled 1/mammalian target of rapamycin complex 1,miR-216a-5p/TPT1/mTORC1)信号通路抑制良性前列腺增生(benign prostatic hyperplasia,BPH)。方法将25只大鼠随机分为对照组(等体积生理盐水),QLTC低(56.25 mg/mL)、中(112.50 mg/mL)、高(225.00 mg/mL)剂量组,LBSC组(168.75 mg/mL),每组5只。每组灌胃1 mL/次,2次/d,连续5 d。各组大鼠麻醉后制备含药血清。根据实验目的不同,将CP-H022细胞分5步做实验处理,每部分实验进行独立分组。将miR-216a-5p过表达和沉默表达,及TPT1过表达进行对照研究;RT-qPCR法检测正常和BPH模型CP-H022细胞内miR-216a-5p表达量,并观察不同浓度QLTC处理的BPH细胞中miR-216a-5p表达量的差异;细胞集落形成实验检测细胞增殖能力;CCK-8法检测BPH模型细胞增殖;RT-qPCR法检测miR-216a-5p、TPT1 mRNA表达水平;流式细胞术检测细胞凋亡;生信分析、双荧光素酶实验验证miR-216a-5p与TPT1的靶向关系;过表达TPT1后,Western blot法检测BPH细胞中TPT1/mTORC1信号通路相关分子表达情况。结果与对照组1比较,模型组1的CP-H022细胞内miR-216a-5p表达量下调(P<0.05);不同浓度的QLTC均能上调miR-216a-5p表达量(P<0.05);根据本实验结果,本研究将选用QLTC(高剂量)组CP-H022细胞进行后续实验。与模型组2比较,QLTC组2细胞增殖减少、凋亡增加(P<0.05),B细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2)表达降低(P<0.05),Bcl-2关联X蛋白单克隆抗体(monoclonal antibody to Bcl-2 associated X protein,Bax)、cleaved Caspase-3表达升高(P<0.05)。敲低miR-216a-5p后,与模型组4比较,QLTC组4细胞增殖增强、凋亡减少(P<0.05),Bcl-2表达升高(P<0.05),Bax、cleaved Caspase-3表达降低(P<0.05)。与mimic-NC组比较,miR-216a-5p mimic组TPT1表达量降低(P<0.05);QLTC处理后,细胞Objective To investigate whether Qianlongtong Capsule(QLTC)can inhibit benign prostatic hyperplasia(BPH)by regulating the signaling pathway of miR-216a-5p/tumor protein translationally controlled 1/mammalian target of rapamycin complex 1(miR-216a-5p/TPT1/mTORC1)through cell experiments.Methods A total of 25 rats were randomized into control(equal volume of normal saline),low-(56.25 mg/mL),medium-(112.50 mg/mL),and high-dose(225.00 mg/mL)QLTC,and LBSC groups(168.75 mg/mL),with five rats in each group.The rats in each group were given corresponding drug 1 mL by gavage,twice a day,for continual five days.Drug-containing serum was prepared from rats in each group after anesthesia.According to different experimental purposes,CP-H022 cells were treated experimentally by five steps,and each part of the experiment was grouped independently.The comparative study on overexpression and silencing expression of miR-216a-5p,and TPT1 overexpression was performed;RT-qPCR was used to determine the expression levels of miR-216a-5p in normal and BPH model CP-H022 cells,and observe the differences of miR-216a-5p expression levels in BPH cells treated with different concentrations of QLTC;cell colony formation assay was used to examine cell proliferation;CCK-8 was used to test the proliferation of BPH model cells;RT-qPCR was used to determine the expression levels of miR-216a-5p and TPT1mRNA;cell apoptosis was checked by flow cytometry;bioinformatics analysis and dual luciferase assay were used to verify the targeting relationship between miR-216a-5p and TPT1;after overexpression of TPT1,Western blot was used to examine the expression of TPT1/mTORC1 signaling pathway related molecules in BPH cells.Results Compared with the control group,the expression level of miR-216a-5p in CP-H022 cells in the model group 1 downregulated(P<0.05);different concentrations of QLTC can upregulate the expression of miR-216a-5p;based on the results of this experiment,QLTC(high-dose)group CP-H022 cells will be selected for subsequent experiments in this s

关 键 词：前癃通胶囊 良性前列腺增生 细胞实验 miR-216a-5p 肿瘤蛋白翻译控制1 哺乳动物雷帕霉素靶蛋白复合物1 信号通路 

分 类 号：R277.5[医药卫生—中医学]