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黄鸿宇,郭子莘,朱文雄,袁轶峰,贺菊乔,刘涛,谭梅鑫,杨金玉,曹雨昙,张熙.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[J].湖南中医药大学学报,2024,44(3):374-382[点击复制] |
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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 |
黄鸿宇,郭子莘,朱文雄,袁轶峰,贺菊乔,刘涛,谭梅鑫,杨金玉,曹雨昙,张熙 |
(湖南省脑科医院(湖南省第二人民医院), 湖南 长沙 410021;湖南中医药大学中西医结合学院, 湖南 长沙 410208;湖南中医药大学第一附属医院, 湖南 长沙 410007) |
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TPT1、p-mTORC1表达均降低(P<0.05);过表达TPT1后BPH细胞增殖功能增强(P<0.05),凋亡减少(P<0.05),Bcl-2表达升高(P<0.05),Bax、cleaved Caspase-3表达下降(P<0.05)。结论 QLTC可通过介导miR-216a-5p下调TPT1/mTORC1通路,进而抑制BPH。 |
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 |
DOI:10.3969/j.issn.1674-070X.2024.03.005 |
投稿时间:2023-11-17 |
基金项目: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)。 |
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Experimental study of Qianlongtong Capsule on mediating miR-216a-5p/TPT1/mTORC1 pathway to regulate benign prostate hyperplasia |
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 First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China) |
Abstract: |
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 study. Compared with the model group 2, cell proliferation decreased and apoptosis increased in QLTC group 2 (P<0.05), Bcl-2 expression decreased (P<0.05), and expressions of Bax and cleaved Caspase-3 increased (P<0.05); compared with the model group 4,after knocking down miR-216a-5p, the QLTC group 4 showed higher cell proliferation and lower apoptosis (P<0.05), increased expression of Bcl-2 (P<0.05), and decreased expressions of Bax and cleaved Caspase-3 (P<0.05). Compared with the mimic-NC group, the expression of TPT1 of miR-216a-5p mimic group decreased (P<0.05); after QLTC treatment, the expressions of TPT1 and p-mTORC1 in cells decreased (P<0.05); after overexpression of TPT1, the proliferation function of BPH cells was higher (P<0.05), apoptosis was lower (P<0.05), Bcl-2 expression increased (P<0.05), and expressions of Bax and Cleaved Caspase-3 decreased (P<0.05). Conclusion QLTC can inhibit benign prostatic hyperplasia by mediating miR-216a-5p to downregulate TPT1/mTORC1 pathway. |
Key words: Qianlongtong Capsule benign prostatic hyperplasia cell experiment miR-216a-5p tumor protein translationally controlled 1 mammalian target of rapamycin complex 1 signaling pathway |
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