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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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