miRNA-138-5p靶向调控胱天蛋白酶3减缓吸烟所致睾丸支持细胞TM4细胞凋亡  

miR-138-5p Alleviates Cigarette Smoking-induced Apoptosis of Testicular Sertoli Cells Through Caspase-3

作　　者：徐华[1] 买尔哈巴·阿不力孜 赵婉竹 何丽娟 XU Hua;ABULIZI Maierha-Ba;ZHAO Wuan-Zhu;HE Li-Juan(Reproductive Pregnancy Assistance Center of the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China;Department of Social Medicine,College of Public Health,Xinjiang Medical University,Urumqi 830054,China;Department of Health Toxicology,College of Public Health,Xinjiang Medical University,Urumqi 830054,China;Postdoctoral Research Center,School of Pharmacy,Xinjiang Medical University,Urumqi 830054,China)

机构地区：[1]新疆医科大学第一附属医院生殖助孕中心,乌鲁木齐830054 [2]新疆医科大学公共卫生学院社会医学教研室,乌鲁木齐830054 [3]新疆医科大学公共卫生学院卫生毒理学教研室,乌鲁木齐830054 [4]新疆医科大学药学院博士后流动站,乌鲁木齐830054

出　　处：《中国生物化学与分子生物学报》2023年第2期285-294,共10页Chinese Journal of Biochemistry and Molecular Biology

基　　金：新疆特殊环境与健康研究重点实验室开放课题项目(No.SKL-SEHR-2021-04);国家自然科学基金(No.82260651);博士后科研启动金资助。

摘　　要：近年来研究表明,长期大量吸烟可致睾丸出现不可逆性损伤进而出现精子发生受阻,凋亡信号通路在其中发挥着关键作用,其分子调控机制仍有待深入研究。本研究旨在探讨miRNA-138-5p在香烟烟雾所致小鼠睾丸支持细胞TM4细胞(正常小鼠睾丸Sertoli细胞)损伤中的靶向调控机制。MTT法、乳酸脱氢酶法和TUNEL法结果显示,CSE干预后的TM4细胞存活率显著降低(P<0.05),细胞毒性显著增加(P<0.05),细胞凋亡率显著增加(P<0.05);RT-PCR和Western印迹结果证明,10%CSE干预TM4细胞后,凋亡前体基因p53、促凋亡基因Bak和胱天蛋白酶3(caspase-3)的mRNA和蛋白质表达水平显著上调(P<0.05);在TM4细胞中转染miRNA-138-5p过表达质粒后同样进行CSE干预,结果显示,支持细胞存活率较转染前显著升高(P<0.05),凋亡阳性细胞数较转染前显著降低(P<0.05),凋亡前体p53基因、促凋亡基因Bak和Caspase-3的mRNA和蛋白质表达较转染前显著下调(P<0.05);沉默miRNA-138-5p后,Bak、胱天蛋白酶3的mRNA和蛋白质表达水平显著增加(P<0.05);在线数据库分析显示,miRNA-138-5p与胱天蛋白酶3具有较高的匹配预测值,双荧光素酶报告基因结果显示,Bak未结合至miRNA-138-5p,而胱天蛋白酶3可以靶向结合至miRNA-138-5p。以上结果提示,miRNA-138-5p可靶向调控胱天蛋白酶3,减缓吸烟所致睾丸支持细胞凋亡,具有支持细胞的保护作用。Recent studies have shown that long-term heavy smoking can cause irreversible damage to the testis and then block spermatogenesis,in which the apoptotic signaling pathway plays a key role.However,its molecular regulation mechanism remains to be further studied.The purpose of this study is to explore the targeting regulation mechanism of miRNA-138-5p in the injury of testicular Sertoli cells(TM4)induced by cigarette smoking.The MTT assay,lactate dehydrogenase(LDH)assay and TUNEL assay showed that after cigarette smoke exposure(CSE)intervention,the survival rate of TM4 cells was significantly decreased(P<0.05),LDH activity was significantly increased(P<0.05),and apoptosis rate was significantly increased(P<0.05).RT-PCR and Western blotting results showed that the mRNA and protein expression levels of p53,Bak and caspase-3 were significantly up-regulated after 10%CSE intervention(P<0.05).CSE intervention was also performed after transfection of miRNA-138-5p overexpression plasmids.The results showed that the survival rate of Sertoli cells was significantly increased(P<0.05),the number of apoptotic positive cells was significantly decreased(P<0.05),and the mRNA and protein expressions of p53,Bak and caspase-3 were significantly down-regulated compared with those before transfection(P<0.05).After silencing miRNA-138-5p,the mRNA and protein expression levels of Bak and Caspase-3 were significantly increased(P<0.05).Online database analysis showed that miRNA-138-5p and caspase-3 had high matching prediction values.The double luciferase reporter gene results showed that Bak did not bind to miRNA-138-5p,while caspase-3 could bind to miRNA-138-5p.These results suggest that miRNA-138-5p can target the regulation of Caspase-3 and slow down the apoptosis of Sertoli cells induced by cigarette smoking,which has a protective effect on Sertoli cells.

关 键 词：香烟烟雾暴露 TM4细胞(正常小鼠睾丸Sertoli细胞) miRNA-138-5p 

分 类 号：R114[医药卫生—卫生毒理学]