LncRNA AC000061.1调控CFTR在非梗阻性无精子症发病机制中的作用  

LncRNA AC000061.1 regulates CFTR in the pathogenesis of nonobstructive azoospermia

作　　者：杨慧敏 傅赟星 王飞苗[2] 王亚飞 季静 李嘉玲 胡蓉[2] Yang Huimin;Fu Yunxing;Wang Feimiao;Wang Yafei;Ji Jing;Li Jialing;Hu Rong(Ningxia Medical University,Yinchuan 750000,China;Reproductive Medicine Center of General Hospital of Ningxia Medical University,Yinchuan 750000,China)

机构地区：[1]宁夏医科大学,银川750000 [2]宁夏医科大学总医院生殖医学中心,银川750000

出　　处：《中华生殖与避孕杂志》2021年第9期822-831,共10页Chinese Journal of Reproduction and Contraception

基　　金：国家自然科学基金(8196277,81660257);宁夏自然科学基金(2020AAC03381);宁夏回族自治区重点研究开发计划重大(重点)项目(2019BFG02005);2016领军人才(宁夏);宁夏医科大学校级项目(XY201807)。

摘　　要：目的探讨长链非编码RNA(long non-coding RNA,LncRNA)AC000061.1调节CFTR基因表达在非梗阻性无精子症(nonobstructive azoospermia,NOA)发病机制中的作用。方法基因芯片检测梗阻性无精子症(obstructive azoospermia,OA)组(50例)、NOA组(50例)及对照组(50例)患者的睾丸组织中差异表达的LncRNA并对其对应的靶向mRNA进行生物信息学分析,用qRT-PCR和Western blotting法检测三组患者的睾丸组织凋亡基因Bcl-2表达差异。qRT-PCR验证三组患者睾丸组织、血清、精浆中LncRNA AC000061.1和CFTR mRNA表达水平。酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测血清和精浆中CFTR蛋白浓度。构建LncRNA AC000061.1过表达(H-LncRNA组)、沉默(Si-LncRNA组)及空载对照组载体转染至睾丸癌细胞系(NTERA-2),qRT-PCR验证LncRNA AC000061.1及CFTR mRNA的表达,Western blotting检测CFTR蛋白水平,CCK-8检测细胞增殖能力,流式细胞术及TUNEL凋亡试剂盒检测细胞凋亡率。结果芯片检测和qRT-PCR显示,与对照组相比,NOA组睾丸组织LncRNA AC000061.1和CFTR表达降低(P=0.033,P=0.042),OA组LncRNA AC000061.1表达无差异,CFTR低表达(P=0.039);OA组和NOA组凋亡基因Bcl-2表达水平依次增高(P=0.031,P=0.008)。三组血清中LncRNA AC000061.1 mRNA和CFTR mRNA及蛋白表达水平差异均无统计学意义(P均>0.05)。在精浆中,与对照组相比,NOA组和OA组LncRNA AC000061.1和CFTR mRNA及蛋白含量依次降低(P=0.002,P=0.038和P=0.006,P=0.026),且LncRNA AC000061.1与CFTR mRNA呈正相关(r=0.169,P=0.039)。质粒转染NTERA-2细胞后,沉默Si-LncRNA组LncRNA AC000061.1 mRNA及CFTR mRNA和蛋白表达均降低(P=0.005,P=0.003),过表达H-LncRNA组LncRNA AC000061.1 mRNA及CFTR mRNA和蛋白表达均显著增高(P=0.002,P=0.009)。沉默Si-LncRNA组细胞增殖能力显著下降(P=0.003),细胞凋亡率明显增高(P=0.001);过表达H-LncRNA组细胞增殖能力增加(P=0.017),细胞凋亡率降低(P=0.017)。结论NOA睾丸组织中LncRNA AC00006.1通过调控CObjective To explore the role of long non-coding RNA(LncRNA)AC000061.1 involved in the pathogenesis of nonobstructive azoospermia(NOA)by regulating the CFTR gene expression.Methods Bioinformatics analysis was conducted with the use of gene microarray to screen the differentially expressed LncRNAs and corresponding mRNAs in the testicular tissue of patients in three groups including obstructive azoospermia(OA)group(n=50),NOA group(n=50),and control group(n=50).The expression of apoptosis-related gene Bcl-2 in the testicular tissue of patients in these three groups was detected by qRT-PCR and Western blotting.Additionally,qRT-PCR was performed to determine the expression of LncRNA AC000061.1 and CFTR mRNA in the testicular tissue,serum,and seminal plasma of patients in the three groups,and enzyme-linked immunosorbent assay(ELISA)was conducted to detect the CFTR protein level in the serum and seminal plasma.LncRNA AC000061.1 overexpression(H-LncRNA group)and silencing vectors(Si-LncRNA group)and empty vectors were constructed and transfected into the testicular cancer cell line NTERA-2.Subsequently,the expression of LncRNA AC000061.1 and CFTR mRNA was determined by qRT-PCR,and CFTR protein expression was measured by Western blotting assay.Cell proliferation was assessed using CCK-8 and cell apoptosis was evaluated by flow cytometry and TUNEL.Results Microarray analysis and qRT-PCR showed that the expression of LncRNA AC006100.1 and CFTR decreased in NOA group compared with control group(P=0.033 and P=0.042).But there was no difference in the expression of LncRNA AC000061.1 between OA group and control group,while CFTR in OA group was lowly expressed compared with that in control group(P=0.039).Bcl-2 expression was sequentially upregulated in OA and NOA groups relative to normal control group(P=0.031 and P=0.008).No significant difference was noted in the serum levels of LncRNA AC000061.1 and CFTR mRNA and protein among the three groups(P>0.05).The results also showed sequentially decreased levels of LncRNA AC000061.

关 键 词：长链非编码RNA CFTR基因 非梗阻性无精子症 增殖 凋亡 

分 类 号：R698.2[医药卫生—泌尿科学]