miR-140-3p靶向AGT5调控鼻咽癌细胞增殖、侵袭、迁移及自噬研究

miR-140-3p regulates proliferation,invasion,migration and autophagy of nasopharyngeal carcinoma cells by targeting AGT5

目的探讨miR-140-3p对鼻咽癌细胞增殖、侵袭、迁移和自噬的影响及其与自噬相关基因5(AGT5)的靶向关系。方法选择人鼻咽癌细胞CNE2细胞、人鼻黏膜上皮细胞HNEpC,根据转染不同质粒分为miR-140-3p组(转染miR-140-3p mimic质粒)、si-miR-140-3p组(转染miR-140-3p inhibitor质粒)、miR-空白对照(NC)组(转染miR-140-3p mimic空载体)、NC组(转染miR-140-3p inhibitor空载体)、si-miR-140-3p+OE组(转染miR-140-3p inhibitor质粒、过表达AGT5空载体)及si-miR-140-3p+AGT5组(转染miR-140-3p inhibitor质粒、过表达-AGT5质粒)。CCK-8实验检测细胞增殖能力,Transwell实验检测细胞侵袭、迁移能力,荧光定量聚合酶链式反应(RT-PCR)检测细胞AGT5 mRNA及miR-106b-5p表达水平,Western blot检测细胞微管关联蛋白轻链3Ⅰ(LC3Ⅰ)、微管关联蛋白轻链3Ⅱ(LC3Ⅱ)、AGT5、自噬关键分子酵母Atg6同系物1(Beclin1)及p62蛋白表达水平,TargetScan在线网站预测miR-140-3p与AGT5结合位点,双荧光素酶报告基因实验验证miR-140-3p与AGT5的靶向关系。结果CNE2细胞miR-140-3p、AGT5蛋白及AGT5 mRNA表达水平明显低于HNEpC细胞(0.21±0.03 vs 0.87±0.11、0.21±0.06 vs 0.87±0.12、0.32±0.05 vs0.74±0.08)(P<0.01)。上调miR-140-3p的CNE2细胞增殖、侵袭及迁移能力降低,LC3Ⅱ/LC3Ⅰ、Beclin1、AGT5蛋白表达水平升高,p62蛋白表达水平降低(P<0.01);下调miR-140-3p的CNE2细胞增殖、侵袭及迁移能力升高,LC3Ⅱ/LC3Ⅰ、Beclin1、AGT5蛋白表达水平降低,p62蛋白表达水平升高(P<0.01)。上调AGT5可逆转敲低miR-140-3p对CNE2细胞增殖、侵袭、迁移的促进作用及自噬的抑制作用。miR-140-3p与AGT5具有靶向调控关系。结论miR-140-3p对CNE2细胞增殖、侵袭、迁移具有抑制作用,对自噬具有促进作用,机制可能与正性调控AGT5有关。

Objective To investigate the effects of miR-140-3p on proliferation,invasion,migration and autophagy of na-sopharyngeal carcinoma cells and its targeting relationship with autophagy-associated gene 5(AGT5).Methods The human nasopharyngeal carcinoma cell CNE2 and human nasal epithelial cell HNEpC were selected and divided into miR-140-3p group(transfected with miR-140-3p mimic plasmid),si-miR-140-3p group(transfected with miR-140-3p inhibitor plasmid),miR-negative control(NC)group(transfected with miR-140-3p mimic empty vector),NC group(transfected with miR-140-3p inhibitor empty vector),si-miR-140-3p+overexpression(OE)group(transfected with miR-140-3p inhibitor plasmid,OE-AGT5 empty vector)and si-miR-140-3p+AGT5 group(transfected with miR-140-3p inhibitor plasmid,OE-AGT5 plasmid)according to transfection of different plasmids.The cell counting kit-8(CCK-8)assay was performed to detect proliferation ability;Transwell assay detect invasion and migration ability,the fluorescence quantitative polymerase chain reaction(RT-PCR)detect cellular AGT5 mRNA and miR-106b-5p expression level.The protein expression levels of microtubule-associated protein light chain 3 I(LC3I),microtubule-associated protein light chain 3 II(LC3II),AGT5,autophagy key molecule yeast Atg6 homo-logue 1(Beclin1)and p62 were detected by Western blot.The binding site of miR-140-3p and AGT5 was predicted by TargetScan online website,and targeting relationship between miR-140-3p and AGT5 was verified by dual luciferase reporter gene experiment.Results The expression levels of miR-140-3p,AGT5 protein and AGT5 mRNA in CNE2 cells were significantly lower than those in HNEpC cells(0.21±0.03 vs 0.87±0.11,0.21±0.06 vs 0.87±0.12,0.32±0.05 vs 0.74±0.08)(P<0.01).The proliferation,invasion and migration ability of CNE2 cells with up-regulated miR-140-3p were decreased,the expression levels of LC3II/LC3 I,Beclin1 and AGT5 proteins were increased,and the expression level of p62 protein was decreased(P<0.01).The proliferation,invasion and migration ability of CNE2 cells with down-regulation of miR-140-3p were increased,the expression levels of LC3II/LC3I,Beclin1 and AGT5 proteins were decreased,and the expression level of p62 protein was increased(P<0.01).The up-regulation of AGT5 was reversed the promotion of proliferation,invasion,migration and autophagy inhibition of CNE2 cells by knockdown of miR-140-3p.There was targeted regulatory relationship between miR-140-3p and AGT5.Conclusion It is demonstrated that miR-140-3p showed inhibitory effects on CNE2 cell proliferation,invasion and migration,and promotional effects on autophagy,the mechanism may be related to positive regulation of AGT5.