miRNA-185-5p通过靶向YWHAZ对人非小细胞肺癌细胞A549恶性生物学行为的影响

Effects of miRNA-185-5p on malignant biological behaviors of non-small cell lung cancer cell A549 by targeting YWHAZ

目的探讨微RNA(miRNA)-185-5p通过调控酪氨酸3/色氨酸5-单加氧酶激活蛋白(YWHAZ)对人非小细胞肺癌(NSCLC)细胞系A549增殖、侵袭凋亡和上皮间质转化的影响。方法获取GSE152702数据集,识别在NSCLC患者和健康者血液中差异性表达的miRNA,收集2018年7月至2019年9月在浙江大学附属第二医院确诊为NSCLC患者的癌组织及癌旁组织,使用RT-qPCR法检测miRNA-185-5p在NSCLC癌组织和细胞系中的表达情况。采用脂质体转染法,分别将miRNA-NC、miRNA-185-5p模拟物、miRNA-185-5p抑制剂和miRNA-185-5p模拟物+pcDNA3.0-YWHAZ转染至人NSCLC细胞A549中,Transwell实验检测各组A549细胞的侵袭能力,CCK-8实验检测各组A549细胞的增殖能力,脱氧核糖核酸末端转移酶介导的缺口末端标记(TUNEL)检测各组A549细胞的凋亡率,Western blot实验检测各组细胞中YWHAZ和上皮间质转化相关蛋白E-cadherin和Vimentin的表达情况。检索StarBase、Targetscan和Human protein Atlas数据库,韦恩图绘制与miRNA-185-5p含潜在互补结合位点及与NSCLC患者预后不良有关的基因。检索GEPIA数据库识别YWHAZ表达与肺鳞癌、肺腺癌患者预后的关系。生物学信息技术预测和双荧光素酶报告基因实验验证miRNA-185-5p和YWHAZ之间的关系。结果GSE152702数据集显示miRNA-185-5p在NSCLC患者中明显下调,miRNA-185-5p在NSCLC癌组织中的表达水平低于癌旁组织(P<0.01)。miRNA-185-5p在NSCLC细胞株中的表达水平低于正常人肺上皮细胞株(P<0.05),与miRNA-NC组比较,miRNA-185-5p模拟物组miRNA-185-5p表达水平明显升高(P<0.05),YWHAZ表达水平明显降低(P<0.01),miRNA-185-5p抑制剂组中miRNA-185-5p表达水平明显降低(P<0.05),YWHAZ表达水平明显升高(P<0.01)。过表达miRNA-185-5p能明显抑制A549细胞增殖、侵袭和上皮间质转化,并促进细胞凋亡(P<0.05)。GEPIA数据库显示,YWHAZ在肺鳞癌和肺腺癌患者中明显上调,且与患者预后不良有关。YWHAZ可与miRNA-185-5p靶向结合,过表达YWHAZ可部分逆转miRNA-185-5p模拟物对A549细胞增殖,侵袭和上皮间质转化的抑制作用,以及对细胞凋亡的促进作用。结论miRNA-185-5p在NSCLC癌组织及细胞中明显下调,过表达miRNA-185-5p能明显抑制A549细胞增殖、侵袭和上皮间质转化的能力,并促进细胞凋亡,其机制可能与调控YWHAZ的表达有关。

Objective To investigate the effects of microRNA(miRNA)-185-5p on the proliferation,invasion,apoptosis and epithelial-mesenchymal transition of non-small cell lung cancer(NSCLC)cell line(A549)by regulating tyrosine 3/tryptophan 5-mono-oxygenase activator protein(YWHAZ)expression.Methods The GSE152702 dataset was obtained to identify differentially expressed miRNAs between the blood of NSCLC patients and the blood of normal person.The cancer tissues and paracancerous tissues of the patients with difinitely diagnosed NSCLC in this hospital from July 2018 to September 2019 were collected.The expression of miRNA-185-5p in NSCL C cancer tissues and cell lines was detected by RT-qPCR.miRNA-NC,miRNA-185-5p mimics,miRNA-185-5p inhibitors and miRNA-185-5p mimics+pcDNA3.0-YWHAZ were transfected into NSCLC A549 cells,respectively.The Transwell assay was used to detect the invasion ability of A549 cells,the CCK-8 assay was used to detect the proliferation ability of A549 cells,and the TUNEL assay was used to detect the apoptosis rate of A549 cells in each group.The Western blot assay was used to detect the expressions of YWHAZ,E-cadherin and Vimentin in each group.The StarBase,Targetscan,and Human Protein Atlas databases were searched,and the Venn diagram was used to obtain the genes that contained potential complementary binding sites with miRNA-185-5p and were associated with poor prognosis of NSCLC patients.The GEPIA database was retrieved to identify the relationship between the YWHAZ expression and the prognosis of the patients with lung squamous cell carcinoma and lung adenocarcinoma.The relationship between miRNA-185-5p and YWHAZ was predicted by bioinformatics analysis and verified by the dual luciferase reporter assay.Results The GSE152702 dataset showed that miRNA-185-5p was significantly down-regulated in the patients with NSCLC.In addition,the expression level of miRNA-185-5p in NSCLC tissues was lower than that in paracancerous tissues(P<0.01).The expression of miRNA-185-5p in NSCLC cell lines was lower than that in normal lung epithelial cell lines(P<0.05).Compared with the miRNA-NC group,the expression level of miRNA-185-5p in the miRNA-185-5p mimic group was significantly increased(P<0.05).The expression level of YWHAZ was significantly decreased(P<0.01),the expression level of miRNA-185-5p in the miRNA-185-5p inhibitor group was significantly decreased(P<0.05),and the expression level of YWHAZ was significantly increased(P<0.01).Overexpression of miRNA-185-5p significantly inhibited the proliferation,invasion and epithelial-mesenchymal transformation of A549 cells,and promoted cell apoptosis(P<0.05).The GEPIA database showed that YWHAZ was significantly upregulated in the patients with lung squamous cell carcinoma and lung adenocarcinoma,moreover was associated with the poor prognosis.YWHAZ could be targeted to bind to mir-185-5p,and over-expressing YWHAZ could partially reverse the inhibitory effects of miRNA-185-5p mimics on the proliferation,invasion and epithelial-mesenchymal transition of A549 cells,as well as the promotion effect on apoptosis.Conclusion miRNA-185-5p is significantly down-regulated in NSCLC tissues and cells.Overexpression of miRNA-185-5p can significantly inhibit the proliferation,invasion and epithelial-mesenchymal transformation of A549 cells,and promote cell apoptosis.The mechanism may be related to the regulation of YWHAZ expression.