背景:肺癌(LC)是全球最常见的癌症之一,以及癌症相关死亡的主要原因。非小细胞肺癌(NSCLC,占事件的85%)是LC的主要类型。miRNAs似乎在许多恶性肿瘤的发生和进展中发挥作用。根据挂载数据。miRNA调控NSCLC细胞生物学活性和进展的潜在机制,另一方面,仍在调查中。
方法:QRT-PCR检测非小细胞肺癌中miR-185-5p和YWHAZmRNA的表达。CCK-8测定用于确定肿瘤细胞的增殖能力。使用经纬仪测定来测试细胞的迁移和侵入特性。使用流式细胞术检测细胞凋亡。酪氨酸3-单加氧酶/色氨酸5-单加氧酶激活蛋白ζ(YWHAZ),E-Cadherin,使用蛋白质印迹评估N-钙黏着蛋白和切割的caspase3蛋白表达。生物信息学分析软件StarBase2.0预测miR-185-5p下游靶标。为了确认miR-185-5p和YWHAZ之间的靶标关联,使用荧光素酶实验。此外,建立了NCl-H1299异种移植模型,以评估miR-185-5p在体内的抗肿瘤作用.免疫组化法检测YWHAZ在小移植瘤模型肿瘤组织中的表达水平。
结果:在NSCLC中观察到miR-185-5p表达水平降低。体外,过表达的miR-185-5p降低了细胞活力,扩散,入侵/迁移,诱导细胞凋亡,同时抑制体内肿瘤生长。双荧光素酶基因实验证实YWHAZ与miR-185-5p结合。过表达YWHAZ部分恢复了miR-185-5p对细胞行为的抑制作用。
结论:MiR-185-5p在非小细胞肺癌中下调,过表达的miR-185-5p通过负调控YWHAZ抑制细胞的恶性行为和肿瘤生长。
BACKGROUND: Lung cancer (LC) is one of the most frequent cancers worldwide, as well as the leading cause of cancer-related death. Non-small cell lung cancer (NSCLC, which accounts for 85% of occurrences) is the main type of LC. MiRNAs appear to play a role in the occurrence and progression of many malignancies, according to mounting data. The underlying mechanism of miRNAs in regulating NSCLC cell biological activity and progression, on the other hand, is still being investigated.
METHODS: QRT-PCR were used to detect miR-185-5p expression and
YWHAZ mRNA in NSCLC. The CCK-8 assay was used to determine the tumor cells\' ability to proliferate. Transwall assay was used to test the migratory and invasive properties of cells. Cell apoptosis was detected using flow cytometry. Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (
YWHAZ), E-Cadherin, N-Cadherin and cleaved-caspase3 protein expression were assessed using Western Blot. The bioinformatics analysis software StarBase2.0 predicted miR-185-5p downstream targets. To confirm the target association between miR-185-5p and
YWHAZ, a luciferase experiment was used. In addition, an NCl-H1299 xenograft model was created to assess the anti-tumor impact of miR-185-5p in vivo. The expression level of YWHAZ in tumor tissues of small xenograft tumor model was detected by immunohistochemistry assay.
RESULTS: Decreased miR-185-5p expression levels were observed in NSCLC. In vitro, over-expressed miR-185-5p decreased cell viability, proliferation, invasion/migration, and induced cell apoptosis, while inhibiting tumor growth in vivo. Dual-luciferase gene experiments confirmed that
YWHAZ binds to miR-185-5p. Overexpression of
YWHAZ partially restored the inhibitory effects of miR-185-5p on cell behaviors.
CONCLUSIONS: MiR-185-5p was down-regulated in NSCLC, and that overexpressed miR-185-5p inhibited malignant behaviors of cells and tumor growth by negatively regulating YWHAZ.