Hangzhou Hospital of Traditional Chinese Medicine (Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University), Hangzhou, Zhejiang Province, China.
Department of Surgery, Tonglu Hospital of Traditional Chinese Medicine, Hangzhou, Zhejiang Province, China.
Kaohsiung J Med Sci. 2022 Oct;38(10):933-949. doi: 10.1002/kjm2.12582. Epub 2022 Aug 23.
This study aimed to identify the molecular mechanism underlying NEAT1 regulation of non-small-cell lung cancer (NSCLC) autophagy and apoptosis. The expression levels of NEAT1, miR-128-3p, and ADAM28 in NSCLC tissues and cells were examined using qRT-PCR. The relationships between NEAT1, miR-128-3p, and ADAM28 expression levels and prognosis of NSCLC patients were investigated using the Kaplan-Meier analysis method. The interactions between NEAT1, miR-128-3p, and ADAM28 were confirmed by dual-luciferase reporter assay or FISH assay. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry assays, respectively. Apoptosis and autophagy-related proteins Bax, cleaved caspase-3, cleaved caspase-9, Bcl-2, LC3, Beclin-1, and p62 were analyzed by western blotting. Finally, an in vivo NSCLC mouse xenograft model was established to confirm the in vitro data. We showed NEAT1 and ADAM28 expression levels were upregulated, while the miR-128-3p level was downregulated in NSCLC tissues and cells. NSCLC patients with high NEAT1 expression levels, low miR-128-3p levels, or high ADAM28 levels had significantly reduced overall survival times. Silencing of NEAT1 inhibited NSCLC cell autophagy and promoted apoptosis by sponging miR-128-3p. MiR-128-3p directly targeted ADAM28 and suppressed ADAM28 expression, which led to deactivation of the JAK2/STAT3 signaling pathway. Furthermore, ADAM28 overexpression attenuated miR-128-3p overexpression-induced apoptosis and autophagy inhibition in NSCLC by increasing the phosphorylation of JAK2 and STAT3. NEAT1 depletion or miR-128-3p overexpression inhibited autophagy and promoted apoptosis in vivo by suppressing ADAM28. In other word, silencing NEAT1 inhibited autophagy and then promoted NSCLC cell apoptosis by deactivating the JAK2/STAT3 signaling pathway through regulation of miR-128-3p/ADAM28 axis.
本研究旨在确定 NEAT1 调节非小细胞肺癌(NSCLC)自噬和凋亡的分子机制。使用 qRT-PCR 检测 NSCLC 组织和细胞中 NEAT1、miR-128-3p 和 ADAM28 的表达水平。使用 Kaplan-Meier 分析方法研究 NEAT1、miR-128-3p 和 ADAM28 表达水平与 NSCLC 患者预后的关系。通过双荧光素酶报告实验或 FISH 实验验证 NEAT1、miR-128-3p 和 ADAM28 之间的相互作用。通过 CCK-8 和流式细胞术分别检测细胞增殖和凋亡。通过 Western blot 分析凋亡和自噬相关蛋白 Bax、cleaved caspase-3、cleaved caspase-9、Bcl-2、LC3、Beclin-1 和 p62。最后,建立 NSCLC 小鼠异种移植模型以验证体外数据。结果显示,NSCLC 组织和细胞中 NEAT1 和 ADAM28 的表达水平上调,而 miR-128-3p 的水平下调。NEAT1 高表达、miR-128-3p 低表达或 ADAM28 水平高的 NSCLC 患者总生存时间明显缩短。沉默 NEAT1 通过海绵 miR-128-3p 抑制 NSCLC 细胞自噬并促进凋亡。miR-128-3p 直接靶向 ADAM28 并抑制 ADAM28 表达,导致 JAK2/STAT3 信号通路失活。此外,ADAM28 过表达通过增加 JAK2 和 STAT3 的磷酸化,减弱了 miR-128-3p 过表达诱导的 NSCLC 细胞凋亡和自噬抑制。NEAT1 耗竭或 miR-128-3p 过表达通过抑制 ADAM28 抑制体内自噬并促进凋亡。换句话说,沉默 NEAT1 通过调节 miR-128-3p/ADAM28 轴抑制自噬,然后通过激活 JAK2/STAT3 信号通路促进 NSCLC 细胞凋亡。