miR-873-5p通过靶...细胞进展和抗血管生成的机制_王永杰.pdf

解剖学研究2023年第45卷第1期Anat Res,2023,Vol.45,No.1【摘要】目的探讨微小 RNA（miRNA）8735p 通过靶向自噬相关基因 Beclin1 抑制肺腺细胞进展和抗血管生成的机制研究。方法通过双荧光素酶报告验证miR8735p靶向Beclin1。将人肺癌细胞系A549随机分为4组：对照组、Mimic组、Mimic+Beclin1组和Beclin1组。通过质粒转染技术过表达miR8735p以及Beclin1。实时荧光定量PCR（qPCR）和Western blot 检测RNA 或蛋白的表达水平。分别通过CCK8、克隆形成实验和流式细胞术检测细胞活力、增殖和凋亡，细胞划痕实验、Transwell实验和管生成实验检测各组的细胞迁移、侵袭和血管生成能力。结果MiR8735p直接靶向Beclin1。过表达miR8735p抑制Beclin1 mRNA和蛋白的表达水平，过表达 Beclin1 显著逆转 miR8735p 对 Beclin1 的抑制作用。Mimic 组的细胞活力和细胞增殖能力显著低于对照组，凋亡率高于对照组（P0.05），Beclin1组的细胞活力和细胞增殖能力显著高于对照组，细胞凋亡率低于对照组（P0.05），并且Mimic+Beclin1组的细胞活力和细胞增殖能力显著高于Mimic组，细胞凋亡率低于mimc组（P0.05）。Mimic组的细胞迁移和侵袭能力显著低于对照组（P0.05），Beclin1组的细胞迁移和侵袭能力显著高于对照组（P0.05），并且 Mimic+Beclin1 组的细胞迁移和侵袭能力显著高于Mimic组（P0.05）；Mimic组的血管生成能力显著低于对照组（P0.05），Beclin1组的血管生成能力显著高于对照组（P0.05），并且Mimic+Beclin1组的血管生成能力显著高于Mimic组（P0.05）。结论MiR8375p可通过靶向抑制Beclin1的水平抑制肺癌细胞的生长、侵袭和血管生成，从而发挥抑制肺癌转移的作用。【关键词】肺癌；非小细胞肺癌；微小RNA；自噬基因Beclin1；自噬；血管生成DOI：10.20021/ki.16710770.2023.01.05Mechanism of miR8735p inhibiting progression and antiangiogenesis of lung cancer cells by targeting autophagyrelated gene Beclin1WANG Yongjie1，FANG Xiaoli2，LIU Shiqiang1，WANG Jie1，CHEN Xu1，WANG Hua1，HE Wenwu31Department of Thoracic surgery，Nanchong Central Hospital of Sichuan Province，Nanchong，Sichuan Province1637000，China；2Department of Cardiology Medicine，Nanchong Central Hospital of Sichuan Province，Nanchong，Sichuan Province637000，China；3Department of Thoracic Surgery，Sichuan Cancer Hospital，Chengdu，SichuanProvince 3610000【Abstract】ObjectiveTo explore the mechanism of microRNA（miRNA）8735p inhibition of lung glandcell progression and antiangiogenesis by targeting autophagyrelated gene Beclin1.MethodsThe miR8735p wastargeted to Beclin1 by dual luciferase reporter.Human lung cancer cell line A549 was divided into four groups：control group，Mimic group，Mimic+Beclin1 group and Beclin1 group.miR8735p and Beclin1 were overexpressed byplasmid transfection techniques.Quantitative Realtime PCR（qPCR）and Western blot were used to detect the expression level of Ribonucleic Acid（RNA）or protein.Cell viability，proliferation and apoptosis were detected by cellcounting kit8（CCK8）assay，colony formation assay and flow cytometry，respectively.Cell scratch assay，Transwell assay and tube generation assay were used to detect cell migration，invasion and angiogenesis in each group.ResultsMiR8735p directly targets Beclin1.Overexpression of miR8735p inhibits the expression levels of Beclin1 mRNA and protein.Overexpression of Beclin1 significantly reversed the inhibitory effect of miR8735p on Beclin1.The cell viability and cell proliferation ability of the Mimic group were significantly lower than those of themiR8735p通过靶向自噬相关基因Beclin1抑制肺癌细胞进展和抗血管生成的机制王永杰1，方小丽2，刘仕强1，王杰1，陈旭1，汪华1，何文武31四川省南充市中心医院胸心外科，四川南充1637000；2四川省南充市中心医院心内科，四川南充 2637000；3四川省肿瘤医院胸外科，四川成都3610000论著272解剖学研究2023年第45卷第1期Anat Res,2023,Vol.45,No.1control group，and the apoptotic rate was higher than that of the control group（P0.05）.The cell viability and cellproliferation ability of Beclin1 group were significantly higher than that of the control group，and the apoptosis ratewas lower than that of the control group（P0.05）.The cell viability and cell proliferation ability of Mimic+Beclin1group were significantly higher than that of Mimic group，and the apoptosis rate was lower than that of mimc group（P0.05）.The cell migration and invasion ability of the Mimic group was significantly lower than that of the controlgroup（P0.05）.The cell migration and invasion ability of Beclin1 group was significantly higher than that of thecontrol group（P0.05）.The cell migration and invasion ability of Mimic+Beclin1 group was significantly higherthan that of Mimic group（P0.05）.The angiogenic ability of the Mimic group was significantly lower than that of thecontrol group（P0.05）.The angiogenic ability of Beclin1 group was significantly higher than that of the controlgroup（P0.05）.The angiogenic ability of the Mimic+Beclin1 group was significantly higher than that of the Mimicgroup（P0.05）.ConclusionMiR8375p can inhibit the growth，invasion and angiogenesis of lung cancer cellsby targeting the level of inhibition of Beclin1，thereby inhibiting the metastasis of lung cancer.【Key words】Lung cancer；Nonsmall cell lung cancer；MicroRNA；Autophagy gene Beclin1；Autophagy；Angiogenesis肺癌是全球癌症相关死亡的主要原因，约80%的肺癌在组织病理学上被归类为非小细胞肺癌。其死亡率高，五年生存率约15%，威胁着人类健康1-2。大多数患者在确诊时已发展为侵袭性的疾病，因此疾病往往已到晚期，甚至转移到其他器官，给患者和医生带来了严重的困扰。微小RNA（microRNA，miRNA）是一类小的、非编码的、内源性单 RNA 分子，通过与靶基因 mRNA 的 3UTR 结合，导致 mRNA 裂解或翻译抑制，在基因表达中发挥重要作用3。大量研究表明，miRNAs参与各种生物过程，如细胞分化、细胞生长、死亡和时间发育4。miRNA 表达的异常与癌症等不同疾病有关。迄今为止，约有 50%的 miRNA 位于癌症相关基因区或脆弱区。MiR8735p是一种新发现的肿瘤相关 miRNA，具有促进非小细胞肺腺癌（Nonsmall cell lung adenocarcinoma，NSCLC）细胞增殖的作用，但是其在 NSCLC 转移和血管生成中的作用尚不清楚5。自噬是细胞对抗外部压力和刺激的一种调节方式，研究认为肿瘤细胞自噬的激活会促进其侵袭性并帮助获得耐药性，自噬基因Beclin1在细胞生长发育和维持细胞内环境稳定中发挥重要作用，研究已经发现了Beclin1在促进血管生成中的作用，但是期调节机制仍不清楚6。本文研究主要分析miR8735p通过靶向Beclin1抑制肺腺癌细胞侵袭和抗血管生成的机制。1材料与方法1.1材料与试剂1.1.1细胞株人肺腺癌细胞 A549 购自北京博奥凯美科技有限公司（货号：FNC177）。1.1.2主要仪器与主要试剂本研究所使用的miR8735p mimics、Beclin1 过表达质粒以及阴性对照由南京金斯瑞设计并且合