过表达lncRNA HCG11调控线粒体自噬抑制喉癌细胞恶性生物学行为的机制研究

Study on the mechanism of overexpressed lncRNA HCG11 regulating mitochondrial autophagy to inhibit the malignant biological behaviors in laryngeal cancer cells

摘要:

目的 探讨长链非编码RNA HLA复合物11组(lncRNA HCG11，简称HCG11)对HEp-2喉癌细胞恶性生物学行为的影响及机制。方法 利用质粒转染技术将人喉表皮样癌HEp-2细胞分为HCG11对照组和过表达组，人鼻咽上皮NP69细胞作为正常组；采用定量聚合酶链式反应(qPCR)检测HCG11在3组细胞中表达情况。通过克隆形成实验检测HEp-2细胞生长；划痕实验检测HEp-2细胞迁移；Annexin V/PI染色检测HEp-2细胞凋亡；DCFH-DA荧光探针染色检测HEp-2细胞内活性氧(ROS)；透射电镜观察细胞内线粒体形态；Western blot分析细胞中PINK1、Parkin、LC3Ⅱ/LC3Ⅰ、p62蛋白表达。结果 qPCR结果显示，喉癌细胞比正常细胞HCG11水平表达更低，且成功构建喉癌细胞HCG11过表达模型。与对照组比较，HCG11过表达组HEp-2细胞增殖、迁移及凋亡均被抑制(P<0.05)；细胞内ROS水平升高(P<0.05)；细胞内线粒体形态受损；细胞PINK1、Parkin、LC3Ⅱ/LC3Ⅰ蛋白水平升高，p62蛋白水平降低(P<0.05)。结论 LncRNA HCG11在HEp-2喉癌细胞中表达下调，HCG11过表达可抑制HEp-2喉癌细胞增殖、迁移及凋亡，其机制可能与激活PINK1/Parkin信号通路，从而调控细胞内线粒体发生过度自噬相关。

Abstract:

Objective To explore the effect and mechanism of long non-coding RNA HLA complex 11 group (lncRNA HCG11，abb.HCG11) on the malignant biological behavior of HEp-2 laryngeal cancer cells. Methods Through plasmid transfection, HEp-2 cells from human laryngeal squamous cell carcinoma were divided into a HCG11 control group and an overexpression group, with NP69 nasopharyngeal epithelial cells as the normal control. Expression levels of HCG11 in these groups were assessed by quantitative polymerase chain reaction (qPCR). Cellular proliferation was measured via cloning formation assays, migration was monitored using scratch tests, apoptosis was identified through Annexin V/PI staining, and intracellular reactive oxygen species (ROS) levels were quantified with the DCFH-DA fluorescent probe. Mitochondrial structure was visualized under transmission electron microscope, and the protein expressions of PINK1, Parkin, the LC3 II/I ratio, and p62 were determined by Western blot. Results The qPCR results revealed that the expression level of HCG11 in laryngeal cancer cells was lower than that in normal cells, and an HCG11 overexpression model in laryngeal cancer cells was successfully constructed. Compared with the control group, the proliferation, migration, and apoptosis of HEp-2 cells in the HCG11 overexpression group were all inhibited (P<0.05), the intracellular ROS level increased (P<0.05), the mitochondrial morphology inside the cells was damaged, the protein levels of PINK1, Parkin, LC3Ⅱ/LC3Ⅰin the cells increased, while the protein level of p62 decreased (P<0.05). Conclusion The expression of lncRNA HCG11 is downregulated in HEp-2 laryngeal cancer cells. Overexpression of HCG11 can inhibit the proliferation, migration and apoptosis of HEp-2 laryngeal cancer cells. The underlying mechanism may be related to the activation of the PINK1/Parkin signaling pathway, thereby regulating excessive autophagy of intracellular mitochondria.