lncRNA LOC101928477在食管鳞状细胞癌中的作用研究

Role of lncRNA LOC101928477 in esophageal squamous cell carcinoma

摘要:

目的 探讨lncRNA LOC101928477对人食管鳞状细胞癌（ESCC）ECA109细胞增殖、迁移、凋亡及自噬的生物学作用，并探索其相关分子调控关联。方法 将ECA109细胞分为5组：A组（空白对照，仅培养ECA109细胞）、B组（空载病毒对照，ECA109+oe-NC）、C组（LOC101928477过表达，ECA109+oe-LOC101928477）、D组（miR-139-5p抑制剂，ECA109+miR-139-5p抑制剂）、E组（联合干预，ECA109+miR-139-5p抑制剂+oe-LOC101928477）。CCK-8法检测细胞活性，流式细胞术分析细胞凋亡，划痕试验评估细胞迁移能力，实时荧光定量聚合酶链反应检测LOC101928477和miR-139-5p的mRNA表达，Western blotting检测自噬相关蛋白（Beclin1、LC3-Ⅱ/LC3-Ⅰ、P62）的表达，酶联免疫吸附试验（ELISA）检测肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）水平。结果 A组与B组ECA109细胞活力、细胞凋亡率、细胞迁移能力、LOC101928477 mRNA及Beclin1、LC3-Ⅱ/LC3-Ⅰ、P62蛋白表达，以及TNF-α、IL-6水平比较，差异均无统计学意义（P >0.05）；与B组相比，C组ECA109细胞活力、细胞迁移能力降低，细胞凋亡率升高，Beclin1蛋白表达及LC3-Ⅱ/LC3-Ⅰ显著上调，P62蛋白表达下调，TNF-α和IL-6水平升高（P <0.05）；与C组相比，D组上述LOC101928477诱导的表型效应及自噬激活均逆转（P <0.05）；与D组相比，E组细胞活力、细胞迁移能力进一步降低，凋亡率及自噬活性进一步升高，TNF-α和IL-6水平也进一步升高（P <0.05）。结论 lncRNA LOC101928477可激活ECA109细胞自噬，抑制ECA109的增殖与迁移，促进细胞凋亡，并上调炎症因子TNF-α和IL-6水平；其发挥上述抑癌作用的分子机制与调控miR-139-5p的表达相关，可为ESCC的靶向治疗提供新的理论依据。

Abstract:

Objective To investigate the biological effects of long non-coding RNA (lncRNA) LOC101928477 on proliferation, migration, apoptosis and autophagy of human esophageal squamous cell carcinoma (ESCC) ECA109 cells, and to explore the associated molecular regulatory mechanisms.Methods ECA109 cells were divided into five groups: Group A (blank control, ECA109 cells untreated), Group B (empty vector control, ECA109 cells treated with oe-NC), Group C (LOC101928477 overexpression, ECA109 cells treated with oe-LOC101928477), Group D (miR-139-5p inhibition, ECA109 cells treated with miR-139-5p inhibitor), and Group E (combined intervention, ECA109 cells treated with both miR-139-5p inhibitor and oe-LOC101928477). Cell viability was detected by the Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was analyzed by flow cytometry. Cell migration ability was assessed by the scratch assay. The mRNA expressions of LOC101928477 and miR-139-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The expressions of autophagy-related proteins (Beclin1, LC3-Ⅱ/LC3-I, P62) were detected by Western Blotting. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined by enzyme-linked immunosorbent assay (ELISA).Results Compared with Group A, there were no significant differences in all detected indicators in Group B (P > 0.05). Compared with Group B, the cell viability and migration ability of ECA109 cells in Group C were significantly decreased, the apoptosis rate was significantly elevated, the expression of Beclin1 and the ratio of LC3-Ⅱ/LC3-Ⅰ were remarkably upregulated, the protein expression of P62 was prominently downregulated, and the levels of TNF-α and IL-6 were significantly increased (all P < 0.05). Compared with Group C, LOC101928477-induced phenotypic effects and autophagy activation as mentioned above were significantly reversed in Group D (all P < 0.05). Compared with Group D, the cell viability and migration ability in Group E were further decreased, accompanied by further increases in the apoptosis rate, autophagic activity, and levels of TNF-α and IL-6 (all P < 0.05).Conclusion The long non-coding RNA LOC101928477 activates autophagy in human esophageal squamous cell carcinoma ECA109 cells, inhibits cell proliferation and migration, promotes apoptosis, and upregulates the levels of inflammatory cytokines TNF-α and IL-6. The tumor-suppressive effects of LOC101928477 may be mediated through regulation of miR-139-5p expression. These findings provide a novel theoretical basis for targeted therapy in esophageal squamous cell carcinoma.