南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (7): 1012-1021.doi: 10.12122/j.issn.1673-4254.2021.07.07

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miR-424通过调控ATG14的表达抑制肝癌细胞的自噬和增殖

赵梓丹，黄 为，和君建，冯 超   

  • 出版日期:2021-07-20 发布日期:2021-07-19

MicroRNA-424 inhibits autophagy and proliferation of hepatocellular carcinoma cells by targeting ATG14

  • Online:2021-07-20 Published:2021-07-19

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摘要： 目的 研究miR-424通过调控ATG14表达影响自噬和肝癌细胞增殖。方法 收集肝癌患者因手术切除的肝癌组织及其癌旁组织，采用 qRT-PCR 法检测肝癌组织中 miR-424-5p，ATG14 的表达。培养人肝癌细胞系 HepG2、SMMC-7721、Huh-7、MHCC97H、HCCLM3和人正常肝细胞LO2，qRT-PCR法检测上述各细胞系的miR-424-5p表达，选取表达量较高的Huh-7和表达量较低的HCCLM3肝癌细胞为研究对象，Huh-7细胞实验分组（n=3）：干扰miR-424-5p表达阴性对照组（in-NC组），干扰miR-424-5p表达组（in-miR-424-5p组）；HCCLM3细胞实验分组（n=3）：过表达miR-424-5p组（mi-miR-424-5p组），另设过表达miR-424-5p阴性对照组（mi-NC组）；过表达miR-424-5p阴性对照+过表达ATG14阴性对照组（mi-NC+NC组）；过表达miR-424-5p阴性对照+过表达ATG14组（mi-NC+ ATG14组）；过表达miR-424-5p +过表达ATG14阴性对照组（mi-miR-424-5p+NC组）；过表达miR-424-5p +过表达ATG14组（mi-miR-424-5p +ATG14组）。qRT-PCR法及Western blot法验证各组miR-424-5p和ATG14的转染情况。采用MTT法检测各组细胞的细胞增殖，流式细胞术检测各组细胞的细胞凋亡水平，Western blot检测各组细胞的ATG14和自噬相关蛋白LC3、Beclin1和P62的表达水平，双荧光素酶报告基因实验检测miR-424-5p和ATG14之间的相互作用。结果 在肝癌组织和细胞中ATG14高表达，miR-424-5p低表达。与相应对照组相比，过表达miR-424-5p抑制肝癌细胞增殖和促进凋亡（P<0.05）；干扰miR-424-5p表达及过表达ATG14促进肝癌细胞增殖和抑制凋亡（P<0.05）；miR-424可能通过靶向ATG14发挥抑癌的作用。干扰miR-424-5p表达及过表达ATG14均能提高肝癌细胞自噬体标志蛋白LC3-ΙΙ/LC3-Ι、Beclin1的表达水平（P<0.05），降低自噬受体蛋白P62的表达水平（P<0.05）；过表达miR-424-5p则降低肝癌细胞自噬体标志蛋白LC3-ΙΙ/LC3-Ι、Beclin1的表达水平（P<0.05），提高自噬受体蛋白P62的表达水平（P<0.05）。结论 miR-424调控ATG14表达影响自噬，从而抑制肝癌细胞增殖。

关键词: 肝癌；miR-424-5p；ATG14；自噬；细胞增殖；细胞凋亡

Abstract: Objective To determine whether miR- 424 affects cancer cell proliferation and autophagy through ATG14 in hepatocellular carcinoma (HCC) cells. Methods We detected miR-424-5p and ATG14 expression levels in surgical specimens of HCC and adjacent tissues and in different HCC cell lines (HepG2, SMMC-7721, Huh-7, MHCC97H, and HCCLM3) and normal human hepatocyte LO2 cells using qRT-PCR and Western blotting. In the cell transfection experiments, we observed the effects of miR-424-5p knockdown in Huh-7 cells and the effects of overexpression miR-424-5p and ATG14 in HCCLM3 cells on the proliferation, cell cycle, apoptosis and expression levels of autophagy-related proteins (LC3, Beclin1 and p62). Dual luciferase reporter assay was used to verify the possible interaction between miR-424-5p and ATG14. Results In HCC tissues and cells, ATG14 was highly expressed and miR- 424- 5p expression was downregulated. In HCC cells, overexpression of miR- 424-5p obviously suppressed cell proliferation and promoted cell apoptosis (P<0.05), while inhibiting miR-424-5p or overexpressing ATG14 significantly promoted cell proliferation and inhibited cell apoptosis (P<0.05). Dual luciferase reporter assay indicated that miR-424-5p inhibits HCC cells by targeting ATG14. In addition, inhibition of miR-424-5p and overexpression of ATG14 both enhanced the expressions of LC3-ΙΙ/LC3-Ι and Beclin1 and decreased p62 expression (P<0.05), but miR-424-5p overexpression reduced the expressions of LC3-ΙΙ/LC3-Ι and Beclin1 and increased p62 expression (P<0.05). Conclusion MiR-424 inhibits HCC cell autophagy and proliferation through regulating ATG14.

Key words: hepatocellular carcinoma; miR-424-5p; ATG14; autophagy; proliferation; apoptosis