LncRNA ZFAS1靶向miR-373导致肝癌细胞顺铂耐药的机制研究#br#

目的　探究长链非编码RNA（LncRNA）锌指结构反义转录本1（ZFAS1）靶向微小RNA（miR）-373导致肝癌细胞顺铂（DDP）耐药的机制。方法　HepG2细胞设正常培养组、si-NC组、si-ZFAS1组，实时荧光定量PCR（qPCR）检测细胞中ZFAS1、miR-373表达水平。在si-NC组、si-ZFAS1组基础上分别添加0、50、100、200、300 μmol/L DDP处理细胞12 h，CCK-8检测细胞增殖情况，Transwell检测细胞侵袭情况，蛋白免疫印迹检测细胞中基质金属蛋白酶（MMP）2、MMP9蛋白表达水平。双荧光素酶验证ZFAS1与miR-373的靶向关系。在si-ZFAS1组基础上添加inhibitor miR-373，检测细胞中MMP2、MMP9蛋白表达水平。结果　si-ZFAS1组细胞中ZFAS1表达水平均低于正常培养组和si-NC组，miR-373表达水平均高于正常培养组和si-NC组（P＜0.05）。si-NC组和si-ZFAS1组经不同浓度顺铂处理后细胞增殖率、侵袭数量、侵袭蛋白MMP2、MMP9表达水平基本呈降低趋势；si-ZFAS1组上述指标分别低于其对应浓度的si-NC组（P＜0.05）。Starbase分析发现，miR-373与ZFAS1存在互补的结合位点并经双荧光素酶验证。si-ZFAS1+inhibitor miR-373组细胞MMP2、MMP9蛋白表达水平高于si-ZFAS1组和si-ZFAS1+inhibitor NC组（P＜0.05）。结论　ZFAS1可能降低肝癌细胞DDP耐药，其机制可能与调控miR-373有关。

Study on the mechanism of LncRNA ZFAS1 targeting miR-373 to induce cisplatin resistance in hepatocellular carcinoma cells

Objective　To explore the mechanism of long non-coding RNA (LncRNA) zinc finger antisense 1 (ZFAS1) targeting microRNA (miR)-373 to induce cisplatin (DDP) resistance in hepatoma cells. Methods　There were the normal culture group, the si-NC group and the si-ZFAS1 group in this study. The levels of ZFAS1 and miR-373 were detected by real-time quantitative PCR (qPCR). The cells were treated with DDP (0, 50, 100, 200 and 300 μmol/L) for 12 h on the basis of the si-NC group and the si-ZFAS1 group. CCK-8 was used to detect cell proliferation. Transwell was used to detect cell invasion. Western blot assay was used to detect the protein levels of matrix metalloproteinase (MMP)2 and MMP9. Double luciferase was used to verify the targeting relationship between ZFAS1 and miR-373. Inhibitor miR-373 was added on the basis of the si-ZFAS1 group, and the expression levels of MMP2 and MMP9 were detected. Results　The expression level of ZFAS1 was lower in the si-ZFAS1 group than that of the normal culture group and the si-NC group, and the expression level of miR-373 was higher than that of the normal culture group and the si-NC group (P＜0.05). After treatment with different concentrations of cisplatin, the cell proliferation rate, invasion number and the expression levels of invasion proteins MMP2 and MMP9 basically showed a decreasing trend in the si-NC group and the si-ZFAS1 group. The above indexes were lower in the si-ZFAS1 group than their corresponding concentrations of the si-NC group (P＜0.05). Starbase analysis showed that miR-373 and ZFAS1 had complementary binding sites, which were verified by double luciferase. The expression levels of MMP2 and MMP9 proteins were higher in the si-ZFAS1+inhibitor miR-373 group than those in the si-ZFAS1 group and the si-ZFAS1+inhibitor NC group (P＜0.05). Conclusion　ZFAS1 may reduce DDP resistance in liver cancer cells, and the mechanism may be related to the regulation of miR-373.