lncRNA UCA1对口腔鳞状细胞癌细胞增殖、凋亡的影响及其机制研究

The role of long non-coding RNA UCA1 in the proliferation and apoptosis of oral squamous cell carcinoma cell

摘要:

目的 探究长链非编码RNA尿路上皮癌相关基因1（lncRNA UCA1）通过靶向miR-206调控Yes相关蛋白1（YAP1）的表达，介导口腔鳞状细胞癌（OSCC）细胞增殖、凋亡的分子机制。方法 通过实时荧光定量聚合酶链反应（qRT-PCR）检测人正常口腔角质细胞系HOK和人口腔鳞状细胞癌细胞株TSCCA、SCCl5、HN13、CAL27、HSC3和SCC9中lncRNA UCA1和microRNA-206（miR-206）的表达；在HN13细胞中敲除IncRNA UCA1，采用CCK-8法和流式细胞术检测其对HN13细胞增殖和细胞凋亡的影响；通过生物信息学网站starBase预测IncRNA UCA1、YAP1与miR-206的互补结合位点，并通过双荧光素酶实验验证它们之间的结合关系；Western blotting检测YAP1蛋白的表达。结果 qRT-PCR结果显示，相比于人正常口腔角质细胞系HOK，6种OSCC细胞中lncRNA UCA1 mRNA相对表达量上调（P <0.05），而miR-206 mRNA相对表达量均降低（P <0.05）。与转染siNC的对照组相比，siUCA1组细胞活力下降，凋亡比例升高（P <0.05）。生物信息学预测结合双荧光素酶实验证实miR-206与lncRNA UCA1、YAP1均存在互补结合。qRT-PCR和Western blotting检测结果显示，siUCA1组miR-206 mRNA相对表达量较对照组升高（P <0.05），YAP1 mRNA相对表达量较对照组降低（P <0.05），而同时转染siUCA1和miR-206 inhibitor则抑制miR-206表达，恢复YAP1的表达（P <0.05）。结论 lncRNA UCA1在OSCC细胞中高表达，通过抑制miR-206的表达，上调YAP1，从而促进OSCC细胞增殖，抑制凋亡。

Abstract:

Objective To explore the molecular mechanism of long non-coding RNA urothelial carcinoma-associated 1 (lncRNA UCA1) mediating the proliferation and apoptosis of oral squamous cell carcinoma (OSCC) cells, potentially by regulating the expression of Yes-associated protein 1 (YAP1) via targeting miR-206.Methods The expression of lncRNA UCA1 and miR-206 was detected via quantitative real-time polymerase chain reaction (qRT-PCR) in normal human oral keratinocytes (HOKs) and human OSCC cell lines TSCCA, SCCl5, HN13, CAL27, HSC3 and SCC9. The CCK8 assay and flow cytometry were used to assess the cell proliferation and apoptosis of HN13 after lncRNA UCA1 knockdown, respectively. The binding site of lncRNA UCA1 to miR-206, and that of miR-206 to YAP1 were predicted through the bioinformatics website starBase. Dual-luciferase reporter assay was applied to verify the interaction of miR-206 with lncRNA UCA1 and YAP1. Western blotting was performed to determine the protein expression level of YAP1.Results The qRT-PCR revealed that the expression of lncRNA UCA1 was increased (P < 0.05), while the expression of miR-206 was reduced (P < 0.05) in all OSCC cells relative to the normal HOKs. Compared with the control group, the cell viability of the siUCA1 group was lower, and the proportion of cell apoptosis was higher (P < 0.05). The bioinformatic analysis predicted and dual-luciferase reporter assay verified that miR-206 could bind to both lncRNA UCA1 and YAP1. The qRT-PCR and Western blotting showed that miR-206 was up-regulated (P < 0.05) and YAP1 was down-regulated (P < 0.05) in the siUCA1 group compared with the control group. Reversely, miR-206 expression was inhibited and YAP1 expression was restored after the co-transfection of siUCA1 and miR-206 inhibitor (P <0.05).Conclusions lncRNA UCA1 was highly expressed in OSCC cells, which promotes the proliferation and suppresses the apoptosis of OSCC cells via inhibiting the expression of miR-206 and up-regulating the level of YAP1.