Abstract:

Objective: To investigate the effects of long non-coding RNA(lncRNA) growth arrest specific protein 6-antisense RNA1(DLX6-AS1) on the proliferation, migration and invasion of oral squamous cell carcinoma cells and its mechanism. Methods: The expression levels of DLX6-AS1, microRNA-15b (miR-15b) and phospholipase D1(PLD1) mRNA in oral squamous cell carcinoma and adjacent tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR). After DLX6-AS1 small interfering RNA was transfected into oral squamous cell carcinoma cells HSC3, MTT assay, Transwell and Western blot were used to detect the effects of inhibition of DLX6-AS1 expression on the proliferation, migration and invasion of HSC3 cells and the expression of p21, matrix metalloproteinase 2(MMP-2) and E-cadherin protein. Co-transfection of miR-15b inhibitor or PLD1 overexpression vector and DLX6-AS1 small interfering RNA into HSC3 cells. The same methods as above were used to observe the proliferation, migration and invasion and the expression of p21, MMP-2 and E-cadherin protein of HSC3 cells with DLX6-AS1 expression inhibition. The double luciferase reporter gene experiment verified the regulatory relationship between DLX6-AS1 and miR-15b or miR-15b and PLD1. Results: Compared with adjacent tissues, the expression levels of DLX6-AS1 and PLD1 mRNA in oral squamous cell carcinoma tissues were increased (P<0.001), and the expression level of miR-15b was decreased (P<0.001). Inhibition of DLX6-AS1 expression could reduce the survival rate, migration and invasion number of HSC3 cells and the expression level of MMP-2 protein (P<0.001), and increase the expression levels of p21 and E-cadherin protein (P<0.001). Inhibition of miR-15b expression or over-expression of PLD1 could reduce the effects of inhibition of DLX6-AS1 expression on the survival rate, migration and invasion number of HSC3 cells and the expression of p21, MMP-2 and E-cadherin protein. DLX6-AS1 targets negative regulation of miR-15b in HSC3 cells, and miR-15b targets negative regulation of PLD1. Conclusion: Inhibition of DLX6-AS1 expression may inhibit the proliferation, migration and invasion of oral squamous cell carcinoma cells through targeted regulation of miR-15b/PLD1 axis.

Key words: growth arrest specific protein 6-antisense RNA1(DLX6-AS1), microRNA-15b(miR-15b), phospholipase D1(PLD1), oral squamous cell carcinoma, invasion and metastasis

摘要：

目的：探讨长链非编码RNA（long non-coding RNA，lncRNA）生长停滞特异性蛋白6-反义RNA1（growth arrest specific protein 6-antisense RNA1，DLX6-AS1）对口腔鳞状细胞癌（口腔鳞癌）细胞增殖、迁移和侵袭的影响及作用机制。方法：实时荧光定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）检测口腔鳞癌组织及癌旁组织中DLX6-AS1、微小RNA-15b（microRNA-15b，miR-15b）和磷脂酶D1（phospholipase D1，PLD1）mRNA的表达水平。转染DLX6-AS1小干扰RNA至口腔鳞癌细胞HSC3，四甲基偶氮唑盐微量酶反应比色法（MTT法）、Transwell小室和Western印迹法（Western blot）检测抑制DLX6-AS1表达对HSC3细胞的增殖、迁移和侵袭能力，以及对p21、基质金属蛋白酶2（matrix metalloproteinase 2，MMP-2）和E-钙黏附素（E-cadherin）蛋白表达的影响。分别共转染miR-15b抑制剂或PLD1过表达载体与DLX6-AS1小干扰RNA至HSC3细胞，用上述相同方法观察抑制miR-15b或过表达PLD1对DLX6-AS1表达抑制的HSC3细胞增殖、迁移和侵袭能力，以及对p21、MMP-2和E-cadherin蛋白表达的影响。双荧光素酶报告基因实验验证DLX6-AS1与miR-15b及miR-15b与PLD1的调控关系。结果：与癌旁组织比较，口腔鳞癌组织中DLX6-AS1和PLD1 mRNA表达水平升高（P<0.001），miR-15b表达水平降低（P<0.001）。抑制DLX6-AS1表达可降低HSC3细胞存活率、迁移和侵袭，以及MMP-2的蛋白表达水平（P<0.001），提高p21和E-cadherin蛋白表达水平（P<0.001）。抑制miR-15b表达或过表达PLD1均可降低抑制DLX6-AS1表达对HSC3细胞的存活率、迁移和侵袭及P21、MMP-2和E-cadherin蛋白表达的影响。DLX6-AS1在HSC3细胞中靶向负调控miR-15b，miR-15b靶向负调控PLD1。结论：抑制DLX6-AS1表达可能通过靶向调控miR-15b和PLD1轴抑制口腔鳞癌细胞的增殖、迁移和侵袭。

关键词: 生长停滞特异性蛋白6-反义RNA1, 微小RNA-15b, 磷脂酶D1, 口腔鳞状细胞癌, 侵袭转移

CLC Number: 

• R739.8