Abstract:

Objective: To study the role of miR-503 targeting reversion inducing cysteine rich protein with Kazal motifs (RECK) in the progression of oral squamous cell carcinoma (OSCC). Methods: Human OSCC cells SCC-4 were divided into NC inhibitor group (transfected with miR-503 inhibitor negative control sequence), miR-503 inhibitor group (transfected with miR-503 inhibitor), si-NC group (transfected with RECK siRNA negative control sequence), si-RECK group (transfected with RECK siRNA) and miR-503 inhibitor+si-NC group (co-transfected with miR-503 inhibitor and RECK siRNA negative control sequence) and miR-503 inhibitor+si-RECK group (co-transfected with miR-503 inhibitor and RECK siRNA); real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression level of miR-503 and RECK in immortalized human oral keratinocytes RT7 and SCC-4 cells in each group; Western blotting was used to detect the protein expression level of RECK. TargetScan was used to predict the target gene of miR-503 and dual luciferase reporter gene detection was used to verify the targeting relationship between miR-503 and RECK; CCK-8 and EdU staining were used to detect cell proliferation ability in each group; Transwell chamber was used to detect cell invasion ability; flow cytometry was used to detect cell apoptosis in each transfected group. Results: Compared with RT7 cells, the expression level of miR-503 in SCC-4 cells was increased (P<0.05), while the expression level of RECK was decreased (P<0.05); Compared with NC inhibitor group, the mRNA expression level of RECK and the protein expression level of RECK in the miR-503 inhibitor group were significantly increased (P<0.05). In addition, TargetScan database and luciferase reporter gene analysis showed a targeting relationship between RECK and miR-503 (P<0.01), suggesting that miR-503 could target the expression of RECK; compared with NC inhibitor group, the proliferation ability and invasion ability of SCC-4 cells in miR-503 inhibitor group were significantly decreased (P<0.05), while cell apoptosis ability was significantly increased (P<0.01), which indicated that the inhibition of miR-503 expression decreased proliferation, invasion ability and accelerated apoptosis ability of OSCC cells. Compared with si-NC group, the cell proliferation and invasion ability of si-RECK group were significantly increased (P<0.05), while cell apoptosis ability was significantly decreased (P<0.05), which indicated that the knockdown of RECK increased the proliferation, invasion ability and reduced the apoptosis ability of OSCC cells; compared with the miR-503 inhibitor+si-NC group, the expression level of RECK mRNA and the apoptotic ability in the miR-503 inhibitor+si-RECK group were significantly decreased (P<0.05), while the cell proliferation and invasion ability was significantly increased (P<0.05), which indicated that the knockdown of RECK weakened the inhibitory effect of miR-503 inhibitor on biological behavior of OSCC cells. Conclusion: The expression of miR-503 is up-regulated in OSCC cells. Inhibition of miR-503 can weaken its down-regulation of the target gene RECK, thereby reducing the proliferation and invasion of tumor cells and promoting cell apoptosis.

Key words: miR-503, RECK, targeted regulation, oral squamous cell carcinoma, proliferation, invasion, apoptosis

摘要：

目的：通过研究miR-503靶向回复引导半胱氨酸丰富蛋白Kazal基元(reversion inducing cysteine rich protein with Kazal motifs，RECK)在口腔鳞状细胞癌(oral squamous cell carcinoma，OSCC)进展中的作用。方法：将人OSCC细胞SCC-4分为NC inhibitor组(转染miR-503 inhibitor阴性对照序列)、miR-503 inhibitor组(转染miR-503 inhibitor)、si-NC组（转染RECK siRNA阴性对照序列）、si-RECK组(转染RECK siRNA)、miR-503 inhibitor + si-NC组（共转染miR-503 inhibitor和RECK siRNA阴性对照序列）和miR-503 inhibitor + si-RECK组(共转染miR-503 inhibitor和RECK siRNA)。实时定量聚合酶链反应 （real-time quantitative polymerase chain reaction，RT-qPCR）检测miR-503和RECK在各组SCC-4细胞及永生化人口腔角质形成细胞RT7中的表达；Western blotting检测RECK蛋白的表达；TargetScan预测miR-503的靶基因并通过双荧光素酶报告基因检测来验证miR-503与RECK的靶向关系；CCK-8和EdU染色检测各组细胞的增殖能力；Transwell小室检测细胞侵袭能力；流式细胞术检测各转染组细胞凋亡情况。结果：相比RT7细胞，miR-503在SCC-4细胞中高表达（P<0.05），RECK则呈低表达（P<0.05）；与NC inhibitor组相比，miR-503 inhibitor组细胞中RECK mRNA及RECK蛋白的表达水平均显著升高（P<0.05）；TargetScan数据库及荧光素酶报告基因分析显示了RECK 和 miR-503 之间的靶向关系（P<0.01），提示miR-503可靶向抑制RECK的表达；与NC inhibitor组相比，miR-503 inhibitor组SCC-4细胞的增殖能力、侵袭能力均显著下降（P<0.05），而细胞的凋亡则显著增加（P<0.01），抑制miR-503表达降低了OSCC细胞的增殖、侵袭并加速凋亡；与si-NC组相比较，si-RECK组细胞增殖、侵袭能力均显著增加（P<0.05），而细胞凋亡能力显著下降（P<0.05），敲低RECK增加了OSCC细胞的增殖、侵袭并降低凋亡；与miR-503 inhibitor + si-NC组相比较，miR-503 inhibitor + si-RECK组细胞中RECK mRNA的表达水平和细胞凋亡能力均显著下降（P<0.05），细胞增殖、侵袭能力显著增加（P<0.05），敲低RECK可削弱miR-503 inhibitor对OSCC细胞生物学行为的抑制作用。结论：miR-503在OSCC细胞中表达上调，抑制miR-503可削弱其对靶基因RECK的下调，从而降低肿瘤细胞的增殖与侵袭能力，并促进细胞的凋亡。

关键词: miR-503, 回复引导半胱氨酸丰富蛋白Kazal基元, 靶向调控, 口腔鳞状细胞癌, 增殖, 侵袭, 凋亡

CLC Number: 

• R782