miR-1264对喉癌Hep2细胞增殖和迁移的影响

Effect of MiR-1264 on the proliferation and migration of Hep2 cell line

目的 miR-1264在喉癌中表达下调。文中旨在研究miR-1264对喉癌Hep2细胞生物学功能的影响及是否参与下调LCRG1基因的表达。方法 Real-time quantitative PCR检测miR-1264在人喉癌组织中的表达情况;将转染mimic/inhibitor NC Hep2细胞设为对照组,将未转染Hep2细胞设为空白组,将转染miR-1264 mimic/inhibitor设为实验组。利用MTT、Transwell分别观察miR-1264对Hep2细胞增殖、迁移和侵袭能力的影响;luciferase实验验证miR-1264和LCRG1 3'UTR的结合;RT-PCR、Western blot分别检测miR-1264、LCRG1蛋白表达。结果与对照组和空白组相比,Hep2细胞瞬转miR-1264mimic后,活细胞数量明显增加,增殖能力显著增强(P<0.05);而瞬转miR-1264 inhibitor后,活细胞数量明显降低,增殖能力减弱(P<0.05)。与对照组[(80.80±1.07)个]及空白组[(73.60±1.44)个]迁移细胞数量相比,实验组[(97.00±1.41)个]明显增加(P<0.05),迁移能力亦显著增强(P<0.05);与对照组[(80.00±1.11)个]及空白组[(73.60±1.44)个]迁移细胞数量相比,miR-1264 inhibitor组[(71.40±1.21)个]明显降低(P<0.05),迁移能力亦减弱(P<0.05)。与对照组[(43.00±1.41)个]和空白组[(50.60±1.03)个]侵袭细胞数量相比,实验组[(57.00±1.00)个]明显增加(P<0.05),侵袭能力亦显著增强(P<0.05);与对照组[(61.20±1.50)个]和空白组[(50.60±1.03)个]侵袭细胞数量相比,实验组[(27.60±0.93)个]明显降低,侵袭能力受到显著抑制(P<0.05),利用RT-PCR验证瞬转成功后,进一步应用Western blot实验检测LCRG1蛋白表达结果,显示:实验组LCRG1蛋白表达较NC对照组和空白组均无明显差异(P>0.05)。结论 miR-1264在喉癌组织中高表达,miR-1264可能不参与LCRG1蛋白的表达下调,但能增强Hep2细胞的增殖、迁移和侵袭能力。

Objective Previous study shows that the expression of mir-1264 is down-regulated in laryngeal carcinoma.This study aimed to investigate the effect of miR-1264 on the biological function of laryngeal carcinoma Hep2 cells and verify whether miR-1264 could down-regulate the expression of tumor suppressor gene laryngeal carcinoma related gene 1（LCRG1） in laryngeal carcinoma.Methods Real-time quantitative PCR was used to assess the miR-1264 expression in human laryngeal cancer tissues.There are three groups in the experiment：miR-1264 mimic/inhibitor group,mimic/inhibitor NC group and untransfected group.MTT and Transwell were applied to observe the effect of miR-1264 on the proliferation,migration and invasion capabilities of Hep2 cells.Luciferase experiment was used to verify the combination of miR-1264 and LCRG1 3 ＇UTR.RT-PCR and Western blot were used to test the expression of miR-1264 and LCRG1 protein respectively.Results Compared with the adjacent laryngeal tissue,miR-1264 expression in human laryngeal cancer tissues was significantly increased（P0.05）.Compared with NC control group and blank group,the proliferation,migration and invasion capabilities of Hep2 cells were significantly enhanced after they were transiently transfected with miR-1264 mimic（P0.05）; however,after Hep2 cells were transiently transfected with miR-1264 inhibitor,their proliferation,migration and invasion capabilities were significantly inhibited（P0.05）.Luciferase experiment showed miR-1264 mimic could significantly decrease LCRG13＇UTR luciferase activity,which was of statistical significance.After the verification of successful transient transfection,the results of further Western blot on LCRG1 protein expression showed that there was no significant difference between the experimental group and the mimic NC control group as well as the blank group（P 0.05）.Conclusion miR-1264 is highly expressed in laryngeal cancer tissues.Though miR-1264 may not participate in down-regulating the expression of tumor suppressor gene LCRG1 in laryngeal carcinoma,it can promote the proliferation,migration and invasion capabilities of Hep2 cells.