MicroRNA-320a调控唾液腺腺样囊性癌侵袭转移的实验研究

MicroRNA-320a regulates invasion and migration of salivary gland adenoid cystic carcinoma

目的:探讨microRNA-320a(miR-320a)对唾液腺腺样囊性癌侵袭、转移能力的调控作用。方法:以唾液腺腺样囊性癌高转移细胞株ACC-M为实验样品,通过脂质体介导,将miR-320a的拟似物(miR-320a mimics)转染至ACC-M细胞,升高miR-320a的表达水平。采用荧光实时定量RT-PCR检测转染前、后ACC-M中miR-320a的表达变化,通过Transwell实验检测细胞侵袭、迁移能力的改变,并通过细胞计数试剂盒-8检测转染后细胞增殖能力的变化,流式细胞仪检测miR-320a对细胞凋亡的影响,Western印迹检测miR-320a的靶基因整合素β3(integrinβ3,ITGB3)的表达变化。应用SPSS13.0软件包对所得数据进行t检验或单因素方差分析。结果:转染miR-320amimics后的ACC-M中miR-320a的表达明显上调(P<0.001)。ACC-M细胞的侵袭迁移能力显著降低(P<0.001),而细胞的增殖和凋亡无显著变化。Western印迹检测结果显示,Integrinβ3在低转移细胞株ACC-2表达阴性,在ACC-M中高表达;升高miR-320a在ACC-M中的表达,Integrinβ3表达明显降低。结论:低表达miR-320a有助于维持ACC的侵袭转移特性,调高其表达水平能有效抑制ACC-M的侵袭迁移能力。miR-320a可能通过调控其靶基因Integrinβ3的表达而发挥作用。

PURPOSE： To investigate the function of miR-320a via research of cell invasion and migration in salivary gland adenoid cystic carcinoma. METHODS： ACC-M was chosen as the experimental subject, miR-320a mimics were constructed and transfected into ACC-M by Lipofectamine TM2000.The expression of miR-320a was detected by real-time PCR.The changes of cell invasion and migration were detected by Transwell assays.In order to exclude the effect of cell proliferation, CCK-8 was conducted.CCK-8 was used to study the effect of miR-320a on the proliferation of ACC-M cells.Flow cytometry was performed to detect the effect of miR-320a on the apoptosis of ACC-M cells. Western blotting was performed to study the expression of Integrinβ3,which was predicted as target gene of miR-320a. One-way ANOVA or Student/s t test were performed for statistical analysis using SPSS 13.0 software package. RESULTS： After transfection of miR-320a mimics,the expression of miR-320a was significantly up-regulated （P〈0.001）. Transwell assays indicated that the invasion and migration ability of ACC-M were substantially reduced by miR-320a.CCK-8 and flow cytometry indicated that there was no significant change in cell proliferation and cell apoptosis. Western blotting showed that Integrin β3 was negatively expressed in ACC-2,while its expression was positive in ACC-M. With the elevated expression of miR-320a,the expression of Integrin β3 was down regulated substantially in ACC-M. CONCLUSION： Lower expression ofmiR-320a played an important role in the process of invasion and migration in ACC. Overexpression of miR-320a in ACC-M could substantially suppress its ability of cell invasion and migration, which may play its role by regulating its target gene integrin β3.