体外沉默ICMT基因对人唾液腺腺样囊性癌细胞侵袭和迁移的影响  

Effects of ICMT gene silencing on the invasion and migration of human salivary adenoid cystic carcinoma cells in vitro

作　　者：陆洲 宫文红 许晓 陈正岗[2] LU Zhou;GONG Wenhong;XU Xiao;CHEN Zhenggang(College of Stomatology,Weifang Medical University,Weifang 261021,China;Department of Stomatology,Qingdao Municipal Hospital,Qingdao University,Qingdao 266071,China)

机构地区：[1]潍坊医学院口腔医学院,山东潍坊(261021 [2]青岛大学附属青岛市市立医院口腔医学中心,山东青岛(266071

出　　处：《口腔疾病防治》2023年第6期400-407,共8页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：山东省自然科学基金青年项目(ZR2022QC034)。

摘　　要：目的 探讨异戊二烯基半胱氨酸羧基甲基转移酶(isoprene cysteine carboxymethyl transferase,ICMT)基因对唾液腺腺样囊性癌(salivary adenoid cystic carcinoma,SACC)细胞迁移和侵袭的影响及相关机制,为SACC的分子靶向治疗提供实验依据。方法 体外培养腺样囊性癌细胞SACC-LM和SACC-83,采用脂质体载体瞬时转染的方法,将ICMT siRNA转染至人SACC-LM和SACC-83细胞中(实验组),并分别设置空白对照组和阴性对照组(转染NC-siRNA)。通过qRT-PCR检测转染后各组细胞中ICMT和RhoA的m RNA表达并明确沉默效率;Western blot检测各组的ICMT、膜RhoA、总RhoA、Rho关联含卷曲螺旋结合蛋白激酶1(Rho associations contain curly helical binding protein kinase 1,ROCK1)、基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)蛋白表达;通过CCK-8实验检测SACC细胞的增殖能力;通过比较细胞划痕实验的相对愈合面积和Transwell实验细胞穿膜数目,分别检测SACC细胞的迁移和侵袭能力。结果 SACC-LM和SACC-83细胞转染ICMT-siRNA后,实验组相较于空白对照组和阴性对照组,ICMT mRNA和蛋白表达显著下降(P<0.05),但RhoA mRNA和RhoA总蛋白表达均无显著性差异(P>0.05);膜RhoA、ROCK1、MMP-2、MMP-9的蛋白表达显著下降(P<0.05),细胞增殖能力明显下降(P<0.05),迁移和侵袭能力明显降低(P<0.05)。结论 体外沉默ICMT基因可有效抑制人SACC-LM和SACC-83细胞迁移及侵袭能力,其机制可能与RhoA-ROCK信号通路有关。Objective To investigate the effect of isoprene cysteine carboxymethyltransferase(ICMT) gene on the migration and invasion of salivary adenoid cystic cancer cells(SACC) and the related mechanism,to provide experimental evidence for molecular targeted therapy of SACC.Methods Adenoid cystic cancer cells SACC-LM and SACC-83were cultured in vitro,and siRNA was transfected into human SACC-LM and SACC-83 cells(experimental group) by transient transfection of a liposome vector.A blank control group and negative control group were set up respectively(transfected NC-siRNA).qRT-PCR was peformed to measure the mRNA expression of ICMT and RhoA in each group after transfection and to determine the silencing efficiency.The expression of ICMT,membrane RhoA,total RhoA,matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9) and Rho associated with coiled helical binding protein kinase 1(ROCK1) in each group was detected by Western blot.The proliferation abilityies of SACC cells was detected by CCK-8 assay.The migration and invasion ability of SACC cells were detected by comparing the relative healing area of cell scratch assay and the number of Transwell assay cells.Results After transfection of ICMT-siRNA into SACC-LM and SACC-83 cells,the expression of ICMT gene and protein in the experimental group was significantly decreased compared with the negative control group and blank control group(P<0.05),but there were no significant differences in the expression of RhoA gene and total protein among all groups(P>0.05).The expression of RhoA membrane proteins,ROCK1,MMP-2,MMP-9 in the experimental group was significantly decreased compared with that in the negative control group and blank control group(P<0.05).Cell proliferation ability was significantly decreased(P<0.05).The migration and invasion abilities were significantly decreased(P<0.05).Conclusion In vitro silencing of ICMT gene can effectively inhibit the migration and invasion of human SACC-LM and SACC-83 cells,and the mechanism may be related to RhoA-ROCK signali

关 键 词：腺样囊性癌 RHOA 异戊二烯基半胱氨酸羧基甲基转移酶 小干扰RNA 侵袭 迁移 Rho关联含卷曲螺旋结合蛋白激酶1 基质金属蛋白酶-2 基质金属蛋白酶-9 RhoA-ROCK信号通路 

分 类 号：R78[医药卫生—口腔医学]