摘要目的 探讨反义微小RNA-21寡核苷酸(antisense oligonucleotide micro RNA-21,AS-miRNA-21)抑制Tb3.1人舌鳞状细胞癌增殖的效果和机制.方法 实验分3组,①空白对照组;②无义寡核苷酸转染组;③AS-miRNA-21转染组.寡核苷酸介导转染反义寡核苷酸敲低Tb3.1细胞miRNA-21表达.使用荧光实时定量聚合酶链反应(PCR)鉴定转染后Tb 3.1细胞miRNA-21表达水平;甲基噻唑基四唑(MTT)法检测转染后Tb 3.1细胞生存率;流式细胞术检测转染后Tb3.1早期凋亡;Matrigel基质生长实验检测转染后Tb 3.1细胞生长形成球形集落能力;Transwell体外迁移实验检测转染后Tb 3.1细胞迁移能力;蛋白质印迹法检测转染后Tb3.1细胞增殖核抗原(antigen KI-.67,Ki67)、B细胞淋巴瘤2(B cell lymphoma 2,Bcl-2)、人第10号染色体缺失的磷酸酶及张力蛋白同源的基因(phosphatase and tensin homolog,PTEN)、基质金属蛋白酶2、9(matrix metalloproteinase 2/9,MMP-2、MMP-9)和组织基质蛋白酶抑制因子蛋白1(tissue inhibitor of metalloproteinase 1,TIMP-1)蛋白表达.结果 荧光实时定量PCR显示转染后miRNA-21表达水平下调;转染第4天,AS-miRNA-21转染组肿瘤细胞生长速度[(53.43±11.83)%]低于其他两组[(91.32±8.02)%和100%](F=27.02,P=0.00);细胞凋亡率显著升高[(12.23±2.92)%,F=26.641,P=0.001];AS-miRNA-21转染组细胞生长不能形成球形克隆且通过Transwell小室聚碳酸酯膜的细胞数小于空白对照组(F=268.231,P=0.000);Ki67、Bcl-2、MMP-2和MMP-9蛋白表达下调,PTEN和TIMP-1蛋白表达上调.结论 敲低miRNA-21后Tb3.1人舌癌细胞增殖与侵袭能力被抑制,并为探索miRNA-21调控人舌癌发生机制提供实验依据.

abstractsObjective To investigate the effect of micro RNA-21 (miRNA-21) knocking on the Tb3.1 human tongue squamous cell carcinoma growth. Methods Anti-sense miRNA-21 oligonucleotide was delivered with oligofectamine to suppress Tb 3. 1 tongue cancer cell growth in vitro. Real-time polymerase chain reaction (PCR) was conducted to detect the miRNA-21 expression after transfection. Methyl thiazolyl tetrazolium(MTT) assay was used to determine Tb 3. 1 cell survival rate. Apoptosis were examined by flowcytometry. Matrigel matrix and transwell assay were used to determine Tb 3.1 cell colony formation and migration ability. Antigen KI-67 (Ki67), B cell lymphoma (Bcl-2), phosphatase and tensin homolog (PTEN), matrirx metalloproteinase 2(MMP-2, MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) protein expression in Tb 3. 1 cell were measured by Western blotting. Results miRNA-21 expression was decreased in miRNA-21 antisense oligonucleotide (ASODN) group. The survival rate of Tb 3. 1 cells with AS-miRNA-21 transfection was significantly suppressed (F=27.02, P = 0.00) and early phase apoptosis(F =26. 641 ,P = 0. 001) induced in Tb 3.1 cell. Ki67, Bcl-2, MMP-2 and MMP-9 protein weredown regulated while PTEN and TIMP-1 protein expression was increased. Conclusions Blocking miRNA-21 expression in Tb3.1 cell could suppress cancer cell growth in vitro and miRNA-21 can serve as a novel target candidate for human tongue cancer gene therapy.