CAJ | 학술논문

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.
목적 탐토반의미소RNA-21과핵감산(antisense oligonucleotide micro RNA-21,AS-miRNA-21)억제Tb3.1인설린상세포암증식적효과화궤제.방법 실험분3조,①공백대조조;②무의과핵감산전염조;③AS-miRNA-21전염조.과핵감산개도전염반의과핵감산고저Tb3.1세포miRNA-21표체.사용형광실시정량취합매련반응(PCR)감정전염후Tb 3.1세포miRNA-21표체수평;갑기새서기사서(MTT)법검측전염후Tb 3.1세포생존솔;류식세포술검측전염후Tb3.1조기조망;Matrigel기질생장실험검측전염후Tb 3.1세포생장형성구형집락능력;Transwell체외천이실험검측전염후Tb 3.1세포천이능력;단백질인적법검측전염후Tb3.1세포증식핵항원(antigen KI-.67,Ki67)、B세포림파류2(B cell lymphoma 2,Bcl-2)、인제10호염색체결실적린산매급장력단백동원적기인(phosphatase and tensin homolog,PTEN)、기질금속단백매2、9(matrix metalloproteinase 2/9,MMP-2、MMP-9)화조직기질단백매억제인자단백1(tissue inhibitor of metalloproteinase 1,TIMP-1)단백표체.결과 형광실시정량PCR현시전염후miRNA-21표체수평하조;전염제4천,AS-miRNA-21전염조종류세포생장속도[(53.43±11.83)%]저우기타량조[(91.32±8.02)%화100%](F=27.02,P=0.00);세포조망솔현저승고[(12.23±2.92)%,F=26.641,P=0.001];AS-miRNA-21전염조세포생장불능형성구형극륭차통과Transwell소실취탄산지막적세포수소우공백대조조(F=268.231,P=0.000);Ki67、Bcl-2、MMP-2화MMP-9단백표체하조,PTEN화TIMP-1단백표체상조.결론 고저miRNA-21후Tb3.1인설암세포증식여침습능력피억제,병위탐색miRNA-21조공인설암발생궤제제공실험의거.
Objective 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.