小干扰RNA下调畸胎瘤衍生生长因子-1基因表达对黑素瘤细胞黏附、侵袭和尿激酶纤溶酶原激活物的影响  

Effects of small interfering RNA tareting teratocarcinoma-derived growth factor-1 gene expression on cell adhesion and invasion and urokinase-type plasminogen activator of human malignant mela-noma cell

作　　者：方娜[1] 丁克云[2] 范钰[1] 

机构地区：[1]江苏大学附属人民医院肿瘤研究所, 江苏镇江212002 [2]江苏大学附属人民医院皮肤科

出　　处：《中华实验外科杂志》2014年第6期1309-1311,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81250035）;镇江市社会发展基金资助项目（SH2013048）

摘　　要：目的观察畸胎瘤衍生生长因子-1（TDGF-1）基因小干扰RNA对黑素瘤细胞黏附和侵袭的影响。方法培养人黑素瘤A-375、C-918及M14细胞株，以实时荧光定量聚合酶链反应（FQ—PCR）方法检测TDGF—1基因mRNA水平，筛选出TDGF-1表达最高者。采用TDGF-1基因小干扰RNA（siRNA）转染黑素瘤细胞株，分别以FQ—PCR和免疫荧光方法观察TDGF-1基因mRNA和蛋白水平，然后以噻唑蓝（MMT）法检测细胞黏附，以Boyden方法检测癌细胞侵袭力。结果FQ-PCR方法显示，A-375、C-918及M14细胞株TDGF-1 mRNA水平分别为0．589±0．081、0．712±0．065、1．517±0．085；以TDGF-1 siRNA转染黑素瘤M14细胞后，癌细胞TDGF-1基因mRNA和蛋白表达明显下降，且呈浓度依赖性（P〈0．01）；细胞黏附结果显示，Con-A、Con—B、3．125nmol／L、6．250nmol／L和12．500nmol/L组吸光度A值分别为0．89±0．15、0．85±0．12、0．62±0．09、0．51±0．08、0．33±0．06。Boyden小室试验结果显示，Con-A、Con-B、3．125nmol／L、6．250nmol／L和12．500nmol／L组穿膜细胞数分别为（37．8±1．6）、（36．9±1．5）、（21．8±1．2）、（11．6±0．8）和（5．6±0．5）个。酶联免疫吸附试验（ELISA）结果显示，Con—A、Con-B、3．125nmol／L、6．250nmol／L和12．500nmol／L组尿激酶型纤溶酶原激活物（uPA）含量分别为（85．2±1．8）、（84．8±1．5）、（51．6±1．2）、（35．6±0．8）、（17．8±0．6）ng／L。结论TDGF-1基因在黑素瘤细胞黏附和侵袭中发挥着重要作用；以siRNA转染黑素瘤细胞，可抑制黑素瘤细胞黏附和侵袭能力，抑制uPA是其重要机制之一。Objective To study the effects of teratocarcinoma-derived growth factor-1 （TDGF-1） geue small interfering RNA （siRNA） on adhesion and invasion of human melanoma cell. Methods Realtime fluorescent quantitative polymerase chain reaction（FQ-PCR） was used to evaluate the TDGF-1 mRNA expression of human melanoma cell lines A-375, C-918 and M14. The highest expression of TDGF-1 was transfected with different dose of TDGF-1 siRNA. The expression of TDGF-1 mRNA and protein were were determined by Real-time quantitative PCR and Western blotting, respectively. Cell adhesion was exmined by methyl thiazol tetrazolium （MTT） assay, and cell invasion was evaluated by boyden chamber, respectively. The matrix metalloproteinase-13 （MMP-13） were examined by enzyme linked immunosorbent assay （EL1SA） assay. Results The results from FQ-PCR showed that the TDGF-1 mRNA of A-375, C-918 and M14 cell line is 0. 589 ±0. 081,0. 712 ±0. 065 ,and 1. 517 ±0. 085. After M14 cell line was transfected by TDGF-1 siRNA, the results of the MTT assay showed that the A Values of Con-A, Con-B, 3. 125 nmol/L, 6. 250 nmoL/L, and 12. 500 nmol/L siRNA were 0. 89 ± 0. 15,0. 85 ± 0. 12,0. 62 ± 0. 09, 0. 51 ± 0. 08, 0. 33 ± 0.06, respectively （ P 〈 0. 05 ） ; the results of Boyden assay showed that the membrane cell number of Con-A, Con-B, 3. 125 nmol/L siRNA,6. 250 nmol/L siRNA, and 12. 500 nmol/L siRNA were 37.8 ± 1.6, 36.9±1.5, 21.8±1.2, 11.6 ±0.8,andS. 6 ±0.5,respectively （P〈0.05）. The results from ELISA showed that urokinase-type plasminogen activator （uPA） content of Con-A, Con-B, 3. 125 nmoL/L siRNA ,6. 250 nmol/L siRNA,and 12. 500 nmoL/L siRNA were （85.2 ± 1.8）, （84. 8 ± 1.5）, （51.6 ± 1.2）, （35.6 ± 0. 8） , （ 17. 8 ± 0. 6 ） ng/L. Conclusion TDGF-1 gene might play an important play in adhesion and invasion of human melanoma cancer cell. The siRNA targeted TDGF-1 could effectively inhibit adhesion and invasion of human melanoma cancer cell through downregulation uPA.

关 键 词：黑素瘤 畸胎瘤衍生生长因子-1 黏附 侵袭 尿激酶纤溶酶原激活物 

分 类 号：R739.5[医药卫生—肿瘤]