特异性小干扰RNA下调中期因子基因表达对黑素瘤细胞黏附和侵袭的影响

中华皮肤科杂志 ›› 2011, Vol. 44 ›› Issue (7): 497-500.

特异性小干扰RNA下调中期因子基因表达对黑素瘤细胞黏附和侵袭的影响

周永静¹,龚丹丹¹,邱志远¹,彭辉勇¹,范钰²

1.
2. 江苏大学附属人民医院

收稿日期:2010-07-16 修回日期:2011-03-05 出版日期:2011-07-15 发布日期:2011-07-12
通讯作者: 范钰 E-mail:zjfany36@gmail.com
基金资助:
中国博士后科学基金

Down-regulation of midkine gene expression by small interfering RNA affects melanoma cell adhesion and invasion

Received:2010-07-16 Revised:2011-03-05 Online:2011-07-15 Published:2011-07-12
Contact: Yu Fan E-mail:zjfany36@gmail.com

摘要/Abstract

摘要：

目的探讨中期因子（midkine, MK）基因小干扰RNA（siRNA）对黑素瘤细胞侵袭的影响。方法根据MK基因mRNA序列特点，设计并用化学方法合成3个siRNA，转染人黑素瘤A375细胞后，以荧光实时定量PCR方法筛选出效果最好的siRNA，以此siRNA转染人黑素瘤A375细胞。同时设空白对照组（不予任何处理）和空载对照组（仅用脂质体转染）。分别以荧光实时定量PCR和Western印迹方法观察MK基因mRNA和蛋白水平，然后以噻唑蓝法检测细胞黏附率，以Boyden小室模型方法检测细胞侵袭力。结果所设计的siRNA均能有效下调MK基因mRNA表达水平，以S3号作用最强。以该siRNA转染A375细胞后，荧光实时定量PCR结果显示，转染组A375细胞MK基因mRNA水平明显下降，且呈浓度（24 h时r = -0.906；48 h时r = -0.922；72 h时r = -0.939，P均 < 0.01）和时间（3.125 nmol/L r = -0.889；6.25 nmol/L r = -0.935；12.5 nmol/L r = -0.928，P均 < 0.01）依赖性。细胞黏附试验显示，3.125、6.25和12.5 nmol/L siRNA组黏附率分别为73.66% ± 2.25%、49.36% ± 2.16%和28.35% ± 1.68%，呈浓度依赖性下降（r = -0.936，P < 0.01）。Boyden小室试验结果显示，3.125、6.25和12.5 nmol/L siRNA组穿过滤膜的细胞数分别为23.9 ± 1.6、12.1 ± 1.5、5.6 ± 1.2，而空白对照组为36.8 ± 1.5，穿膜细胞数呈浓度依赖性下降（r = -0.915，P < 0.05）。结论 MK基因在黑素瘤细胞黏附、侵袭中发挥重要作用；以siRNA转染黑素瘤细胞抑制该基因表达可抑制黑素瘤细胞黏附、侵袭能力。

关键词: 侵袭

Abstract:

Objective To study the effects of midkine（MK） gene-targeting small interfering RNA （siRNA） on the invasion of melanoma cells. Methods Three MK gene-targeting siRNAs（S1, S2 and S3） were designed, constructed, and transfected into human A375 melanoma cells. Real-time PCR was performed to measure the expression of MK gene and to screen the siRNA with best efficacy. Then, A375 cells were transfected with the optimal siRNA of various doses （3.125, 6.25 and 12.5 nmol/L） followed by additional culture of various durations （24, 48, 72 hours）. Some A375 cells remaining untreated served as the blank control group, and some transfected only with liposomes served as the vector control group. Reverse transcription （RT）-PCR and Western blot were conducted to detect the mRNA and protein expression of MK, respectively, MTT assay to observe the adhesion of A375 cells, and Boyden chamber was used to evaluate cell invasion. Results The expression of MK mRNA was downregulated by all the three siRNAs, especially by the siRNA S3, which was used in the following transfection experiment. Real-time quantitative PCR revealed that the MK mRNA expression was reduced by the siRNA in a dose- （r 24 hours = -0.906, r 48 hours = -0.922, r 72 hours = -0.939, all P < 0.01） and time-dependent （r 3.125 nmol/L = -0.889, r 6.25 nmol/L = -0.935, r 12.5 nmol/L = -0.928, all P < 0.01） manner. MTT assay showed that the percentage of adhesing cells was 73.66% ± 2.25%, 49.36% ± 2.16% and 28.35% ± 1.68% in A375 cells transfected with the siRNA of 3.125, 6.25 and 12.5 nmol/L, respectively. The number of cells migrating across the chamber filter was 23.9 ± 1.6, 12.1 ± 1.5, 5.6 ± 1.2 among A375 cells transfected with the siRNA of 3.125, 6.25 and 12.5 nmol/L, respectively, significantly lower than that in the blank control group （36.8 ± 1.5）. The percentage of adhesing cells and number of migrating cells decreased with the dose of siRNA （r = -0.936, -0.915, P < 0.01, 0.05, respectively）. Conclusions MK gene might play an important role in the adhesion and invasion of melanoma cells. To down-regulate the expression of MK gene by siRNA may suppress the adhesion and invasion of melanoma cells.

Key words: invasion

周永静龚丹丹邱志远彭辉勇范钰. 特异性小干扰RNA下调中期因子基因表达对黑素瘤细胞黏附和侵袭的影响[J]. 中华皮肤科杂志, 2011, 44(7): 497-500.

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