miRNA干扰乙酰肝素酶基因对胆囊癌细胞侵袭、迁移的影响

Effects of heparanase gene silencing by miRNA interference on invasion and migration of gallbladder carcinoma cells

目的观察miRNA干扰沉默乙酰肝素酶（HPA）基因表达对人胆囊癌GBC—SD细胞侵袭和迁移的影响。方法根据人HPA基因序列设计并合成4对miRNA寡聚单链DNA，同时合成1对阴性对照质粒表达载体。采用阳离子脂质体转染法将以上质粒转染人人胆囊癌GBC—SD细胞，采用逆转录一聚合酶链反应（RT—PCR）检测转染前后HPAmRNA表达的变化，选出对HPA沉默效果最佳的质粒，使用抗生素Blasticidin2ms／L加压筛选阳性克隆并扩增培养传代，获得稳定表达细胞株，供下一步实验。通过划痕损伤实验和Transwell小室细胞侵袭、迁移实验检测干扰沉默HPA表达后对人胆囊癌GBC．SD细胞侵袭和迁移能力的影响。结果质粒pcDNA6．2-GW／EmGFP—miR-HPA-2沉默人胆囊癌GBC—SD细胞HPAmRNA表达的效果最好。pcDNA6．2-GW／EmGFP-miR-HPA-2组中穿过Transwell小室基质的胆囊癌GBC—SD细胞数明显低于阴性对照组（P〈0．05）和空白对照组。胆囊癌GBC—SD细胞的迁移距离在0、12、24hpcDNA6．2-GW／EmGFP—miR—HPA-2组明显低于阴性对照组和空白对照组（P〈0．05）。结论miRNA干扰沉默人胆囊癌GBC—SD细胞中HPAmRNA表达，从而抑制人胆囊癌GBC-SD细胞的侵袭和迁移能力。

Objective To investigate the effects of heparanase （HPA） gene silencing by miRNA interference on invasion and migration of GBC-SD cells. Methods Recombinant plasmid four pairs of single-stranded miRNA oligo-DNA and a pair of negative control plasmid expression vector targeting human HPA gene sequence were respectively transfected into GBC-SD cells by LipofectamineTM 2000, Reverse transcription-polymerase chain reaction （RT-PCR） were used to detect the mRNA expression changes of HPA before and after transfection and adopt to select the recombinant plasmid which showed the best silencing effect. Stable transfectants GBC-SD cells can be selected using 2 mg/L antibiotics Blasticidin pressure screening and positive clones for further experiments. Matrigel invasion assays and wound closrue assays were performed to examine the migration and invasive ability of the GBC-SD cells. Results Recombinant plasmid pcDNA6. 2-GW/EmGFP-miR-HPA-2 is the best one to silence mRNA expressions of HPA. Howerer, level of recombinant plasmid pcDNA6. 2-GW/EmGFP-miR-HPA-2 group GBC-SD cells is the most decreased by transwell invasion assay, compared with the negative control and blank, the difference was significant （P 〈 0. 05）. GBC-SD cells of recombinant plasmid pcDNA6. 2-GW/EmGFP-miR-HPA-2 group migration distance at 0, 12, 24 h was the most lowest compared the negative control and blank, the difference was significant （P 〈 0. 05）. Conclusion HPA mRNA of human gallbladder carcinoma GBC-SD cells are silenced by miRNA interference. （ Invasion and migration of human gallbladder carcinoma GBC-SD cells are inhibited afterHPA gene is silenced. ） The recombinant plasmid pcDNA6. 2-GW/EmGFP-miR can effectively suppress HPA mRNA expression, inhibit cellular invasion and migration in GBC-SD cells in vitro.