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目的:探讨siRNA干扰沉默乙酰肝素酶(heparanase,HPA)基因对人腺样囊性癌SACC-M细胞侵袭和迁移能力的影响。方法:根据人HPA基因序列设计并合成质粒表达载体转染至人涎腺腺样囊性癌(salivary adenoid cystic carcinoma,SACC)细胞,采用逆转录-聚合酶链反应(RT-PCR)和Western blot检测转染前后HPA mRNA和蛋白的表达变化,选出对HPA沉默效果最佳的质粒,获得稳定表达细胞株。通过划痕损伤实验和Transwell小室细胞侵袭实验检测RNA干扰沉默HPA表达后对SACC-M细胞侵袭和迁移能力的影响。结果:重组质粒pGPH1/GFP/Neo/HPA-siRNA显著降低HPA mRNA和蛋白的表达水平,干扰组中穿透Transwell小室基质的SACC-M细胞数明显低于对照组(P<0.05),划痕损伤实验结果显示,干扰组与对照组细胞迁移距离比较,24、48 h内差异有统计学意义(P<0.05)。结论:沉默人SACC-M细胞中HPA的mRNA和蛋白表达,可抑制SACC-M细胞的侵袭和迁移能力。HPA可能是治疗人类涎腺腺样囊性癌的一个新靶点。
Objective: To investigate the effect of silencing heparanase (siRNA) on invasion and migration of human adenoid cystic carcinoma SACC-M cells. METHODS: Plasmid expression vector was designed and synthesized according to human HPA gene sequence and transfected into human salivary adenoid cystic carcinoma (SACC) cells. RT-PCR and Western blot The expression of HPA mRNA and protein was detected before and after transfection, and the plasmid with the best silencing effect on HPA was selected to obtain a stably expressing cell line. The effects of RNA interference on the invasion and migration of SACC-M cells were detected by scratch injury assay and Transwell cell invasion assay. Results: The recombinant plasmids pGPH1 / GFP / Neo / HPA-siRNA significantly decreased the expression of HPA mRNA and protein. The number of SACC-M cells penetrating the Transwell matrix in the interference group was significantly lower than that in the control group (P <0.05) The results of injury experiments showed that there was significant difference between the interference group and the control group in migration distance within 24 and 48 h (P <0.05). Conclusion: The silencing of HPA mRNA and protein expression in SACC-M cells can inhibit the invasion and migration of SACC-M cells. HPA may be a new target for the treatment of human salivary adenoid cystic carcinoma.