【摘要】 目的观察RNA干扰沉默缺氧诱导因子-1α(HIF-1α)基因对人舌癌细胞Tca8113侵袭和迁移的影响。方法将3个shRNA序列质粒导入pGCsi-H1/Hygro/GFPshRNA质粒表达载体,并将重组质粒经脂质体介导转染人舌癌细胞Tca8113(siHIF-1α组),转染阴性对照质粒的细胞作为空载组。半定量RT-PCR及Western blot法检测HIF-1α的mRNA及蛋白,细胞侵袭实验和划痕实验检测Tca8113的侵袭和迁移能力,同时采用Western blot法检测其上皮—间质转化(EMT)相关标志蛋白和黏着斑激酶(FAK)、磷酸化FAK(P-FAK)。结果测序证实成功构建了HIF-1αshRNA重组质粒(分别为sh1、sh2、sh3),将sh3,转染Tca8113后进行低氧培养,HIF-1α基因表达显著被抑制。与空载组相比,转染sh3的siHIF-1α组穿透Matrigel胶的细胞数明显减少,且迁移能力也显著下降,两组相比,P均<0.05。与对照组(未转染质粒的Tca8113)相比,siHIF-1α组EMT相关标志蛋白和p-FAK蛋白表达下降,两组相比,P均<0.05。结论 RNA干扰沉默HIF-1α基因能有效抑制舌癌细胞的侵袭和迁移,在一定程度上可以逆转舌癌细胞的恶性表型。更多还原

【Abstract】 Objective To investigate the effect of RNA interference( RNAi) targeting hypoxia-inducible factor-1α( HIF-1α) gene on the invasion and migration of human tongue cancer cell line Tca8113. Methods The three kinds of HIF-1α-specific short hairpin RNA( shRNA) templates were cloned into pGCsi-H1 / Hygro / GFPshRNA expression vector, the recombinant plasmids were stably transfected into cultured human tongue cancer cells Tca8113 via liposome( siHIF-1α group),cells transfected by negative control plasmids were taken as the no-load group. The inhibition effects on HIF-1α gene were determined by semi-quantitative RT-PCR and Western blotting. The invasive and migratory abilities were detected with transwell invasion assay and migration assay. Additionally,the expression of epithelial-mesenchymal transition( EMT) related biomarker protein,focal adhesion kinase( FAK) and phosphorylated FAK( p-FAK) was measured by Western blotting. Results The successful construction of HIF-1α-specific shRNA recombinant plasmids( sh1,sh2 and sh3) were identified by sequencing,and then sh3 was transfected into tongue cancer cells Tca8113 and cultured under hypoxia condition. The expression of both HIF-1α mRNA and HIF-1α protein significantly decreased in tongue cancer cells. The number of penetrating matrigel cells was significantly less and the migratory ability of cells was significantly decreased in the siHIF-1α group as compared with the no-load group( all P < 0. 05). The expression of EMT related biomarker protein and p-FAK in human tongue cancer cells was down-regulated in the siHIF-1α group,and statistical analysis indicated that the difference was significant between two groups( all P < 0. 05). Conclusion RNA interference targeting HIF-1α gene could suppress invasion and migration of human tongue cancer cells effectively,and reverse the malignant phenotypes of human tongue cancer cells to some extent.更多还原