Effect of GASP-1-ShRNA on proliferation and apoptosis of lung cancer cells and tumor formation in nude mice
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摘要:
目的 探讨shRNA干扰G蛋白偶联受体相关分选蛋白1(GASP-1)对肺癌细胞增殖凋亡和裸鼠成瘤影响。 方法 将GASP-1-shRNA1、2和3用Lipofectamine 2 000转染到肺癌A549细胞。选择转染GASP-1-shRNA1的细胞,建立空白对照组、空载体转染组和GASP-1-shRNA1转染组,采用菌落形成试验和流式细胞术分别检测肺癌A549细胞增殖和调亡;采用Western Blot检测ki67、cleaved caspase-3、Bax和Bcl-2蛋白表达量。将转染GASP-1-shRNA1细胞注射裸鼠后腿建立原位异种移植小鼠模型。检测肿瘤的生长,并通过免疫组化检测ki67和caspase-3蛋白表达量。 结果 GASP-1-shRNA1、2和3转染A549细胞后GASP-1 mRNA和蛋白表达量均显著下调,尤其转染GASP-1-shRNA1组。GASP-1下调能抑制A549细胞增殖,并促进A549细胞凋亡;在体内试验中,下调GASP-1能抑制体内肿瘤生长,同时也能抑制细胞增殖,并促进细胞凋亡。 结论 GASP-1的下调可抑制肺癌细胞增殖、促进凋亡,并抑制肿瘤生长。 -
关键词:
- shRNA干扰G蛋白偶联受体相关分选蛋白1(GASP-1) /
- 肺癌 /
- 增殖 /
- 凋亡
Abstract:Objective To explore the effect of G-protein coupled receptor-associated sorting protein 1-short-hairpin RNA (GASP-1-ShRNA) on proliferation and apoptosis of lung cancer cells and tumor formation in nude mice. Methods GASP-1-shRNA1, GASP-1-shRNA2 and GASP-1-shRNA3 were transfected into lung cancer A549 cells with Lipofectamine 2 000. Colony formation test and flow cytometry were used to detect proliferation and apoptosis of A549 cells from groups of GASP-1-shRNA1 transfection, blank control, and empty vector transfection, respectively. The A549 cells′ expressions of Ki67, cleaved caspase-3, B cell lymphoma-2 protein (Bcl-2) and Bcl-2-associated X protein (Bax) were determined with Western blot. An orthotopic xenograft mouse model was established through injection of GASP-1-shRNA1-transfected cells into hind leg tissues in nude mice. The tumor growth of the model mice were observed and the expressions of ki67 and caspase-3 protein in tumor tissues were detected with immunohistochemistry. Results The mRNA and protein expression of GASP-1 in A549 cells were down-regulated after transfections of GASP-1-shRNA1, GASP-1-shRNA2, and GASP-1-shRNA3, especially in the GASP-1-shRNA1 transfected A549 cells. The down-regulation of GASP-1 could inhibit proliferation and promote cell apoptosis of the A549 cells. In vivo experiments, down-regulated GASP-1 could restrain tumor growth by inhibiting proliferation and promoting apoptosis of the A549 cells. Conclusion Down-regulation of GASP-1 could inhibit proliferation and promote apoptosis of lung cancer A549 cells. -
表 1 ShRNA转染对GASP-1 mRNA和蛋白表达影响(
$\overline x $ ± s,n = 8)组别 GASP-1 mRNA表达量 GASP-1蛋白表达量 对照组 1.00 ± 0.00 0.131 ± 0.024 shRNA-NC 0.99 ± 0.05 0.125 ± 0.033 GASP-1-shRNA 1 0.15 ± 0.07 a 0.006 ± 0.005 a 2 0.64 ± 0.09 b 0.048 ± 0.014 b 3 0.41 ± 0.06 b 0.260 ± 0.015 b 注:与shRNA-NC组比较,a P < 0.01,b P < 0.05。 -
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