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目的构建靶向存活素(survivin)基因的短发夹干扰RNA(shRNA)表达载体,导入人大肠癌细胞SW480中,研究靶向抑制survivin基因对SW480细胞侵袭和转移的影响。方法根据siRNA设计原则,在survivin序列中选取设计含19个核苷酸(19nt)的靶序列,间以9个核苷酸的茎环序列,两端分别加上对应的酶切位点,形成shRNA的DNA模板并克隆到shRNA表达载体pRNAT-U6.1/Neo中,获得靶向抑制survivin基因的sihNA表达载体pRNAT-U6.1/Neo-survivin;采用Lipofectamine2000TM转染试剂将干扰质粒pRNAT-U6.1/Neo-survivin导入到大肠癌细胞SW480中;用Westernblotting从蛋白水平检测干扰效果;分别采用肿瘤侵袭粘附实验和明胶酶谱分析法检测pRNAT-U6.1/Neo-survivin对SW480细胞的侵袭和转移潜能的影响。结果Survivin基因的蛋白表达均得到显著抑制;沉默SW480的survivin基因可以显著抑制SW480细胞的侵袭和转移潜能,而且survivin基因表达被抑制后,SW480细胞分泌基质金属蛋白酶明显减少。结论Survivin基因可能在SW480的侵袭和转移潜能中起重要作用,沉默SW480的survivin基因可以显著抑制其侵袭、转移和基质金属蛋白酶分泌,因而survivin影响SW480的侵袭和转移可能与调控基质金属蛋白酶分泌密切相关。
Objective To construct short hairpin RNA (shRNA) expression vector targeting survivin gene into human colorectal cancer cell line SW480 and study the effect of targeted inhibition of survivin gene on the invasion and metastasis of SW480 cells. Methods According to the principle of siRNA design, a target sequence of 19 nucleotides (19 nt) in length was selected in the survivin sequence and a 9 nucleotide stem-loop sequence was selected. The corresponding restriction sites were added to both ends to form shRNA plasmid was cloned into the shRNA expression vector pRNAT-U6.1 / Neo to obtain the sihNA expression vector pRNAT-U6.1 / Neo-survivin targeting the survivin gene. The recombinant plasmid pRNAT-U6 The expression of pRNAT-U6.1 / Neo-survivin was detected by Western blotting in SW480 cells. Western blotting was used to detect the interference effect of pRNAT-U6.1 / Neo-survivin on SW480 cells Impact of invasion and metastasis potential. Results The survivin gene silencing SW480 significantly inhibited the invasion and metastasis potential of SW480 cells, and the expression of survivin gene was significantly inhibited after SW480 cells were treated with SW480 cells. Conclusion Survivin may play an important role in invasion and metastasis of SW480. Silencing the survivin gene of SW480 can significantly inhibit its invasion, metastasis and secretion of matrix metalloproteinases. Survivin may play an important role in the invasion and metastasis of SW480 by regulating the expression of matrix metalloproteinase Related.