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为了在细胞学水平研究靶向抑制MAP3K1(mitogen-activated protein kinase kinase kinase 1)基因对小鼠乳腺癌4T1细胞黏附、运动及侵袭能力等生物学行为的影响,试验用体外构建靶向“沉默”MAP3K1基因的amiRNA(artificial microRNA)干扰载体,以X-treme GENE HP转染4T1细胞(干扰组转染amiRNA干扰载体,对照组转染空载体,空白组未转染质粒)对MAP3K1基因mRNA及蛋白表达水平进行检测,以测定干扰效率;分别以黏附试验检测小鼠乳腺癌4T1细胞的黏附能力,用划痕-愈合试验与Transwell侵袭试验测定小鼠乳腺癌4T1细胞的运动与侵袭能力。结果表明:小鼠乳腺癌4T1细胞转染MAP3K1 amiRNA-3干扰载体与空载体后,干扰4T1细胞MAP3K1 mRNA水平及蛋白水平均被有效抑制,干扰效率高达70%(P<0.01)。试验组4T1细胞黏附能力极显著低于对照组(P<0.01);干扰组4T1细胞的运动与侵袭能力均显著低于对照组(P<0.05)。说明构建的amiRNA干扰载体能够有效抑制靶基因MAP3K1的表达,并能够显著抑制小鼠乳腺癌4T1细胞的黏附、运动与侵袭能力;靶向抑制MAP3K1基因能够广泛地影响小鼠乳腺癌4T1细胞的生物学行为。
In order to study the effect of mitogen-activated protein kinase kinase kinase 1 (MAP3K1) on the biological behaviors of mouse breast cancer cell line 4T1, such as adhesion, motility and invasion, MAP3K1 gene was transfected into 4T1 cells by X-treme GENE HPP (interference group transfected with amiRNA interference vector, control group transfected with empty vector and blank group untransfected plasmids) MAP3K1 mRNA And the protein expression levels were measured to determine the interference efficiency. The adhesive ability of 4T1 cells in mouse breast cancer cells was detected by adhesion test. The ability of invasion and migration of 4T1 cells in mouse breast cancer cells was determined by scratch-healing assay and Transwell invasion assay. The results showed that the MAP3K1 mRNA and protein levels in 4T1 cells were significantly inhibited after 4T1 cells were transfected with MAP3K1 amiRNA-3 interfering vector and empty vector. The interference efficiency was as high as 70% (P <0.01). The adhesion capacity of 4T1 cells in the experimental group was significantly lower than that in the control group (P <0.01). The activity and invasiveness of 4T1 cells in the interference group were significantly lower than those in the control group (P <0.05). The results showed that the constructed amiRNA interference vector can effectively inhibit the expression of the target gene MAP3K1 and significantly inhibit the adhesion, motility and invasion ability of the mouse breast cancer cell line 4T1. Targeted inhibition of the MAP3K1 gene can widely affect the biological characteristics of mouse breast cancer 4T1 cells Academic behavior.