PAK6低表达对乳腺癌细胞增殖、迁移的影响及机制

Effect and mechanism of low expression of PAK6 gene on proliferation and metastasis of breast cancer cells

目的观察p21激活激酶6(PAK6)低表达对乳腺癌细胞增殖、迁移的影响,并探讨其作用机制。方法常规培养人正常乳腺上皮细胞(MCF-10A)及乳腺癌细胞系(MDA-MB-435S、MCF-7、MDA-MB-361),用qRT-PCR法检测各细胞中PAK6 mRNA表达,乳腺癌细胞中PAK6 mRNA相对表达量均高于MCF-10A,其中MCF-7中最高,将MCF-7作为实验细胞。将MCF-7随机分为si-NC组、si-PAK6组,分别用脂质体2000转染NC siRNA(对照小干扰RNA)和抑制PAK6表达的小干扰RNA(PAK6 siRNA)。转染24 h收集细胞,用qRT-PCR法检测细胞内PAK6 mRNA表达,CCK-8法检测细胞增殖能力,Transwell实验检测细胞转移能力,Western blotting法检测细胞内磷酸化PI3K(pPI3K)、磷酸化AKT(pAKT)蛋白表达。结果si-NC组、si-PAK6组PAK6 mRNA相对表达量分别为1.06±0.12、0.45±0.08。与si-NC组比较,si-PAK6组PAK6 mRNA相对表达量低(P<0.05)。si-NC组、si-PAK6组细胞增殖率分别为(100.00±0.00)%、(47.37±7.41)%。与si-NC组比较,si-PAK6组细胞增殖率低(P<0.05)。si-NC组、si-PAK6组穿膜细胞数分别为(89.63±8.37)、(28.58±5.21)个。与si-NC组比较,si-PAK6组穿膜细胞数少(P<0.05)。pPI3K蛋白在si-NC组、si-PAK6组的相对表达量分别为1.42±0.20、0.75±0.11,pAKT蛋白相对表达量分别为1.59±0.18、0.81±0.14。与si-NC组比较,si-PAK6组pPI3K、pAKT蛋白相对表达量均低(P均<0.05)。结论抑制PAK6表达可阻止乳腺癌细胞增殖、迁移,其作用机制与抑制PI3K/AKT信号通路激活有关。

Objective To investigate the effects of low expression of p21-activated kinase 6(PAK6)on the proliferation and metastasis of breast cancer cells,and to explore its mechanism.Methods Human normal breast epithelial cells(MCF-10A)and breast cancer cell lines(MDA-MB-435S,MCF-7,and MDA-MB-361)were routinely cultured.The expression of PAK6 mRNA was detected by qRT PCR.The relative expression of PAK6 mRNA in the breast cancer cells was higher than that in the MCF-10A cells,and MCF-7 cells were the highest to be used as experimental cells.MCF-7 cells were randomly divided into the si-NC group and si-PAK6 group,which were transfected with NC siRNA(control siRNA,control effect)and PAK6 siRNA(PAK6 siRNA,inhibition of PAK6 expression)by liposome 2000.The cells were collected at 24 hours after transfection,and the expression of PAK6 mRNA was detected by qRT PCR.CCK-8 assay was used to detect cell proliferation,Transwell assay was used to detect cell metastasis,and Western blotting was used to detect the expression of pPI3K and pAKT.Results The expression levels of PAK6 in the si-NC group and si-PAK6 group were 1.06±0.12 and 0.45±0.08,respectively.Compared with the si-NC group,the relative expression of PAK6 mRNA in the si-PAK6 group was lower(P<0.05).The cell proliferation rates of the si-NC group and si-PAK6 group were 100.00%±0.00%and 47.37%±7.41%,respectively.Compared with the si-NC group,the proliferation rate of si-PAK6 group was lower(P<0.05).The number of penetrating cells in the si-NC group and si-PAK6 group was 89.6±8.37 and 28.58±5.21,respectively.Compared with the si-NC group,the number of penetrating cells in the si-PAK6 group was less(P<0.05).The relative expression levels of pPI3K protein in the si-NC group and si-PAK6 group were 1.42±0.20 and 0.75±0.11,and the relative expression levels of pAKT protein were 1.59±0.18 and 0.81±0.14,respectively.Compared with the si-NC group,the relative expression levels of pPI3K and pAKT in the si-PAK6 group were lower(both P<0.05).Conclusion Silencing PAK6 gene can prevent the proliferation and metastasis of breast cancer cells,and its mechanism is related to the inhibition of PI3K/Akt signaling pathway activation.