沉默糖原合成激酶3β表达对胰腺癌PANC1细胞生长及上皮间质转化的影响

Effects of glycogen synthase kinase3β silencing on cell growth and epithelial-mesenchymal transition in pancreatic cancer PANC1 cells

目的观察沉默糖原合成激酶3β（GSK3β）的表达对胰腺癌PANC1细胞增殖及上皮间质转化（EMT）的影响，探讨其作用机制。方法设计并合成靶向GSK3β的3个siRNA（siRNA-GSK3β）及阴性对照siRNA（siRNA-NC），分别转染PANC1细胞。通过RT-PCR法检测各组细胞GSK3β mRNA表达量，筛选基因沉默效果最佳的siRNA进行后续实验。采用MTT法检测转染细胞的增殖。采用RT-PCR法检测转染细胞Wnt/β-catenin通路成员Wnt、β-catenin、C-myc、CyclinD1mRNA；Hedgehog通路成员Shh、Smo mRNA；PI3K/Akt/mTOR通路成员PI3K、Akt、mTOR、4E-BP1、p70S6K mRNA以及EMT相关分子Slug、Snail、Twist、E-cadherin、N-cadherin、Vimentin mRNA的表达。结果转染siRNA-NC的PANC1细胞GSK3β mRNA表达量为1.00±0.15，3个转染siRNA-GSK3β的GSK3β mRNA表达量分别为0.25±0.08、0.62±0.09、0.70±0.11，转染siRNA-GSK3β细胞均显著低于转染siRNA-NC细胞，差异有统计学意义（P值均〈0.0001 ）。转染后siRNA-GSK3β组细胞的增殖显著低于转染siRNA-NC组细胞，差异有统计学意义（P〈0.05）。转染后siRNA-GSK3β组细胞的Wnt、β-catenin、C-myc、CyclinD1、PI3K、Akt、mTOR、4E-BP1、p70S6K、Slug、Snail、N-cadherin、Vimentin mRNA表达量分别为0.28±0.04、0.47±0.05、0.37±0.05、0.62±0.08、0.22±0.03、0.47±0.06、0.65±0.08、0.39±0.04、0.56±0.07、0.33±0.05、0.46±0.07、0.55±0.06、0.38±0.04，均显著低于转染siRNA-NC组细胞，差异有统计学意义（P值均〈0.05）；Shh、Smo mRNA表达量分别为1.10±0.13、1.05±0.11，与转染siRNA-NC组细胞的差异无统计学意义；Twist mRNA表达量为0.62±0.08，低于转染siRNA-NC组细胞，E-cadherin mRNA表达量为2.12±0.25，高于转染siRNA-NC组细胞，但差异均无统计学意义。结论沉默PANC1细胞GSK3β基因表达后能够通过Wnt/β-catenin、PI3K/Akt通路抑制胰腺癌细胞的增殖，并抑制其上皮间质转化过程。

Objective To study the effect of inhibiting glycogen synthase kinase3β （GSK3β） on cell proliferation and epithelial mesenchymal transition （EMT） in pancreatic cancer PANC1 ceils. Methods The siRNA trageting GSK3β （siRNA-GSK3β） and negative control siRNA （siRNA-NC） were synthesized to transfect pancreatic cancer PANC1 cells. MTT assay was used to detect cell proliferation, mRNA expression of Wnt, β-catenin, C-myc, CyclinD1 in Wnt/13-catenin signaling, Shh and Smo in Hedgehog pathway, PI3K, Akt, roTOR, 4E-BP1, p70S6K in PI3K/Akt/mTOR signal pathway and Slug, Snail, Twist, E-cadherin, N-cadherin, Vimentin involved in EMT were detected by RT-PCR. Results GSK3β mRNA expression was 1in cells transfected with siRNA - NC, which was 0.25 ±.08, 0.62 ±.09, 0.70 ±.11 in cells with 3 different siRNA-GSK313s, respectively. GSK313 mRNA was significantly decreased by transfection of siRNA- GSK3β（P〈0.0001 ）. The cell proliferation of siRNA-GSK315 group was significantly lower than that of siRNA-NC group （P 〈0.05）. Wnt, β-catenin, C-myc, CyclinD1, PI3K, Akt, roTOR, 4E-BP1, p70S6K, Slug, Snail, N-cadherin, Vimentin mRNA in cells transfected with siRNA-GSK3β was 0.2g ±.04, 0.47 ±.05, 0.37±05, 0.62±08, 0.22±03, 0.47±06, 0.65±08, 0.39±04, 0.56±07, 0.33 ±.05, 0, 46 ±.07, 0.55 ±.06, 0.38 ±.04, which were greatly lower than those in ceils transfected with siRNA-NC, and the difference was statistically significant （all P 〈0.05 ）. Shh and Smo mRNA was 1. 10 ±13 and 1.05 ±.11, which was comparable with those in cells with siRNA-NC. Twist mRNA was 0.62 ±.08, which was lower than that in ceils with siRNA-NC, while E-cadherin mRNA was 2.12 ±25, which was higher than that in ceils with siRNA-NC, but the differences were not statistically significant. Conclusions GSK3 βsilencing in PANC1 cells can suppress cell proliferation and EMT of pancreatic cancer cells by regulating Wnt/β-catenin and PI3K/Akt pathway.