磷脂酰肌醇3激酶/蛋白激酶B/核转录因子E2相关因子2信号通路对高糖状态下人甲状腺乳头状癌K1细胞增殖的作用及机制

目的研究核转录因子E2相关因子2(Nrf2)及磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路对高糖状态下人甲状腺乳头状癌K1细胞增殖的影响,并探讨糖代谢相关酶在Nrf2及PI3K/Akt影响K1细胞增殖中的作用。方法根据不同处理因素将K1细胞分为5组:正糖组(正糖5.5 mmol/L培养48 h)、高糖组(正糖5.5 mmol/L培养24 h后,换25 mmol/L高糖继续培养24 h)、Nrf2siRNA组(正糖条件下siRNA转染细胞24 h后,换高糖继续培养24 h)、NcsiRNA组(正糖条件下阴性转染细胞24 h后,换高糖继续培养24 h)、LY294002组(正糖条件下培养24 h后,加50μmol/L LY294002预孵育0.5 h再换用高糖培养24 h)。K1细胞按照上述分组处理后分别用以下方法检测相关指标:MTT法检测细胞增殖,细胞免疫荧光法检测Nrf2蛋白的分布,Western blot检测PI3K、p-PI3K、Akt、p-Akt、核Nrf2、浆Nrf2、葡萄糖-6-磷酸脱氢酶(G6PD)、丙酮酸激酶M2(PKM2)蛋白的表达水平。两组数据比较采用独立样本t检验,多组数据比较采用单因素方差分析。结果与正糖组比较,高糖组K1细胞增殖率、PI3K、p-Akt/Akt、Nrf2、G6PD、PKM2表达显著升高[分别为(87.31±3.67)%比(126.64±5.41)%、0.272±0.039比0.425±0.019、0.168±0.035比0.446±0.021、0.308±0.026比0.597±0.014、0.421±0.024比0.626±0.026、0.198±0.023比0.314±0.023,t=6.109~16.951,均P<0.05],Nrf2在细胞核分布的比例显著升高[(21.6±4.5)%比(91.2±3.5)%,χ2=98.497,P<0.01];与高糖组比较,Nrf2siRNA组K1细胞增殖率明显下降,G6PD、PKM2的蛋白表达明显下调,差异有统计学意义(t=8.936、7.056、8.843,均P<0.01),LY294002组也呈现相似的趋势(t=7.228、6.351、7.910;均P<0.01);与高糖组比较,LY294002组K1细胞PI3K、Akt蛋白水平无明显改变,而p-PI3K、p-Akt、核Nrf2、浆Nrf2蛋白水平及Nrf2核浆蛋白比值均被显著抑制(t=5.748~23.572,均P<0.01)。结论高糖可激活PI3K/Akt信号通路,上调Nrf2表达与核转位,上调磷酸戊糖和糖酵解途径相关酶PKM2、G6PD的表达,促进K1细胞增殖。

Effect of phosphatidylinositoll 3-kinase/protein kinase B/nuclear factor erythroid 2-related factor 2 signaling pathway on proliferation of papillary thyroid cancer K1 cells under high glucose environmental conditions

Objective To investigate the effects of nuclear factor erythroid 2-related factor 2(Nrf2)and phosphatidylinositoll 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway on proliferation of human papillary thyroid carcinoma K1 cells under high glucose environmental conditions,and explore the role of glucose metabolism-related enzymesin the proliferation of K1 cells.Methods According to different treatment factors,K1 cells were divided into five groups:normal glucose group(5.5 mmol/L glucose cultured for 48 h),high glucose group(5.5 mmol/L glucose cultured for 24 h,then 25 mmol/L glucose cultured for 24 h),Nrf2siRNA group(transfected cells in 5.5 mmol/L glucose for 24 h,then cultured in 25 mmol/L glucose for 24 h),NcsiRNA group(negativly transfected cells for 24 h under 5.5 mmol/L glucose conditions,then cultured in 25 mmol/L glucose for 24 h),LY294002 group(negativly transfected cells for 24 h under 5.5 mmol/L glucose conditions,plus 50μmol/L LY294002 pre-incubation for 0.5 h and then cultured in 25 mmol/L glucose for 24 h).Cell proliferation was detected by MTT assay,distribution of Nrf2 in K1 cells was detected by immunofluorence staining,and the expression levels of PI3K,p-PI3K,Akt,p-Akt,Nrf2(nuclear and cytoplasmic protein),G6PD(glucose-6-phosphate dehydrogenase)and PKM2(pyruvate kinase M2)proteins were detected by Western blot.The two groups of data were compared by independent sample t test,and the multiple groups of data were compared by one-way anova.Results Compared with the normal glucose group,the proliferation rate of K1 cells,protein expression of PI3K,p-akt/Akt,Nrf2,G6PD and PKM2[(87.31±3.67)%vs(126.64±5.41)%,0.272±0.039 vs 0.425±0.019,0.168±0.035 vs 0.446±0.021,0.308±0.026 vs 0.597±0.014,0.421±0.024 vs 0.626±0.026,0.198±0.023 vs 0.314±0.023,t=6.109-16.951,all P<0.05],as well as the proportion of Nrf2 distribution in the nucleus[(21.6±4.5)%vs(91.2±3.5)%,χ2=98.497]were significantly increased in the high glucose group(P<0.01).Compared with the high glucose group,the proliferation rate of K1 cells in Nrf2 siRNA group was significantly decreased,and the protein expression of G6PD and PKM2 was down-regulated,the difference was statistically significant(t=8.936,7.056,8.843,all P<0.01),and the LY294002 group also showed a similar trend(t=7.228,6.351,7.910,all P<0.01).Compared with the high glucose group,the protein PI3K and Akt levels of K1 cells in LY294002 group were not significantly changed,p-PI3K,p-Akt,nuclear Nrf2,cytoplasmic Nrf2 protein levels and the ratio of nuclear and cytoplasmic Nrf2 were significantly inhibited(t=5.748-23.572,all P<0.01).Conclusion High glucose can activate PI3K/Akt signaling pathway,up-regulate Nrf2 expression and nuclear translocation,and up-regulate the expression of pentose phosphate and glycolytic pathway-related enzymes PKM2 and G6PD to promote the proliferation of K1 cells.