Objective: To determine the sensitivity of cervical cancer HeLa cells to cGMP-dependent protein kinase（ PKG） inhibitor KT-5823 and explore the effects and mechanisms of KT-5823 on the viability,apoptosis and autophagy of HeLa cells. Methods:Different concentrations of KT-5823 were used to intervene HeLa cells for 24 hours. The cell viability and PKG1 expression level were detected by CCK-8 and Western blotting respectively. Then the subsequent drug stimulation concentration was determined.HeLa cells were divided into two groups,namely DMSO group（ control group） and KT-5823 group（ experimental group）. Apoptosis was detected by flow cytometry. The number of autophagy spots was detected by GFP-LC3B fluorescence spot test. The mRNA expression level of PKG1,microtubule associated protein 1 light chain 3-Ⅱ（ LC3Ⅱ） and Beclin1 was detected by real-time fluorescent quantitative PCR（ RT-qPCR）.The expression levels of PKG1,autophagy related protein LC3Ⅱ/Ⅰ,Beclin1,autophagy related pathway protein kinase B（ Akt）, phosphorylated protein kinase B（ p-Akt）, mammalian rapamycin target protein（ mTOR）,phosphorylated mammalian rapamycin target protein（ p-mTOR）,apoptosis related protein B-cell Lymphoma 2 family protein（ Bcl-2）,Bcl-2 related X protein（ Bax） and Caspase 3 protein were detected by Western blotting. The autophagy inhibitor SBI-0206965 group was increased to observe the role of autophagy in this process. Results:Under the stimulation concentration of 3-100 μmol/L,the activity of HeLa cells reduced in the experimental group,compared with the control group（ t=10.23-14.83,all P＜0.05）. The experimental group decreased PKG1 protein expression levels gradually（ t=10.01-14.17,all P＜0.05） compared with the control group. Final selection 3 μ mol/L was the stimulation concentration for subsequent study. Compared with the control group, apoptosis rates increased（ t =10.78,P =0.004）,number of fluorescent autophagy spots increased（ t =10.12,P =0.000 5）,expression levels of PKG1 mRNA and protein of HeLa cells decreased（ t=13.56,P=0.000 2;t=5.461,P=0.005 5）,LC3Ⅱ and Beclin1 mRNA（ t=8.359,P=0.001 1;t=5.782,P=0.004 4） and protein（ t=6.924,P=0.002 3;t=11.84,P=0.000 3） express level increased.The proteins of P-Akt/Akt（ t=5.194,P=0.006 5）,p-mTOR/mTOR（ t=12.78,P=0.000 2）,Bcl-2/Bax（ t=13.79,P =0.000 2） ratios decreased and Caspase 3 expression（ t=9.341,P=0.000 7） increased in the experimental group. Compared with the experimental group,the autophagy inhibitor SBI-0206965 group（ 30 、50 、70 、100 μmol/L）increased apoptosis（ t=7.616,P=0.001 6;t=15.43,P=0.000 1;t=11.01,P=0.000 4;t=15.39,P=0.000 1）. Conclusion:PKG inhibitor KT-5823 can effectively inhibit the proliferation and apoptosis of HeLa cells. At the same time,it can induce autophagy by suppressing Akt/mTOR pathway and the autophagy induced by KT-5823 plays a protective role in this process. PKG may become a new target for clinical treatment of cervical cancer.

葡萄糖是一种重要的营养物质,在维持机体正常生理活动过程中起重要作用.当机体由于糖代谢紊乱或限制碳水化合物摄入而处于高血糖或低血糖状态时,可影响细胞自噬.自噬有益或有害,细胞在不利环境压力下通过适度自噬维持稳态,但超过自噬阈值细胞会发生损伤甚至死亡.近年来,葡萄糖影响自噬具体机制的研究取得了一定成果,同时靶向自噬治疗疾病也成为一种有前景的发展方向.因此,全面总结葡萄糖水平影响自噬的相关文献不仅可为自噬机制研究提供新的理论依据,也可为临床治疗疾病提供新思路.

目的 探究miR-130b-3p对糖饥饿诱导宫颈癌细胞自噬性死亡的影响及机制.方法 将宫颈癌细胞分别置于低糖(0.1 mmol/L)和正常糖含量(5 mmol/L)、低糖+DMSO和低糖+雷帕霉素的培养液中培养,利用碘化丙啶(propidium iodide,PI)染色实验、GFP-LC3B自噬泡形成检测实验、实时荧光半定量PCR(RT-qPCR)实验和Western blot实验,依次检测细胞死亡、自噬水平,细胞内miR-130b-3p、腺苷单磷酸激活激酶(AMP-activated protein kinase,AMPK)mRNA和AMPK蛋白水平;分别转染miR-130b-3p抑制物及AMPK过表达质粒,再利用自噬抑制剂SBI-0206965(SBI)检测miR-130b-3p、ampk对细胞死亡和自噬的影响;生物信息学工具预测miR-130b-3p的靶基因并利用荧光报告基因实验进行验证.结果 低糖诱导宫颈癌细胞发生自噬、miR-130b-3p水平升高,AMPK表达水平降低(均P<0.05),死亡率无显著变化(均P>0.05);与低糖+DMSO组相比,低糖+雷帕霉素使得宫颈癌细胞死亡率显著增加(均P<0.05);转染miR-130b-3p抑制物后,低糖条件下宫颈癌细胞自噬水平和自噬性死亡率显著增加(均P<0.05);过表达AMPK能促进宫颈癌细胞自噬和自噬性死亡率显著升高(均P<0.05);荧光报告基因表达水平检测结果证实,AMPK是miR-130b-3p的直接靶基因(P<0.05).结论 低糖条件下,miR-130b-3p靶定AMPK基因抑制宫颈癌细胞自噬性死亡.