丝胶蛋白通过自噬通路调控人胃癌MKN45细胞的增殖

Sericin regulates proliferation of human gastric cancer MKN45 cells through autophagic pathway

目的探索丝胶蛋白对胃癌MKN45细胞增殖活性的影响及作用机制。方法用LC3双荧光自噬病毒转染胃癌MKN45细胞,用嘌呤霉素筛选LC3双荧光自噬病毒的MKN45稳转株。实验分为3组,空白对照组(Blank)、丝胶蛋白阳性组(Sericin)、丝胶蛋白加自噬抑制剂组(Sericin+3-MA)。培育48 h后用cck-8试剂测定细胞增殖活性,根据所测数据得出丝胶蛋白半数致死浓度IC50,按此浓度处理细胞并培育48h,用流式细胞仪分别检测凋亡、周期,电镜检测细胞自噬,Western blotting检测LC3、p62、Beclin蛋白表达。将种植胃癌的裸鼠分为2组,即对照组(Saline)、丝胶蛋白阳性组(Sericin),每组5只;分别注射生理盐水和丝胶蛋白,测量肿瘤体积和质量。结果丝胶蛋白阳性组(Sericin)与空白对照组(Blank)相比,MKN45细胞增殖活性明显受到抑制,且细胞凋亡增加(P<0.01),细胞阻滞于G2/M期(P<0.01)。相对于空白组,丝胶蛋白处理后细胞在电镜观察到自噬小体明显增多;Western blotting检测到LC3-2表达上调,Beclin表达增加,p62表达逐渐下调;丝胶蛋白加自噬抑制剂组(Sericin+3-MA)实验结果则介于两者之间。动物实验中,丝胶蛋白阳性组(Sericin)与对照组(Saline)相比,肿瘤体积和质量有显著下降。结论丝胶蛋白可通过调控胃癌MKN45细胞自噬影响细胞增殖活性。

Objective To investigate the effect of sericin on the proliferation of human gastric cancer MKN45 cells and explore the underlying molecular mechanism.Methods MKN45 cells were transfected by LC3 double fluorescent autophagic virus,and the positive cells screened by puromycin were divided into blank group,sericin group and sericin ＋ 3-MA group.After incubation with sericin for 48 h,the cells were examined for proliferation,apoptosis and cell cycle using CCK-8 assay and flow cytometry.Cell autophagy was detected by transmission electron microscopy（TEM） and fluorescent inverted microscope,and the autophagy-related markers including LC3,p62 and Beclin proteins were detected with Western blotting.Nude mice bearing gastric cancer xenograft were treated with normal saline or sericin injections（n=5） and the changes in the tumor volume and weight were measured.Results Compared with the blank group,MKN45 cells with sericin treatment showed significantly inhibited proliferation both in vitro and in nude mice.Autophagosomes were observed in sericin-treated cells under TEM and fluorescent inverted microscope.Sericin treatment of the cells significantly increased the cell apoptosis（P〈0.01）,caused obvious cell cycle arrest in G2/M phase（P〈0.01）,up-regulated the expressions of both LC3-2 and Beclin,and down-regulated the expression of p62.The autophagy inhibitor 3-MA obviously antagonized the effects of sericin on cell apoptosis,cell cycle and autophagic protein expressions.Conclusion Sericin can inhibit the proliferation of human gastric cancer MKN45 cells by regulating cell autophagy to serve as potential anti-tumor agent.