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.
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。