摘要
目的:分析灰树花多糖(Grifola frondose polysaccharide,GFP)对胆囊癌细胞增殖、迁移和侵袭的影响及机制.方法:将对数期GBC-SD细胞随机分组:对照组、GFP组(0.5、1、2 μg/mL GFP)、shNC组、shHULC 组、miR-NC 组、miR-186-5p mimics 组、GFP+pcDNA 组、GFP+pcDNA-HULC 组.CCK-8 法检测细胞抑制率;Transwell小室法检测GBC-SD细胞迁移和侵袭;Western Blot印迹检测Cyclin D1、P21、MMP-2、MM-9蛋白水平;qRT-PCR检测LncRNA HULC和miR-186-5p的水平;Starbase在线软件预测HULC与miR-186-5p的结合位点,双荧光素酶报告实验检测LncRNA HULC和miR-186-5p的靶向关系.裸鼠移植瘤实验检测GFP对GBC-SD细胞移植瘤体内生长的影响.结果:0.25～8 μg/mL GFP对胆囊癌GBC-SD细胞具有一定的抑制作用;与对照组比较,0.5、1、2 μg/mL的GFP(极)显著(P＜0.05,P＜0.01)抑制GBC-SD细胞迁移和侵袭,(极)显著降低 Cyclin D1、MMP-2 和 MM-9 水平(P＜0.05,P＜0.01),(极)显著(P＜0.05,P＜0.01)增加 P21 水平,极显著(P＜0.01)下调 LncRNA HULC,上调 miR-186-5p 的表达(P＜0.05,P＜0.01):Starbase 在线软件预测HULC与miR-186-5p存在结合位点,与miR-NC组比较,miR-186-5p mimics组HULC-WT的荧光素酶活性极显著降低(P＜0.01),而HULC-MUT荧光素酶活性无统计学差异(P＞0.05);与shNC组比较,shHULC组HULC相对表达水平极显著(P＜0.01)降低,miR-186-5p相对表达水平极显著(P＜0.01)增加,细胞存活率、迁移数目、侵袭数目极显著(P＜0.01)降低,Cylin Dl、MMP-2和MM-9水平极显著下调(P＜0.01);与GFP+pcDNA组比较,GFP+pcDN-HULC组抑制率显著降低(P＜0.05),迁移数目、侵袭数目极显著增加(P＜0.01),Cylin D1、MMP-2和MM-9水平显著上调(P＜0.05);与GFP+pcDNA给药组比较,GFP+pcDN-HULC给药组瘤体体积、质量极显著增加(P＜0.01),miR-186-5p水平极显著下调(P＜0.01).结论:GFP可抑制胆囊癌细胞的增殖、侵袭和迁移,其机制与调控LncRNA HULC/miR-186-5p有关.
Abstract
Objective:To analyze the effects of Grifola frondose polysaccharide(GFP)on proliferation,migration and invasion of gallbladder carcinoma cells and its mechanism.Methods:Logarithmic phase GBC-SD cells randomized:Control group,GFP group(0.5,1,2 μg/mL GFP),shNC group,shHULC group,miR-NC group,miR-186-5p mimics group,GFP+pcDNA group,GFP+pcDNA-HULC group.Cell inhibition rate was detected by CCK-8 method.The migration and invasion of GBC-SD cells were detected by Transwell cell assay.The protein levels of Cyclin D1,P21,MMP-2 and MM-9 were detected by Western Blot.LncRNA HULC and miR-186-5p levels were detected by qRT-PCR.Starbase online software predicted the binding sites of HULC and miR-186-5p,dual luciferase reporting assay was used to detect the targeting relationship between LncRNA HULC and miR-186-5p.The effect of GFP on the endogenetic growth of GBC-SD cells was detected by tumor transplantation in nude mice.Results:0.25～8 μg/mL GFP had definite inhibitory effect on GBC-SD cells of gallbladder carcinoma.Compared with the control group,0.5,1 and 2 μg/mL of GFP(extremely)significant(P＜0.05,P＜0.01)inhibited the migration and invasion of GBC-SD cells,and(extremely)significant(P＜0.05,P＜0.01)decreased the levels of Cyclin D1,P21,MMP-2 and MM-9,extremely significant increased the levels of P21(P＜0.05,P＜0.01).The expression of LncRNA HULC was extremely significant down-regulated and miR-186-5p was extremely significant up-regulated(P＜0.05,P＜0.0l).Starbase online software predicted that there was a binding site between HULC and miR-186-5p,and compared with miR-NC group,the luciferase activity of HULC-WT in miR-186-5p mimics group was extremely significant(P＜0.01)decreased.There was no significant difference in the luciferase activity of HULC-MUT(P＞0.05).Compared with shNC group,the relative expression level of HULC in shHULC group was extremely significant(P＜0.01)decreased,the relative expression level of miR-186-5p was extremely significant(P＜0.01)increased,the cell survival rate,migration number and invasion number were significantly(P＜0.05)decreased,respectively,the levels of MMP-2,MMP-2 and MM-9 were extremely significant decreased(P＜0.01).Compared with GFP+pcDNA group,the inhibition rate of GFP+pcDN-HULC group significantly decreased(P＜0.05),and the number of migration and invasion was significantly increased(P＜0.01),the CylinDl,MMP-2 and MM-9 levels were significantly increased(P＜0.05,P＜0.01).Compared with GFP+pcDNA administration group,tumor volume and mass in GFP+pcDN-HULC administration group were significantly(P＜0.05)increased,and miR-186-5p levels were extremely significantly decreased(P＜0.01).Conclusion:GFP could inhibit proliferation,invasion and migration of gallbladder cancer cells,and its mechanism was related to the regulation of LncRNA HULC/miR-186-5p.
基金项目
河南省二0二二年科技发展计划(222102310443)
漯河医学高等专科学校2023年度科技创新项目(2023ZD04)
国家科技期刊平台
NSTL
万方数据