摘要
目的探讨微小RNA-19a(miR-19a)在胃癌组织和血清中的表达及其对人胃癌AGS细胞增殖、侵袭能力的影响和血小板反应蛋白1(thrombospondin 1,THBS1)表达的调控作用。方法应用实时荧光定量PCR(RT-qPCR)法检测22份胃癌组织、相应癌旁组织、血清及22份健康人群血清中miR-19a的表达量。将体外培养的人胃癌AGS细胞分为对照、inhibitor NC、mimics NC、miR-19a inhibitor、miR-19a mimics、miR-19a inhibitor+si-NC和miR-19a inhibitor+siTHBS1组,每组设3个复孔。对照组不做任何处理,其余组采用Lipofectamine^(TM)2000脂质体分别转染inhibitor NC、mimics NC、miR-19a inhibitor、miR-19a mimics、miR-19a inhibitor+si-NC和miR-19a inhibitor+si-THBS1,转染24 h后,分别采用RT-qPCR、细胞计数试剂盒-8(cell counting kit-8,CCK-8)、5-乙炔基-2'脱氧尿嘧啶核苷(5-ethynyl-2'-deoxyuridine,EdU)、Transwell小室、Western blot法分别检测各组细胞miR-19a的表达、细胞活力、增殖率、侵袭数及相关蛋白表达水平。结果胃癌组织中miR-19a的表达水平明显高于相应癌旁组织(t=5.061,P<0.001),胃癌患者血清中miR-19a的表达水平明显高于健康人群血清(t=6.299,P<0.001)。miR-19a inhibitor组AGS细胞中miR-19a的表达水平显著低于inhibitor NC组(t=11.120,P<0.001),miR-19a mimics组miR-19a的表达水平显著高于mimics NC组(t=11.637,P<0.001),miR-19a inhibitor+si-THBS1组THBS1 mRNA和蛋白的表达水平显著低于miR-19a inhibitor+si-NC组(t分别为10.070和13.164,P分别为0.001和<0.001)。与inhibitor NC组相比,miR-19a inhibitor组AGS细胞活力、增殖率、侵袭数及细胞周期蛋白D1(Cyclin D1)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白表达水平均显著降低(t分别为16.679、10.072、17.737、11.173、11.549,P均<0.001),THBS1蛋白表达水平显著升高(t=16.774,P<0.001);与mimics NC组相比,miR-19a mimics组AGS细胞活力、增殖率、侵袭数及Cyclin D1、PCNA蛋白表达水平均显著升高(t分别为15.004、22.746、13.349、11.484、19.022,P均<0.001),THBS1蛋白表达水平显著降低(t=16.384,P<0.001);与miR-19a inhibitor+si-NC组相比,miR-19a inhibitor+si-THBS1组AGS细胞活力、增殖率、侵袭数及Cyclin D1、PCNA蛋白表达水平均显著升高(t分别为13.180、8.669、14.493、6.828、13.587,P均<0.001),THBS1蛋白表达水平显著降低(t=14.824,P<0.001)。结论miR-19a在胃癌组织及胃癌患者血清中呈高表达状态,敲低miR-19a可能通过激活THBS1表达抑制人胃癌AGS细胞的增殖和侵袭能力。
Objective To investigate the expression of micro RNA-19a(miR-19a)in gastric cancer tissues and serum and its effect on the proliferation,invasion and regulation of thrombospondin 1(THBS1)expression in human gastric cancer AGS cells.Methods Real-time fluorescence quantitative PCR(RT-qPCR)was used to measure the expression of miR-19a in 22 gastric cancer tissues,corresponding adjacent noncancerous tissues,serum,and the serum of 22 healthy individuals.Then,the human gastric cancer AGS cells were cultured in vitro and divided into control,inhibitor NC,mimics NC,miR-19a inhibitor,miR-19a mimics,miR-19a inhibitor+si-NC and miR-19a inhibitor+si-THBS1 groups,three repeated wells for each group.The control group was without intervention,and the other groups were transfected with inhibitor NC,mimics NC,miR-19a inhibitor,miR-19a mimics,miR-19a inhibitor+si-NC and miR-19a inhibitor+si-THBS1 by using Lipofectamine^(TM)2000 liposomes respectively.After 24 h of transfection,each group was detected for the miR-19a expression,cell viability,proliferation rate,invasion number and the related proteins expression by RT-qPCR,cell counting kit-8(CCK-8),5-ethynyl-2'-deoxyuridine(EdU),Transwell chamber and Western blot,separately.Results The expression level of miR-19a in gastric cancer tissues was significantly higher than that in corresponding adjacent tissues(t=5.061,P<0.001),and the expression level of miR-19a in serum of patients with gastric cancer was significantly higher than that of healthy people(t=6.299,P<0.001).In vitro,the expression level of miR-19a in AGS cells in the miR-19a inhibitor group was significantly lower than that in the inhibitor NC group(t=11.120,P<0.001),the expression level of miR-19a in the miR-19a mimics group was significantly higher than that in the mimics NC group(t=11.637,P<0.001),and the mRNA and protein expression levels of THBS1 in the miR-19a inhibitor+si-THBS1 group were significantly lower than those in the miR-19a inhibitor+si-NC group(t=10.070 and 13.164,P=0.001 and<0.001,respectively).Compared with the inhibitor NC group,the AGS cell viability,proliferation rate,invasion number,and the protein expression levels of Cyclin D1 and proliferating cell nuclear antigen(PCNA)in the miR-19a inhibitor group significantly decreased(t=16.679,10.072,17.737,11.173,and 11.549,respectively,each P<0.001),and the THBS1 protein expression level increased significantly(t=16.774,P<0.001).Compared with the mimics NC group,the AGS cells in the miR-19a mimics group had significantly higher viability,proliferation rate,invasion number,and the expression levels of Cyclin D1 and PCNA proteins(t=15.004,22.746,13.349,11.484,and 19.022,respectively,each P<0.001),while the expression level of THBS1 protein significantly decreased(t=16.384,P<0.001).Compared with miR-19a inhibitor+si-NC group,the viability,proliferation rate,invasion number,and the protein expression levels of Cyclin D1 and PCNA of AGS cells in miR-19a inhibitor+si-THBS1 group significantly increased(t=13.180,8.669,14.493,6.828,and 13.587,respectively,each P<0.001),while THBS1 protein expression level significantly decreased(t=14.824,P<0.001).Conclusion The miR-19a is highly expressed in gastric cancer tissues and serum of patients with gastric cancer,and knockdown of miR-19a may inhibit the proliferation and invasion of human gastric cancer AGS cells by activating the expression of THBS1.
作者
单彪
卞良
李书君
王佩显
吴殿超
雷秋香
刘登湘
SHAN Biao;BIAN Liang;LI Shujun;WANG Peixian;WU Dianchao;LEI Qiuxiang;LIU Dengxiang(Xingtai People's Hospital,Xingtai 054001,Hebei Province,China)
出处
《中国生物制品学杂志》
2025年第1期34-41,47,共9页
Chinese Journal of Biologicals
基金
2023年度河北省医学科学研究课题计划(20232013)。
