POU2F1调控miR-433-3p抑制胃癌细胞增殖、迁移的实验研究

Experimental Study of POU2F1 Regulating miR-433-3p to Inhibit the Proliferation and Migration of Gastric Cancer Cells

目的探讨第2类POU结构域转录因子1(POU2F1)调控miR-433-3p对胃癌细胞增殖、迁移能力的影响。方法选取2021年6月-2022年4月在河北北方学院附属第一医院收集的45例胃癌组织、癌旁组织和人胃黏膜上皮细胞GES-1及人胃癌细胞株BGC-823、MKN-28、SGC-7901为研究对象,采用qRT-PCR检测胃癌组织、癌旁组织和GES-1、BGC-823、MKN-28、SGC-7901细胞中miR-433-3p、POU2F1 mRNA表达;采用蛋白印迹法检测胃癌组织、癌旁组织和GES-1、BGC-823、MKN-28、SGC-7901细胞中POU2F1蛋白表达;对BGC-823细胞进行转染,分为空白组(细胞未转染)、POU2F1小干扰RNA(siRNA)组(POU2F1 siRNA转染细胞)、POU2F1 siRNA-NC组(POU2F1 siRNA阴性对照转染细胞)、miR-433-3p siRNA组(miR-433-3p siRNA转染细胞)、miR-433-3p siRNA-NC组(miR-433-3p siRNA阴性对照转染细胞)、miR-433-3p siRNA+POU2F1 siRNA-NC组(miR-433-3p siRNA和POU2F1 siRNA阴性对照共转染细胞)、miR-433-3p siRNA+POU2F1 siRNA组(miR-433-3p siRNA和POU2F1 siRNA共转染细胞),采用MTT法和Transwell实验分别检测BGC-823细胞增殖、迁移能力;采用qRT-PCR和蛋白印迹法分别检测BGC-823细胞中miR-433-3p、POU2F1 mRNA和POU2F1蛋白表达;采用双荧光素酶报告基因实验验证POU2F1与miR-433-3p启动子区结合情况。结果与癌旁组织比较,胃癌组织miR-433-3p表达水平显著降低,POU2F1 mRNA和蛋白表达水平显著升高(P<0.05);与GES-1细胞比较,BGC-823、MKN-28、SGC-7901细胞miR-433-3p表达水平均降低,POU2F1 mRNA和蛋白表达水平均升高(P<0.05),其中BGC-823细胞miR-433-3p表达水平最低,POU2F1 mRNA和蛋白表达水平最高。沉默POU2F1可抑制BGC-823细胞增殖、迁移,促进miR-433-3p表达;沉默miR-433-3p可促进BGC-823细胞增殖、迁移;沉默POU2F1可抑制miR-433-3p下调对BGC-823细胞增殖、迁移的作用效果,并可促进miR-433-3p表达。双荧光素酶报告基因实验证实POU2F1通过与miR-433-3p启动子区结合,发挥调控miR-433-3p表达的作用;沉默POU2F1可抑制BGC-823细胞的增殖与迁移能力,逆转下调miR-433-3p表达对BGC-823细胞增殖、迁移的影响。结论POU2F1可与miR-433-3p启动子区结合,沉默POU2F1可促进miR-433-3p表达进而抑制胃癌细胞的增殖与迁移。

Objective To investigate the effects of Recombinant Human POU domain,class 2,transcription factor 1(POU2F1)on the proliferation and migration of gastric cancer cells by regulating miR-433-3p.Methods In total,45 gastric cancer tissues,adjacent tissues,human gastric mucosal epithelial cells GES-1 and human gastric cancer cell lines BGC-823,MK-28 and BGC-7901 were collected in the First Affiliated Hospital of Hebei North University from June 2021 to April 2022.mRNA expressions of miR-433-3p and POU2F1 in gastric cancer tissues,adjacent tissues and GES-1,BGC-823,MN-28,SGC-7901 cells were detected by qRT-PCR.The expression of POU2F1 protein in gastric cancer tissues,adjacent tissues and GES-1,BGC-823,MN-28,SGC-7901 cells was detected by western blot.BGC-823 cells were transfected,and divided into blank group(untransfected cells),POU2F1 small interfering RNA(siRNA)group(POU2F1 siRNA transfected cells),POU2F1 siRNA-NC group(POU2F1 siRNA negative control transfected cells),and miR-433-3p siRNA group(miR-433-3p siRNA transfected cells),miR-433-3p siRNA-NC group(miR-433-3p siRNA negative control transfected cells),miR-433-3p siRNA+POU2F1 siRNA-NC group(miR-433-3p siRNA and POU2F1 siRNA negative control co-transfected cells),and miR-433-3p siRNA+POU2F1 siRNA group(miR-433-3p siRNA and POU2F1 siRNA co-transfected cells).The proliferation and migration of BGC-823 cells were detected by MTT assay and Transwell assay respectively.The mRNA and protein expressions of miR-433-3p and POU2F1 in BGC-823 cells were detected by qRT-PCR and western blotting.Dual luciferase reporter assay was used to verify the binding of POU2F1 to the miR-433-3p promoter region.Results Compared with adjacent tissues,the expression level of miR-433-3p in gastric cancer tissues was significantly decreased,while POU2F1 mRNA and protein expression levels were significantly increased(P<0.05).Compared with GES-1 cells,the expression level of miR-433-3p in BGC-823,MN-28 and SGC-7901 cells was decreased,while the mRNA and protein expression levels of POU2F1 were increased(P<0.05).The expression level of miR-433-3p in BGC-823 cells was the lowest,and POU2F1 had the highest mRNA and protein expression levels.POU2F1 silencing inhibited BGC-823 cell proliferation and migration,and promoted the expression of miR-433-3p.miR-433-3p silencing could promote the proliferation and migration of BGC-823 cells.POU2F1 silencing inhibited the effect of down-regulation of miR-433-3p on the proliferation and migration of BGC-823 cells,and promoted the expression of miR-433-3p.Double luciferase reporter gene assay confirmed that POU2F1 played a role in regulating the expression of miR-433-3p by binding to the miR-433-3p promoter region.POU2F1 silencing inhibited the proliferation and migration of BGC-823 cells and reversed the effect of down-regulating the expression of miR-433-3p on the proliferation and migration of BGC-823 cells.Conclusion POU2F1 binds to the promoter region of miR-433-3p,and POU2F1 silencing can promote the expression of miR-433-3p,thereby inhibiting the proliferation and migration of gastric cancer cells.