miR-106a-5p通过靶向PTPN3影响结肠癌细胞的增殖、迁移和侵袭

miR-106a-5p Affects the Proliferation,Migration and Invasion of Colon Cancer Cells by Targeting PTPN3

目的:探究mi R-106a-5p在结直肠癌(CRC)中的作用和分子机制。方法:采用CancerMIRNome数据库的TCGA-project模块进行mi R-106a-5p在CRC中表达差异性分析和生存预后分析。收集选取陕西省人民医院2022年1月至2023年6月共55例收治并接受手术切除术治疗的CRC患者的癌组织和配对癌旁组织。双荧光素酶报告基因检测实验分析mi R-106a-5p和PTPN3靶向关系。将HCT-116细胞分为Control组,inhibitor-NC组(转染inhibitor-NC),mi R-106a-5p inhibitor组(转染mi R-106a-5p inhibitor),mi R-106a-5p inhibitor+si-NC组(共转染mi R-106a-5p inhibitor和si-NC)和mi R-106a-5p inhibitor+si-PTPN3组(共转染mi R-106a-5p inhibitor和si-PTPN3)。采用MTT法检测细胞增殖活性,采用平板克隆形成实验检测细胞克隆形成数,采用流式细胞术检测细胞周期分布,采用Transwell实验检测细胞侵袭水平,采用划痕愈合实验检测细胞迁移水平,采用qRT-PCR分析mi R-106a-5p和PTPN3表达水平,采用Western blot分析PTPN3、CyclinD1、Cyclin A和CDKN4蛋白表达水平,采用免疫组织化学染色检测CRC组织中PTPN3表达水平。结果:mi R-106a-5p在CRC组织和HCT-116细胞中高表达(P<0.05),PTPN3在CRC组织和HCT-116细胞中低表达(P<0.05),mi R-106a-5p和PTPN3间存在靶向结合位点。与Control组和inhibitor-NC组比较,mi R-106a-5p inhibitor组细胞mi R-106a-5p水平和Cyclin D1、Cyclin A蛋白表达水平降低(P<0.05),细胞克隆形成数、细胞增殖活力、细胞相对迁移率和细胞侵袭数量均降低(P<0.05),PTPN3 m RNA和PTPN3、CDKN4的蛋白表达水平均升高(P<0.05),细胞G0/G1期分布比例升高(P<0.05)。与mi R-106a-5p inhibitor+si-NC组比较,mi R-106a-5p inhibitor+si-PTPN3组细胞Cyclin D1和Cyclin A蛋白表达水平均升高(P<0.05),细胞克隆形成数、细胞增殖活力、细胞相对迁移率和细胞侵袭数量均升高(P<0.05),PTPN3 m RNA和PTPN3、CDKN4的蛋白表达水平均降低(P<0.05),细胞G0/G1期分布比例降低(P<0.05)。结论:mi R-106a-5p在CRC中高表达,PTPN3在CRC中低表达,通过抑制mi R-106a-5p表达可以上调PTPN3水平,进而抑制结肠癌细胞增殖、迁移和侵袭。

Objective:To explore the role and molecular mechanism of miR-106a-5p in colorectal cancer(CRC).Methods:The expression difference and survival prognosis of miR-106a-5p in CRC was analyzed by TCGA-project module of CancerMIRNome database.Cancer tissues and paired paracancer tissues of 55 CRC patients who received surgical resection in Shaanxi Provincial People's Hospital from January 2022 to June 2023 were collected and selected.The targeting relationship between miR-106a-5p and PTPN3 was analyzed by Dual luciferase reporter gene assay.HCT-116 cells were divided into Control group,inhibitor-NC group(transfection with inhibitor-NC),miR-106a-5p inhibitor group(transfection with miR-106a-5p inhibitor),miR-106a-5p inhibitor+si-NC group(co-transfection with miR-106a-5p inhibitor and si-NC)and miR-106a-5p inhibitor+si-PTPN3 group(co-transfection with miR-106a-5p inhibitor and si-PTPN3).Cell proliferation activity was detected by MTT assay,the number of cell clone formation was detected by plate clone formation assay,cell cycle distribution was detected by flow cytometry,cell invasion level was detected by Transwell assay,and cell migration level was detected by scratch healing assay.The mRNA expression levels of miR-106a-5p and PTPN3 were analyzed by qRT-PCR.The expression levels of PTPN3,CyclinD1,Cyclin A and CDKN4 were analyzed by Western blot.And the expression levels of PTPN3 in CRC tissues were detected by immunohistochemical staining.Results:miR-106a-5p was highly expressed in CRC tissues and HCT-116 cells(P<0.05),while PTPN3 was low expressed in CRC tissues and HCT-116 cells(P<0.05),and there was a targeted binding site between miR-106a-5p and PTPN3.Compared with Control group and inhibitor-NC group,miR-106a-5p levels and the protein expression levels of Cyclin D1 and Cyclin A in miR-106a-5p inhibitor group were decreased(P<0.05).Cell clonal formation number,cell proliferation activity,cell relative mobility and number of cell invasion were decreased(P<0.05).PTPN3 mRNA level and the protein expression levels of PTPN3 and CDKN4 were increased(P<0.05),and the proportion of cell G0/G1 phase distribution was increased(P<0.05).Compared with miR-106a-5p inhibitor+si-NC group,the protein expression levels of Cyclin D1 and Cyclin A in miR-106a-5p inhibitor group were increased(P<0.05).Cell clonal formation number,cell proliferation activity,cell relative mobility and number of cell invasion were increased(P<0.05).PTPN3 mRNA level and the protein expression levels of PTPN3 and CDKN4 were decreased(P<0.05),and the proportion of cell G0/G1 phase distribution was decreased(P<0.05).Conclusion:miR-106a-5p is highly expressed in CRC,while PTPN3 is low expressed in CRC.Inhibiting the expression of miR-106a-5p can up-regulate the level of PTPN3,thus inhibiting the proliferation,migration and invasion of colon cancer cells.