摘要
目的 探讨微小RNA-433-3p(miR-433-3p)靶向Ras相关蛋白1A(RAP1A)调控结直肠癌细胞增殖、凋亡、迁移与侵袭的作用及机制。方法 采用实时荧光定量PCR(qPCR)检测结直肠癌细胞的miR-433-3p水平。向SW480细胞转染miR-433-3p模拟物mimics(miR-433-3p mimics组)或无义序列(NC组),另将miR-433-3p mimics和RAP1A过表达质粒共转染SW480细胞(miR-433-3p mimics+RAP1A组),采用CCK-8法、流式细胞术、划痕实验和Transwell小室实验评估SW480细胞的增殖、凋亡、迁移和侵袭情况。双荧光素酶报告基因实验分析miR-433-3p和RAP1A间的靶向关系。qPCR和Western blotting法检测RAP1A、Bax、Bcl-2和基质金属蛋白酶(MMP)-2的mRNA和蛋白水平。结果 SW480、HCT116和LoVo细胞的中miR-433-3p水平低于Caco-2细胞(P<0.05)。RAP1A是miR-433-3p的作用靶点且miR-433-3p能够抑制SW480细胞中RAP1A的表达。与NC组相比,miR-433-3p mimics组SW480细胞增殖、迁移和侵袭能力减弱且凋亡增加并伴有Bcl-2和MMP-2水平降低和Bax水平升高(P<0.05)。相较于miR-433-3p mimics组,miR-433-3p mimics+RAP1A组SW480细胞增殖、迁移和侵袭能力增强且细胞凋亡降低并伴有Bcl-2和MMP-2水平升高和Bax水平降低。结论 结直肠癌中miR-433-3p表达下调且发挥抑癌基因的作用,miR-433-3p/RAP1A轴有望成为结直肠癌的潜在治疗靶点。
Objective To investigate the effect and mechanism of microRNA-433-3p(miRNA-433-3p) on proliferation, apoptosis, migration and invasion of colorectal cancer cells through targeting Ras-related protein 1A(RAP1A). Methods Quantitative reverse-transcription PCR(qPCR) was used to detect miR-433-3p levels of colorectal cancer cells. SW480 cells were transfected with miR-433-3p mimics(miR-433-3p mimics group) or nonsense sequences(NC group), and miR-433-3p mimics and RAP1A overexpression plasmid were co-transfected into SW480 cells(miR-433-3p mimics+RAP1A group). The proliferation, apoptosis, migration and invasion of SW480 cells were evaluated by CCK-8 method, flow cytometry, scratch assay and Transwell chamber assay. The targeting relationship between miR-433-3p and RAP1A was analyzed by double luciferase reporter gene experiment. The mRNA and protein levels of RAP1A, Bax, Bcl-2 and matrix metalloproteinase(MMP)-2 were detected by qPCR and Western blotting. Results The miR-433-3p levels in SW480, HCT116 and LoVo cells were lower than that in Caco-2 cells(P<0.05). RAP1A is the target of miR-433-3p and miR-433-3p can inhibit the expression of RAP1A in SW480 cells. Compared with NC group, the proliferation, migration and invasion abilities of SW480 cells were weakened and apoptosis was increased in miR-433-3p mimics group accompanied by decreased levels of Bcl-2 and MMP-2 and increased level of Bax(P<0.05). Compared with miR-433-3p mimics group, the abilities of proliferation, migration and invasion of SW480 cells in miR-433-3p mimics+RAP1A group was enhanced, and the apoptosis was reduced, accompanied by increased levels of Bcl-2 and MMP-2 and decreased levels of Bax(P<0.05). Conclusion The down-regulated miR-433-3p plays the role of tumor suppressor gene in colorectal cancer. The miR-433-3p/RAP1A axis is expected to become a potential therapeutic target for colorectal cancer.
作者
白雨微
黄小齐
刘航
刘攀
BAI Yuwei;HUANG Xiaoqi;LIU Hang;LIU Pan(Section of Scientific Research,the Affiliated Baoji Hospital of Xi'an Medical University,Baoji 721006,China)
出处
《临床肿瘤学杂志》
CAS
2022年第12期1092-1098,共7页
Chinese Clinical Oncology
基金
陕西省教育厅资助项目(20JK0899)。
