摘要
目的检测微小RNA-646(miR-646)与含杆状病毒凋亡抑制蛋白重复序列蛋白5(BIRC5)基因在结直肠癌组织表达情况,探讨miR-646对结直肠癌细胞(HT-29)增殖、迁移及侵袭的影响。方法 2018年4月-2020年12月在本院行手术治疗的结直肠癌患者72例,手术中取结直肠癌组织及癌旁组织标本,采用实时荧光定量PCR(RT-qPCR)检测miR-646、BIRC5 mRNA表达水平。体外培养结直肠癌细胞系HT-29细胞并随机分为对照组、miR-646阴性对照(NC)组和miR-646模拟物(mimics)组(设正常结肠上皮细胞CCD841对照),RT-qPCR检测各组HT-29细胞miR-646、BIRC5 mRNA的表达;MTT法检测HT-29细胞存活率变化;Transwell小室法检测HT-29细胞迁移与侵袭情况;Western blot检测HT-29细胞凋亡抑制蛋白Survivin、基质金属蛋白酶2(MMP-2)及MMP-9表达的变化;双荧光素酶报告验验证miR-646与BIRC5基因的靶向关系。结果与癌旁组织和正常结肠上皮细胞CCD841细胞比较,结直肠癌组织与HT-29细胞miR-646相对表达水平显著降低,BIRC5 mRNA相对表达水平显著升高(P<0.05)。targetscan数据库预测miR-646与BIRC5基因3’UTR区有结合位点。与BIRC5-3’UTR-WT+miR-646 NC组(0.81±0.09)比较,BIRC5-3’UTR-WT+miR-646 mimics组(0.28±0.04)荧光素酶活性降低(P<0.05);与对照组[(3.04±0.21)个、(3.41±0.24)个、(98.56±0.64)%]和miR-646 NC组[(2.96±0.25)个、(3.32±0.23)个、(97.94±0.57)%]相比,miR-646 mimics组HT-29细胞miR-646表达水平显著升高(P<0.05),细胞存活率[(47.34±1.78)%]、迁移数[(1.35±0.08)个]与侵袭数[(1.48±0.05)个]、BIRC5和Survivin蛋白、MMP-2和MMP-9蛋白表达水平均显著降低(均P<0.05)。结论 miR-646与BIRC5基因存在靶向关系,可能通过靶向抑制BIRC5基因的表达抑制HT-29细胞的增殖、迁移与侵袭。
Objective To study the expression of microRNA-646(miR-646) and the baculoviral inhibitor of apoptosis protein repeat-containing protein 5(BIRC5) gene in colorectal cancer tissues and to explore the effect of miR-646 on the proliferation, migration, and invasion of colorectal cancer cells(HT-29). Methods Subjects were 72 patients with colorectal cancer who underwent surgery at this Hospital from April 2018 to December 2020. Colorectal cancer tissues and adjacent tissues were collected during surgery. The level of expression of miR-646 and BIRC5 in colorectal tissues and adjacent tissues was determined using real-time fluorescence quantitative PCR(RT-qPCR). HT-29 cells and CCD841 normal colon epithelial cells were cultured in vitro. HT-29 cells were randomly divided into a control group, an miR-646 negative control(NC) group, and an miR-646 mimic group. RT-qPCR was used to determine the level of expression of miR-646 and BIRC5 in HT-29 cells. An MTT assay was used to determine the survival rate of HT-29 cells. The Transwell assay was used to detect the migration and invasion of HT-29 cells. Western blotting was used to detect the expression of survivin, matrix metalloproteinase 2(MMP-2), and MMP-9 in HT-29 cells. A dual luciferase reporter assay was used to verify targeting of BIRC5 by miR-646. Results The level of miR-646 expression in colorectal cancer tissues and HT-29 cells was significantly lower than that in adjacent tissues and normal colonic epithelial cells(CCD841 cells), while the expression of BIRC5 was significantly higher(P<0.05). As predicted by the targetscan database, miR-646 had binding sites with the 5’ and 3’utr regions of BIRC. Luciferase activity in the BIRC5-3’UTR-WT + miR-646 mimic group(0.28±0.04) was lower(P<0.05) than that in the BIRC5-3’UTR-WT + miR-646 NC group(0.81±0.09). The level of miR-646 expression was significantly higher(P<0.05) in HT-29 cells in the miR-646 mimic group. The cell survival rate(47.34%±1.78%), the rate of migration(1.35±0.08), and the rate of invasion(1
作者
李红海
吴俊梅
周江浩
LI Hong-hai;WU Jun-mei;ZHOU Jiang-hao(Department of Gastrointestinal Oncology,the First Affiliated Hospital of Hainan Medical College,Haikou,Hainan 570000 China)
出处
《中国病原生物学杂志》
CSCD
北大核心
2021年第8期955-959,共5页
Journal of Pathogen Biology
基金
海南省自然科学基金青年基金项目(No.818QN245)。
