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LncRNA TMPO-AS1调节miR-340-5p/RUNX1轴对结直肠癌细胞增殖、迁移和侵袭的影响

Impacts of LncRNA TMPO-AS1 on the proliferation,migration,and invasion of colorectal cancer cells by regulating the miR-340-5p/RUNX1 axis
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摘要 目的探讨长链非编码RNA TMPO反义RNA1(LncRNA TMPO-AS1)调控miR-340-5p/RUNT相关转录因子1(RUNX1)轴对结直肠癌(CRC)细胞增殖、迁移和侵袭的影响。方法qRT-PCR检测人正常结肠上皮细胞HCoEpiC、人CRC细胞SW480、HCT116、LOVO、HT-29中TMPO-AS1、miR-340-5p、RUNX1表达;将HCT116细胞随机分为:si-ctrl组、si-TMPO-AS1组、mimics NC组、miR-340-5p组、si-TMPO-AS1+anti-miR-ctrl组、si-TMPO-AS1+anti-miR-340-5p组;qRT-PCR检测6组HCT116细胞TMPO-AS1、miR-340-5p、RUNX1水平;CCK-8法检测HCT116细胞活力;EDU法检测HCT116细胞增殖;Transwell检测HCT116细胞迁移和侵袭;western blot检测HCT116细胞RUNX1、PCNA、MMP-2表达;双荧光素酶报告基因实验分别验证TMPO-AS1和miR-340-5p、miR-340-5p和RUNX1的关系。结果与HCoEpiC细胞比较,不同CRC细胞中TMPO-AS1、RUNX1表达显著性升高,miR-424-5p表达显著性降低(P<0.05),且HCT116细胞变化结果更显著,后续实验选择HCT116细胞。与si-ctrl组比较,si-TMPO-AS1组HCT116细胞TMPO-AS1、RUNX1 mRNA水平、细胞活力A值、EDU阳性细胞率、迁移与侵袭细胞数、RUNX1、PCNA、MMP-2蛋白水平均显著性降低,miR-340-5p mRNA水平显著性升高(P<0.05);与mimics NC组比较,miR-340-5p组HCT116细胞RUNX1 mRNA水平、细胞活力A值、EDU阳性细胞率、迁移与侵袭细胞数、RUNX1、PCNA、MMP-2蛋白水平均显著性降低,miR-340-5p mRNA水平显著性升高(P<0.05);与si-TMPO-AS1+anti-miR-ctrl组比较,si-TMPO-AS1+anti-miR-340-5p组HCT116细胞RUNX1 mRNA水平、细胞活力A值、EDU阳性细胞率、迁移与侵袭细胞数、RUNX1、PCNA、MMP-2蛋白水平均显著性升高,miR-340-5p mRNA水平显著性降低(P<0.05);TMPO-AS1靶向负调控miR-340-5p表达,miR-340-5p靶向负调控RUNX1表达。结论沉默TMPO-AS1靶向上调miR-340-5p表达,从而下调RUNX1表达,抑制HCT116细胞增殖、迁移和侵袭。 Objective To investigate the impacts of long non-coding RNA TMPO antisense RNA1(lncRNA TMPO-AS1)on the proliferation,migration and invasion of colorectal cancer (CRC)cells by regulating the miR-340-5p/RUNT related transcription factor 1(RUNX1)axis.Methods The quantitative reverse transcriptase PCR(qRT-PCR)was applied to detect the mRNA levels of TMPO-AS1,miR-340-5p,and RUNX1 in human normal colon epithelial cells HCoEpiC,human CRC cells SW480,HCT116,LOVO,and HT-29.HCT116 cells were transfected with si-ctrl,si-TMPO-AS1,mimics NC,miR-340-5p,si-TMPO-AS1+anti miR-ctrl,and si-TMPO-AS1+anti miR-340-5p.qRT-PCRwas applied to detect the mRNA levels of TMPO-AS1,miR-340-5p,and RUNX1 in transfected HCT116 cells.Cell activity,and proliferation were detected by CCK-8 assay and EdU assay,respectively.Transwell assay was applied to detect migration and invasion of HCT116 cells.Western blot was applied to detect the protein expressions of RUNX1,proliferating cell nuclear antigen(PCNA),and matrix metalloprotease-2(MMP-2)in HCT116 cells.Dual-luciferase reporteRassay was applied to verify the relationship between TMPO-AS1 and miR-340-5p,and that between miR-340-5p and RUNX1.Results Compared with those of HCoEpiC cells,TMPO-AS1 and RUNX1 were significantly upregulated,and miR-424-5p was significantly downregulated in CRC cell lines(P<0.05).Expression changes in HCT116 cells were the most obvious,and therefore,HCT116 cells were selected for subsequent experiments.Compared with those transfected with si-ctrl,HCT116 cells transfected with si-TMPO-AS1 presented significantly lower mRNA levels of TMPO-AS1 and RUNX1,A value of cell viability,EdU-positive cell rate,numbers of migrating and invading cells,and protein levels of RUNX1,PCNA,and MMP-2,but higher mRNA level of miR-340-5p(P<0.05).Compared with those transfected with mimics NC,HCT116 cells transfected with miR-340-5p presented significantly lower mRNA level of RUNX1,A-value of cell viability,EdU-positive cell rate,numbers of migrating and invading cells,and protein levels of RUNX1,
作者 许汉兵 韩建涛 张成鹏 XU Hanbing;HAN Jiantao;ZHANG Chengpeng(Department of General Surgery,Wuhan Third Hospital(Tongren Hospital of Wuhan University),Hubei,Wuhan 430000,China)
出处 《河北医药》 CAS 2024年第2期165-170,共6页 Hebei Medical Journal
基金 武汉市医学科研项目(编号:WX19Q30)。
关键词 LncRNA TMPO-AS1 miR-340-5p RUNX1 结直肠癌细胞 long non-coding RNA TMPO antisense RNA1(lncRNA TMPO-AS1) miR-340-5p RUNT related transcription factor 1(RUNX1) colorectal canceRcells
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