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河北医学  2024, Vol. 30 Issue (9): 1409-1420    DOI: 10.3969/j.issn.1006-6233.2024.09.01
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LncRNA TUSC7通过调控miR-182/TBX5轴抑制结直肠癌的增殖迁移和侵袭
陈从涛, 孙海滨, 董明杰
河北医科大学第二医院急诊外科, 河北 石家庄 050000
LncRNA TUSC7 Inhibits Proliferation Migration and Invasion of Colorectal Cancer by Regulating the miR-182/TBX5 Axis
CHEN Congtao, SUN Haibin, DONG Mingjie
The Second Hospital of Hebei Medical University, Hebei Shijiazhuang 050000, China
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摘要 目的: 探讨长链非编码RNA抑癌候选基因7(long non-coding RNA tumor suppressor candidate 7,LncRNA TUSC7)通过调控微小RNA-182(microRNA-182,miR-182)/T-框蛋白5(T-box5,TBX5)轴对结直肠癌(colorectal cancer,CRC)增殖、迁移和侵袭的影响。方法: 实时荧光定量PCR检测LncRNA TUSC7、miR-182和TBX5在90对CRC腺癌组织及远端正常组织中的表达水平,通过临床样本数据分析三者的相关性,并检测LncRNA TUSC7在人CRC细胞系LoVo、SW1116、CaCO2、HCT116和SW480以及正常人结肠粘膜细胞系NCM460中的表达。RNA荧光原位杂交实验检测LncRNA TUSC7的亚细胞定位。SW480细胞分10组,敲减TUSC7(shTUSC7)组和敲减对照(shNC)组、过表达TUSC7(oeTUSC7)组和过表达对照(oeNC)组、miR-182过表达(miR-182 mimic)组和过表达对照(NC mimic)组、干涉miR-182(miR-182 inhibitor)组和干涉对照(NC inhibitor)组、同时过表达TUSC7和miR-182(oeTUSC7+miR-182 mimic)组及同时过表达TUSC7、miR-182和TBX5(oeTUSC7+miR-182 mimic+oeTBX5)组,分别进行shTUSC7、shNC、oeTUSC7、oeNC、miR-182 mimic、NC mimic、miR-182 inhibitor、NC inhibitor转染及oeTUSC7+miR-182 mimic共转染和oeTUSC7+miR-182 mimic+oeTBX5共转染。CCK8、克隆形成、流式细胞术和Transwell实验测定LncRNA TUSC7、miR-182和TBX5对SW480细胞活力、增殖、凋亡、迁移和侵袭的影响。双荧光素酶报告基因系统实验、RNA免疫沉淀实验、RNA pull-down实验和Western blot验证LncRNA TUSC7、miR-182和TBX5的相互关系。Kaplan-Meier法进行生存分析。将裸鼠随机分为4组(n=12):shTUSC7组、shNC组、oeTUSC7组和oeNC组。分别于右侧腋窝内皮下接种shTUSC7、shNC、oeTUSC7、oeNC转染的SW480细胞。用于在体评价LncRNA TUSC7对CRC肿瘤生长的影响及生存分析。结果: 与癌旁正常组织相比,在CRC组织中,LncRNA TUSC7(1.53±0.84 vs 6.97±3.97)和TBX5(0.58±0.29 vs 3.25±1.51)低表达而miR-182(0.54±0.33 vs 0.09±0.06)高表达(P<0.05),且LncRNA TUSC7与miR-182表达负相关(R2=0.09077)而与TBX5的表达正相关(R2=0.08371),miR-182与TBX5的表达负相关(R2=0.06914;P<0.05)。与NCM460(1.00±0.05)细胞相比,LncRNA TUSC7在LoVo(0.51±0.08)、SW1116(0.41±0.10)、CaCO2(0.42±0.11)、HCT-116(0.53±0.10)和SW480(0.35±0.07)细胞中均显著低表达(P<0.05),且在SW480细胞中表达最低。LncRNA TUSC7定位在在CRC细胞质中表达。与shNC/oeNC组相比,沉默/过表达LncRNA TUSC7促进/抑制CRC细胞增殖、迁移和侵袭,而减少/增加细胞凋亡(P<0.05)。LncRNA TUSC7可直接靶向miR-182,此外,TBX5是miR-182的一个直接的靶点。从机制上讲,LncRNA TUSC7通过靶向miR-182促进TBX5的表达进而抑制CRC细胞的增殖、迁移和侵袭并促进凋亡。与体外实验结果相一致,LncRNA TUSC7可在体抑制CRC肿瘤的生长。结论: LncRNA TUSC7在CRC组织和细胞中低表达,且LncRNA TUSC7通过调控miR-182/TBX5轴抑制CRC的增殖、迁移和侵袭。
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陈从涛
孙海滨
董明杰
关键词 LncRNA TUSC7结直肠癌miR-182/TBX5轴增殖迁移侵袭    
AbstractObjective: To investigate the inhibitory effects of long non-coding RNA tumor suppressor candidate 7 (LncRNA TUSC7) on the proliferation, migration, and invasion of colorectal cancer (CRC) via regulation of microRNA-182 (miR-182)/T-box5 (TBX5) axis.Methods: Quantitative real-time PCR was performed to detect the levels of LncRNA TUSC7, miR-182, and TBX5 in 90 pairs of CRC adenocarcinoma tissues and the corresponding adjacent normal tissues, and their correlations were analyzed in clinical samples, as well as the expressions of LncRNA TUSC7 in human CRC cell lines LoVo, SW1116, CaCO2, HCT116 and SW480 and normal human colon mucosal cell line NCM460. RNA fluorescence in situ hybridization was conducted to detect the subcellular localization of LncRNA TUSC7. SW480 cells were divided into 10 groups:TUSC7 knockdown (shTUSC7) group and knockdown control (shNC) group, TUSC7 overexpression (oeTUSC7) group and overexpression control (oeNC) group, miR-182 overexpression (miR-182 mimic) group and overexpression control (NC mimic) group, interference miR-182 (miR-182 inhibitor) group and interference control (NC inhibitor) group, simultaneous overexpression of TUSC7 and miR-182 (oeTUSC7+miR-182) group and simultaneous overexpression of TUSC7, miR-182, and TBX5 (oeTUSC7+miR-182 mimic+oeTBX5) group, transfected with shTUSC7, shNC, oeTUSC7, oeNC, miR-182 mimic, NC mimic, miR-182 inhibitor, NC inhibitor, oeTUSC7+miR-182 mimic, and oeTUSC7+miR-182 mimic+oeTBX5, respectively. The effects of LncRNA TUSC7, miR-182, and TBX5 on cell viability, proliferation, apoptosis, migration, and invasion of SW480 cells were determined by CCK8, clonal formation, flow cytometry, and Transwell assays. Dual luciferase reporter assay, RNA immunoprecipitation, RNA pull-down assay, and Western blot were performed to verify the relationships in LncRNA TUSC7, miR-182, and TBX5. Kaplan-Meier method was used to determine survival analysis. The nude mice were randomly divided into 4 groups (n=12):shTUSC7 group, shNC group, oeTUSC7 group, and oeNC group, which were inoculated subcutaneously with SW480 cells transfected with shTUSC7, shNC, oeTUSC7, and oeNC, respectively, in the right underarm. The effects of LncRNA TUSC7 on tumor growth of CRC and survival analysis were evaluated by xenograft model in vivo.Results: Compared to adjacent normal tissue, LncRNA TUSC7 (1.53±0.84 vs 6.97±3.97) and TBX5 (0.58±0.29 vs 3.25±1.51) were low-expressed, while miR-182 (0.54±0.33 vs 0.09±0.06) was high-expressed in CRC tissues (P<0.05). LncRNA TUSC7 was negatively correlated with miR-182 (R2=0.09077), but positively related to TBX5 (R2=0.08371). miR-182 were negatively associated with TBX5 (R2=0.06914;P<0.05). Compared with NCM460 (1.00±0.05) cells, LncRNA TUSC7 was significantly lower expressed in LoVo (0.51±0.08), SW1116 (0.41±0.10), CaCO2 (0.42±0.11), HCT-116 (0.53±0.10) and SW480 (0.35±0.07) cells (P<0.05). The expression was lowest in SW480 cells. LncRNA TUSC7 was located in the CRC cytoplasm. Compared with the shNC/oeNC group, silencing/overexpression of LncRNA TUSC7 promoted/inhibited the proliferation, migration, and invasion, while decreased/increased apoptosis of CRC cells. LncRNA TUSC7 directly targeted miR-182, and TBX5 was a direct target of miR-182. Mechanistically, LncRNA TUSC7 promoted the expression of TBX5 by targeting miR-182, thus inhibiting the proliferation, migration, and invasion, but promoting apoptosis of CRC cells (P<0.05). Consistent with the results in vitro, LncRNA TUSC7 could inhibit CRC growth in vivo.Conclusion: LncRNA TUSC7 was low expressed in CRC tissues and cells. LncRNA TUSC7 inhibited the proliferation, migration and invasion of CRC cells by regulating the miR-182/TBX5 axis.
Key wordsLncRNA TUSC7    Colorectal cancer    miR-182/TBX5 axis    Proliferation    Migration    Invasion
    
基金资助:2024年度河北省医学科学研究课题,(编号:20240496)
通讯作者: 董明杰   
引用本文:   
陈从涛, 孙海滨, 董明杰. LncRNA TUSC7通过调控miR-182/TBX5轴抑制结直肠癌的增殖迁移和侵袭[J]. 河北医学, 2024, 30(9): 1409-1420.
CHEN Congtao, SUN Haibin, DONG Mingjie. LncRNA TUSC7 Inhibits Proliferation Migration and Invasion of Colorectal Cancer by Regulating the miR-182/TBX5 Axis. HeBei Med, 2024, 30(9): 1409-1420.
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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2024.09.01     或     http://www.hbyxzzs.cn/CN/Y2024/V30/I9/1409
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