外泌体源性LncRNA ESCCAL-1/miR-874/ITGBL1对结直肠癌细胞增殖凋亡的影响

Effects of exosome-derived LncRNA ESCCAL-1/miR-874/ITGBL1 on the proliferation and apoptosis of colorectal cancer cells

目的本研究旨在探究外泌体源性长非编码RNA(LncRNA)ESCCAL-1调控miR-874/整合素β样因子1(ITGBL1)轴在结直肠癌(CRC)进展中的作用机制。方法运用基因表达综合数据库(GEO)数据库对CRC中的差异表达基因进行分析。应用qRT-PCR检测LncRNA ESCCAL-1、miR-874和ITGBL1在CRC组织和细胞系(SW480、SW620、HCT116和HT29)及癌旁正常组织和NCM460细胞系中的表达;3(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)、克隆形成和流式细胞术分别检测细胞增殖、集落形成和凋亡情况;双荧光素酶报告实验分别验证miR-874与ESCCAL-1、ITGBL1间的互作用关系;荧光原位杂交测定LncRNAESCCAL-1的亚细胞定位。使用Exosome提取试剂盒分离血清中的外泌体。结果ESCCAL-1和ITGBL1在CRC组织和细胞系中的表达高于癌旁正常组织和NCM460细胞系,miR-874则相反(P<0.05)。敲减ESCCAL-1能够抑制CRC细胞增殖和集落形成,促进细胞凋亡。miR-874和ESCCAL-1存在特异性结合位点,miR-874抑制剂能够部分逆转敲减ESCCAL-1在CRC中介导的效应(P<0.05)。ESCCAL-1吸附miR-874上调ITGBL1。CRC患者血清中的ESCCAL-1及exo-ESCCAL-1较对照组上调,血清exo-ESCCAL-1可能是CRC治疗的一个有价值的诊断指标(P<0.05)。结论ESCCAL-1通过调控miR-874/ITGBL1轴促进CRC进展,ESCCAL-1可能是CRC治疗的一个有效分子靶点。

Objective To explore the mechanism of exosome-derived LncRNA ESCCAL-1 regulating the miR-874/ITGBL1 axis in the progression of colorectal cancer(CRC).Methods The differentially expressed genes in CRC were analyzed using the Gene Expression Omnibus(GEO)database.Expressions of LncRNA ESCCAL-1,miR-874 and ITGBL1 in CRC tissues and cell lines(SW480,SW620,HCT116 and HT29)and adjacent normal tissues and NCM460 cell lines were detected by qRT-PCR;3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide(MTT),clone formation and flow cytometry was used to detect cell proliferation,colony formation and apoptosis;dual luciferase reporter assays were used to verify the interaction between miR-874 and ESCCAL-1,ITGBL1;fluorescence in situ hybridization was used to determine the subcellular localization of LncRNAESCCAL-1.Exosomes were isolated from serum using the Exosome extraction kit.Results The expressions of ESCCAL-1 and ITGBL1 in CRC tissues and cell lines were higher than those in adjacent normal tissues and NCM460 cell lines,while the opposite was true for miR-874(P<0.05).Knockdown of ESCCAL-1 can inhibit CRC cell proliferation and colony formation and promote apoptosis.There are specific binding sites for miR-874 and ESCCAL-1,and miR-874 inhibitor could partially reverse the effect of knockdown ESCCAL-1 in CRC(P<0.05).ESCCAL-1 upregulates ITGBL1 by adsorbing miR-874.The serum levels of ESCCAL-1 and exo-ESCCAL-1 in CRC patients were higher than those in the control group.Serum exo-ESCCAL-1 may be a valuable diagnostic indicator for CRC treatment(P<0.05).Conclusion ESCCAL-1 promotes CRC progression by regulating the miR-874/ITGBL1 axis,and ESCCAL-1 may be an effective molecular target for CRC therapy.