LncRNA DLX6-AS1通过miR-181b/IL-6轴影响食管癌细胞的恶性生物学行为

LncRNA DLX6-AS1 affects the malignant biological behavior of esophageal cancer cells through the miR-181b/IL-6 axis

目的探讨长链非编码RNA(long non-coding RNA,lncRNA)同源盒转录因子6反义RNA1(homeobox transcription factor 6 antisense RNA1,DLX6-AS1)调控miR-181b/白细胞介素-6(interleukin-6,IL-6)轴对食管癌Eca-109细胞增殖、迁移及侵袭的影响。方法选取人食管上皮细胞株HEEC和人食管鳞状细胞癌细胞株Eca-109,采用qRT-PCR检测lncRNA DLX6-AS1、miR-181b及IL-6的表达水平。将si-DLX6-AS1、miR-181b mimics、pcDNA-IL-6、pcDNA-IL-6+miR-181b mimics及其相应阴性对照分别转染至Eca-109细胞,采用qRT-PCR检测转染效果,CCK-8法和EdU染色实验检测细胞的增殖能力,Transwell小室实验检测细胞的迁移与侵袭能力,双荧光素酶报告基因实验验证lncRNA DLX6-AS1与miR-181b、miR-181b与IL-6的靶向关系。结果与HEEC细胞相比,Eca-109细胞中lncRNA DLX6-AS1、IL-6 mRNA的表达水平升高(P=0.005,0.006),但miR-181b表达水平降低(P=0.004)。与相应阴性对照组比较,si-DLX6-AS1组和miR-181b mimics组的细胞增殖活性和EdU阳性细胞率均降低(均P<0.05),细胞迁移数目与侵袭数目均减少(均P<0.05),而pcDNA-IL-6组的细胞增殖活性和EdU阳性细胞率升高(均P<0.05),细胞迁移数目与侵袭数目增加(均P<0.05)。lncRNA DLX6-AS1与miR-181b存在靶向结合关系,miR-181b mimics和野生型DLX6-AS1载体共转染后,细胞内的荧光素酶活性明显降低(P=0.006);下调DLX6-AS1表达后Eca-109细胞中miR-181b表达水平升高(P=0.010)。miR-181b与IL-6存在靶向结合关系,miR-181b mimics和野生型IL-6载体共转染后,细胞内的荧光素酶活性明显降低(P<0.05);过表达miR-181b后Eca-109细胞中IL-6的表达水平降低(P<0.05)。与pcDNA-IL-6组比较,pcDNA-IL-6与miR-181b mimics共转染后,Eca-109细胞中IL-6 mRNA和蛋白表达水平下降,细胞增殖活性和EdU阳性细胞率降低,细胞迁移数目与侵袭数目也减少(均P<0.05)。结论LncRNA DLX6-AS1通过miR-181b靶向负调控IL-6表达而促进食管癌Eca-109细胞增殖、迁移及侵袭。

ObjectiveTo investigate the effect of long non-coding RNA(lncRNA) homeobox transcription factor 6 antisense RNA1(DLX6-AS1) regulating miR-181b/interleukin-6(IL-6) axis on the proliferation,migration and invasion of esophageal cancer Eca-109cells.MethodsThe human esophageal epithelial cell line HEEC and human esophageal squamous cell carcinoma cell line Eca-109were selected,and the expression levels of lncRNA DLX6-AS1,miR-181b and IL-6 were detected by qRT-PCR.The si-DLX6-AS1,miR-181b mimics,pcDNA-IL-6,pcDNA-IL-6+miR-181b mimics and their corresponding negative controls were transfected into Eca-109cells,respectively,and the transfection effect was detected by qRT-PCR.The CCK-8 assay and EdU staining assay were used to detect cell proliferation ability.The Transwell assay was used to detect cell migration and invasion ability,and dual luciferase reporter assay was used to verify the targeting relationship between lncRNA DLX6-AS1 and miR-181b,miR-181b and IL-6.ResultsCompared with those of HEEC cells,the expression levels of lncRNA DLX6-AS1 and IL-6 mRNA were increased in Eca-109 cells(P=0.005,0.006),but the expression level of miR-181b was decreased(P=0.004).Compared with the corresponding negative control group,the cell proliferation activity and EdU positive cell rate in the si-DLX6-AS1 group and miR-181b mimics group were decreased(all P<0.05),and the number of cell migration and invasion were decreased(all P<0.05),while the cell proliferation activity and EdU positive cell rate in the pcDNA-IL-6 group were increased(both P<0.05),and the number of cell migration and invasion were increased(both P<0.05).IncRNA DLX6-AS1 had a targeted binding relationship with miR-181b.After co-transfection of miR-181b mimics and wild-type DLX6-AS1 vector,the intracellular luciferase activity was significantly decreased in cells(P=0.006).The expression level of miR-181b in Eca-109 cells was increased after down-regulation of DLX6-AS1 expression(P=0.010).miR-181b had a targeted binding relationship with IL-6.After co-transfection of miR-181b mimics and wild-type IL-6 vector,the intracellular luciferase activity was significantly decreased(P<0.05).The expression level of IL-6 in Eca-109 cells decreased after overexpression of miR-181b mimics(P<0.05).Compared with those of the pcDNA-IL-6 group,the expression levels of IL-6 mRNA and protein in Eca-109 cells,the cell proliferation activity,the EdU positive cell rate,the number of cell migration and invasion were decreased(all P<0.05),after co-transfection of pcDNA-IL-6 and miR-181b mimics.ConclusionsLncRNA DLX6-AS1 promotes the proliferation,migration and invasion of esophageal cancer Eca-109 cells through miR-181b targeting and negatively regulating IL-6 expression.