lncRNA GIHCG靶向miR-429对食管鳞状细胞癌细胞增殖、迁移及侵袭的影响

Effects of lncRNA GIHCG targeting miR-429 on proliferation,migration,and invasion of esophageal squamous cell carcinoma cells

目的探讨长链非编码核糖核酸(lncRNA)GIHCG靶向微小核糖核酸-429(miR-429)对食管鳞状细胞癌(ESCC)细胞增殖、迁移及侵袭的影响。方法常规培养人ESCC细胞系(EC9706、TE1、KYSE150细胞)及正常食管鳞状上皮细胞系(Het-1A细胞),用RT-PCR法筛选实验细胞。将对数生长期实验细胞随机分为si-NC组、si-GIHCG组,分别转染lncRNA敲低质粒阴性对照、lncRNA GIHCG敲低质粒。用RT-PCR法检测细胞中lncRNA GIHCG、miR-429表达,用CCK-8法检测细胞增殖能力[吸光度值(OD值)],用细胞划痕实验检测细胞迁移能力,用Transwell小室实验检测细胞侵袭能力,用Western blotting法检测细胞中细胞增殖表型蛋白[细胞周期蛋白D1(Cyclin D1)、增殖细胞核抗原(PCNA)]和转移表型蛋白[基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)]表达。将对数生长期实验细胞随机分为GIHCG-WT+miR-429 mimics组、GIHCG-WT+NC-mimics组、GIHCG-MUT+miR-429 mimics组、GIHCG-MUT+NC-mimics组,用双荧光素酶报告基因实验验证lncRNA GIHCG与miR-429的靶向关系。结果与Het-1A细胞比较,EC9706、TE1、KYSE150中lncRNA GIHCG相对表达量高(P均<0.05),miR-429相对表达量低(P均<0.05)。由于TE1细胞中lncRNA GIHCG的相对表达表达量最高、miR-429相对表达量最低,因此以TE1细胞作为实验细胞。与si-NC组比较,si-GIHCG组lncRNA GIHCG相对表达量低,miR-429相对表达量高,各时间OD值低,细胞移动距离百分比低,Cyclin D1、PCNA、MMP-2、MMP-9相对表达量低,差异均有统计学意义(P均<0.05)。与GIHCG-WT+NC-mimics组比较,GIHCG-WT+miR-429 mimics组荧光素酶相对活性低(P<0.05);GIHCG-MUT+miR-429 mimics组、GIHCG-MUT+NC-mimics组荧光素酶相对活性比较差异无统计学意义(P>0.05)。结论ESCC细胞中lncRNA GIHCG高表达,miR-429低表达;敲低lncRNA GIHCG通过靶向调控miR-429抑制ESCC细胞增殖、迁移及侵袭。

Objective To investigate the effects of long non-coding RNA(lncRNA)GIHCG targeting microRNA(miR)-429 on the proliferation,migration,and invasion of esophageal squamous cell carcinoma(ESCC)cells.Meth-ods Human ESCC cell lines(EC9706,TE1,and KYSE150 cells)and normal esophageal squamous epithelial cell line(Het-1A cells)were routinely cultured.The expression levels of lncRNA GIHCG and miR-429 in the cells were detected by RT-PCR,and the experimental cells were screened.The experimental cells in the logarithmic growth phase were ran-domly divided into the si-NC group and the si-GIHCG group,which were transfected with lncRNA knockdown plasmid neg-ative control and lncRNA GIHCG knockdown plasmid,respectively.The expression levels of lncRNA GIHCG and miR-429 in cells were detected by RT-PCR.The proliferation ability of the cells[optical density(OD)value]was detected by CCK-8.The migration ability of the cells was detected by cell scratch test.The invasion ability of the cells was detected by Transwell chamber test.The expression levels of cell proliferation phenotype proteins[CyclinD1,proliferating cell nuclear antigen(PCNA)]and metastasis phenotype proteins[matrix metalloproteinase 2(MMP-2),matrix metalloproteinase 9(MMP-9)]in the cells were detected by Western blotting.The experimental cells in the logarithmic growth phase were ran-domly divided into the GIHCG-WT+miR-429 mimics group,the GIHCG-WT+NC-mimics group,the GIHCG-MUT+miR-429 mimics group,and the GIHCG-MUT+NC-mimics group,respectively.The targeted relationship between lncRNA GIHCG and miR-429 was verified by dual luciferase reporter gene assay.Results Compared with Het-1A cells,the rela-tive expression levels of lncRNA GIHCG in EC9706,TE1 and KYSE150 were higher(all P<0.05),and the relative expression levels of miR-429 were lower(all P<0.05).Since the relative expression level of lncRNA GIHCG was the high-est and the relative expression level of miR-429 was the lowest in TE1 cells,TE1 cells were used as the experimental cells.Compared with the si-NC group,the relative expression level of lncRNA GIHCG in the si-GIHCG group was lower,the rel-ative expression level of miR-429 was higher,the OD values were lower,the percentage of cell movement distance was lower,and the relative expression levels of CyclinD1,PCNA,MMP-2 and MMP-9 were lower,and the differences were statistically significant(all P<0.05).Compared with the GIHCG-WT+NC-mimics group,the relative luciferase activity in the GIHCG-WT+miR-429 mimics group was lower(P<0.05);there was no statistically significant difference in the rel-ative luciferase activity between the GIHCG-MUT+miR-429 mimics group and the GIHCG-MUT+NC-mimics group(P>0.05).Conclusions The lncRNA GIHCG is highly expressed and the miR-429 is low expressed in ESCC cells.Knock-down of lncRNA GIHCG inhibits the proliferation,migration and invasion of ESCC cells by targeting miR-429.