Targeted regulation of SHOX2 gene expression by miR-375 in human esophageal squamous cancer cells / 医学研究生学报

ABSTRACT

Objective MiR-375 is lowly expressed in esophageal squamous cancer cells and the downstream target gene of miR-375 remains unclear .This paper discusses the role of miR-375 in regulating the expression of short stature homobox 2 ( SHOX2) in human esophageal squamous cancer cells . Methods The bioinformatics software TargetScan , miRanda, PicTar, miRTarget2, and PITA were used to predict the assumptive targets of miR-375 in SHOX2.Then, two recombinant luciferase gene report plasmids containing wild pSHOX2 3′UTR ( pSHOX2-375-WT ) and mutant pSHOX2 3′UTR ( pSHOX2-375-mut ) were constructed , sequenced , and identified.Human esophageal squamous cancer cells were co-transfected with miR-375 mimics and pSHOX2-375-WT or pSHOX2-375-mut, respectively , and divided into 7 groups pmiR, pSHOX2-375-WT, pSHOX2-375-WT +miR-375, pSHOX2-375-WT +miR-NC, pSHOX2-375-mut, pSHOX2-375-mut+miR-375, and pSHOX2-375-mut+miR-NC, each subjected to the measurement of luciferase activity .The expressions of SHOX 2 mRNA and protein were de-termined after transfection of the esophageal squamous cancer cells with miR-375 mimics, and so were the expressions of miR-375 and SHOX2 in the esophageal squamous carcinoma tissue samples obtained postoperatively . Results Prediction with the five software showed only one conserved function site of miR-375 in SHOX2 3′UTR at 1156-1170 bp.Luciferase activity was significantly lower in the pSHOX2-375-WT+miR-375 group (0.261 ±0.036) than in the pmiR (1.818 ±0.061), pSHOX2-375-WT (1.820 ±0.086), pSHOX2-375-WT+miR-NC (1.851 ±0.094), pSHOX2-375-mut (1.861 ±0.059), pSHOX2-375-mut +miR-375 (1.896 ± 0.048), and pSHOX2-375-mut+miR-NC group (1.760 ±0.062) ( P<0.01).SHOX2 mRNA and protein expressions were sup-pressed by the overexpression of miR-375 in the EC9706 cells.The expression of miR-375 was decreased, while that of SHOX2 in-creased in the esophageal squamous carcinoma tissue as compared with the normal esophageal tissue . Conclusion MiR-375 regu-lates the expression of the SHOX 2 gen in esophageal squamous cancer cells .