摘要
目的 miR-375在食管鳞状细胞癌(简称鳞癌)中低表达,但其调控的下游靶基因仍不明确。文中初步探讨miR-375在调控人食管鳞癌细胞中人矮小同源盒基因2(short stature homobox 2,SHOX2)的表达中的作用。方法运用生物信息学预测软件Target Scan、miRanda、Pic Tar、miRTarget2和PITA预测miR-375在SHOX2基因可能的作用位点。构建野生型SHOX2 3'UTR报告基因载体(p SHOX2-375-WT)及突变型SHOX2 3'UTR报告基因载体(p SHOX2-375-mut),测序鉴定。分7组转染:pmiR组、p SHOX2-375-WT组、p SHOX2-375-WT+miR-375组、p SHOX2-375-WT+miR-NC组、p SHOX2-375-mut组、p SHOX2-375-mut+miR-375组、p SHOX2-375-mut+miR-NC组,分别检测荧光素酶活性。转染miR-375的mimics于食管鳞癌细胞中,SHOX2 mRNA及蛋白表达分析。另检测食管鳞癌组织术后标本miR-375及SHOX2表达。结果 Target Scan、miRanda、Pic Tar、Mir Target2和PITA软件预测结果显示miR-375在SHOX2 3'UTR 1156-1170bp区域有且仅有1个保守作用位点。p SHOX2-375-WT+miR-375组荧光素酶活性(0.261±0.036)显著低于[pmiR(1.818±0.061)、p SHOX2-375-WT组(1.820±0.086)、p SHOX2-375-WT+miR-NC组(1.851±0.094)、p SHOX2-375-mut组(1.861±0.059)、p SHOX2-375-mut+miR-375组(1.896±0.048)、p SHOX2-375-mut+miR-NC组(1.760±0.062)],差异均有统计学意义(P<0.01)。过表达miR-375后EC9706细胞SHOX2 mRNA及蛋白较对照组相比表达受到抑制。食管鳞癌组织中的miR-375较癌旁组织表达降低,而SHOX2表达较癌旁组织表达增强。结论 miR-375在食管鳞癌细胞EC9706中靶向调控SHOX2基因的表达。
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 Target Scan,miRanda,Pic Tar,miRTarget2,and PITA were used to predict the assumptive targets of miR-375 in SHOX2. Then,two recombinant luciferase gene report plasmids containing wild p SHOX2 3＇ UTR（ p SHOX2-375-WT） and mutant p SHOX2 3＇ UTR（ p SHOX2-375-mut） were constructed,sequenced,and identified. Human esophageal squamous cancer cells were cotransfected with miR-375 mimics and p SHOX2-375-WT or p SHOX2-375-mut,respectively,and divided into 7 groups： pmiR,p SHOX2-375-WT,p SHOX2-375-WT ＋ miR-375,p SHOX2-375-WT ＋ miRNC,p SHOX2-375-mut,p SHOX2-375-mut ＋ miR-375,and p SHOX2-375-mut ＋ miR-NC,each subjected to the measurement of luciferase activity. The expressions of SHOX2 mRNA and protein were determined 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 p SHOX2-375-WT ＋ miR-375 group（ 0. 261 ± 0. 036） than in the pmiR（ 1. 818 ± 0. 061）,p SHOX2-375-WT（ 1. 820 ± 0. 086）,p SHOX2-375-WT ＋ miR-NC（ 1. 851 ± 0. 094）,p SHOX2-375-mut（ 1. 861 ± 0. 059）,p SHOX2-375-mut ＋ miR-375（ 1. 896 ±0. 048）,and p SHOX2-375-mut ＋ miR-NC group（ 1. 760 ± 0. 062）（ P 0. 01）. SHOX2 mRNA and protein expressions were suppressed by the overexpression of miR-375 in the EC9706 cells. The expression of miR-375 was decreased,while that of SHOX2 increased in the esophageal squamous carcinoma tissue as compared with the normal esophageal tissue. Conclusion MiR-375 regulates the expression of the SHOX2 gen in esophageal squamous cancer cells.
出处
《医学研究生学报》
CAS
北大核心
2015年第10期1017-1022,共6页
Journal of Medical Postgraduates
基金
国家自然科学基金(81301914)
江苏省自然科学基金(BK2012371)
