医学研究生学报
醫學研究生學報
의학연구생학보
Journal of Medical Postgraduates
2015年
10期
1017-1022
,共6页
金立%易俊%何贞月%宋海珠
金立%易俊%何貞月%宋海珠
금립%역준%하정월%송해주
miR-375%人矮小同源盒基因2%食管鳞癌%靶向调控
miR-375%人矮小同源盒基因2%食管鱗癌%靶嚮調控
miR-375%인왜소동원합기인2%식관린암%파향조공
miR-375%Short stature homobox 2%Esophageal squamous cancer%Targeted regulation
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TargetScan、miRanda、PicTar、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(pSHOX2-375-WT)及突变型SHOX2 3′UTR报告基因载体(pSHOX2-375-mut),测序鉴定. 分7组转染:pmiR 组、pSHOX2-375-WT 组、pSHOX2-375-WT +miR-375 组、pSHOX2-375-WT +miR-NC 组、pSHOX2-375-mut 组、pSHOX2-375-mut+miR-375组、pSHOX2-375-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 TargetScan、miRan-da、PicTar、MirTarget2和PITA软件预测结果显示miR-375 在SHOX2 3′UTR 1156-1170 bp区域有且仅有1个保守作用位点.pSHOX2-375-WT+miR-375组荧光素酶活性(0.261 ±0.036)显著低于[pmiR(1.818 ±0.061)、pSHOX2-375-WT组(1.820 ± 0.086)、pSHOX2-375-WT+miR-NC组(1.851 ±0.094)、pSHOX2-375-mut组(1.861 ±0.059)、pSHOX2-375-mut +miR-375 组(1.896 ±0.048)、pSHOX2-375-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.
目的 miR-375在食管鱗狀細胞癌(簡稱鱗癌)中低錶達,但其調控的下遊靶基因仍不明確. 文中初步探討miR-375在調控人食管鱗癌細胞中人矮小同源盒基因2(short stature homobox 2, SHOX2)的錶達中的作用. 方法 運用生物信息學預測軟件TargetScan、miRanda、PicTar、miRTarget2和PITA預測miR-375在SHOX2基因可能的作用位點. 構建野生型SHOX2 3′UTR報告基因載體(pSHOX2-375-WT)及突變型SHOX2 3′UTR報告基因載體(pSHOX2-375-mut),測序鑒定. 分7組轉染:pmiR 組、pSHOX2-375-WT 組、pSHOX2-375-WT +miR-375 組、pSHOX2-375-WT +miR-NC 組、pSHOX2-375-mut 組、pSHOX2-375-mut+miR-375組、pSHOX2-375-mut+miR-NC組,分彆檢測熒光素酶活性. 轉染miR-375的mimics于食管鱗癌細胞中,SHOX2 mRNA及蛋白錶達分析. 另檢測食管鱗癌組織術後標本miR-375及SHOX2錶達. 結果 TargetScan、miRan-da、PicTar、MirTarget2和PITA軟件預測結果顯示miR-375 在SHOX2 3′UTR 1156-1170 bp區域有且僅有1箇保守作用位點.pSHOX2-375-WT+miR-375組熒光素酶活性(0.261 ±0.036)顯著低于[pmiR(1.818 ±0.061)、pSHOX2-375-WT組(1.820 ± 0.086)、pSHOX2-375-WT+miR-NC組(1.851 ±0.094)、pSHOX2-375-mut組(1.861 ±0.059)、pSHOX2-375-mut +miR-375 組(1.896 ±0.048)、pSHOX2-375-mut +miR-NC組(1.760 ±0.062)],差異均有統計學意義(P<0.01). 過錶達 miR-375 後EC9706細胞SHOX2 mRNA及蛋白較對照組相比錶達受到抑製. 食管鱗癌組織中的miR-375較癌徬組織錶達降低,而SHOX2錶達較癌徬組織錶達增彊. 結論 miR-375在食管鱗癌細胞EC9706中靶嚮調控SHOX2基因的錶達.
목적 miR-375재식관린상세포암(간칭린암)중저표체,단기조공적하유파기인잉불명학. 문중초보탐토miR-375재조공인식관린암세포중인왜소동원합기인2(short stature homobox 2, SHOX2)적표체중적작용. 방법 운용생물신식학예측연건TargetScan、miRanda、PicTar、miRTarget2화PITA예측miR-375재SHOX2기인가능적작용위점. 구건야생형SHOX2 3′UTR보고기인재체(pSHOX2-375-WT)급돌변형SHOX2 3′UTR보고기인재체(pSHOX2-375-mut),측서감정. 분7조전염:pmiR 조、pSHOX2-375-WT 조、pSHOX2-375-WT +miR-375 조、pSHOX2-375-WT +miR-NC 조、pSHOX2-375-mut 조、pSHOX2-375-mut+miR-375조、pSHOX2-375-mut+miR-NC조,분별검측형광소매활성. 전염miR-375적mimics우식관린암세포중,SHOX2 mRNA급단백표체분석. 령검측식관린암조직술후표본miR-375급SHOX2표체. 결과 TargetScan、miRan-da、PicTar、MirTarget2화PITA연건예측결과현시miR-375 재SHOX2 3′UTR 1156-1170 bp구역유차부유1개보수작용위점.pSHOX2-375-WT+miR-375조형광소매활성(0.261 ±0.036)현저저우[pmiR(1.818 ±0.061)、pSHOX2-375-WT조(1.820 ± 0.086)、pSHOX2-375-WT+miR-NC조(1.851 ±0.094)、pSHOX2-375-mut조(1.861 ±0.059)、pSHOX2-375-mut +miR-375 조(1.896 ±0.048)、pSHOX2-375-mut +miR-NC조(1.760 ±0.062)],차이균유통계학의의(P<0.01). 과표체 miR-375 후EC9706세포SHOX2 mRNA급단백교대조조상비표체수도억제. 식관린암조직중적miR-375교암방조직표체강저,이SHOX2표체교암방조직표체증강. 결론 miR-375재식관린암세포EC9706중파향조공SHOX2기인적표체.
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 .
