摘要
目的探讨微小RNA（miRNA，miR）-145调控食管鳞癌（ESCC）细胞增殖与侵袭的分子机制。方法实时定量反转录聚合酶链反应（RT—qPCR）检测miR-145和磷脂酶Csl（PLCEl）mRNA表达；Westernblot检测PLCEl蛋白水平；Targetscan软件预测PLCEl是否为miR-145的靶基因；荧光素酶报告基因检测miR-145是否作用于PLCElmRNA的3’端非编码区域（3’UTR）；噻唑蓝（MTT）和划痕实验检测细胞增殖和侵袭能力。结果4种ESCC细胞miR一145相对表达量分别为0．63±0．06、0．72±0．05、0．55±0．05、0．79±0．04，均明显低于正常食管上皮细胞（P=0．000，P=0．00l，P=0．000，P=0．003）。ESCC组织中miR-145相对表达量为0．68±0．03，较正常组织降低31．9％（P=0．000）。miR-145低表达与肿瘤浸润深度及TNM分期相关（x2=8．380，P=0．039；X2=6．810，P=0．033）。Targetscan预测PLCE1是miR-145的靶基因；荧光素酶报告基因证实miR-145作用于PLCElmRNA的3’UTR。ESCC细胞中PLCE1相对表达量分别为1．69±0．04、1．46±0．06、1。31±0．06、1．60±0．08，均明显高于正常食管上皮细胞（P=0．000，P=0．000，P=0．004，P=0．001）。ESCC组织中PLCEl相对表达量为1．85±0．04，为癌旁正常组织的1．85倍（P=0．000）。PLCEl表达水平与miR-145表达水平负相关（r=-0．828，P=0．002）。过表达miR-145抑制Ecal09增殖（P=0．006），侵袭能力降低22．1％（P=0．013）；过表达PLCE1促进增殖（P=0．003），侵袭能力增强24．9％（P=0．029）；而同时过表达miR-145与PLCE1则可抑制PLCE1的促增殖（P=0．003）和侵袭能力（P=0．001）。结论miR-145通过靶向调控PLCE1抑制ESCC细胞增殖和侵袭。
Objective To investigate the mechanism of microRNA （ miRNA, miR） - 145 in mod- ulating the capacity of proliferation and invasion in esophageal squamous cell carcinoma （ ESCC ）. Meth- ods Real- time quantitative reverse transcriptase- polymerase chain reaction （RT -qPCR） was used to detect the expression of miR - 145 and phospholipase C epsilon 1 （ PLCE1 ） mRNA. PLCE1 protein level was evaluated by Western blotting. The potential target gene of miR - 145 was predicted by targetscan soft- ware. Luciferase reporter gene assay was used to validate the predicted miR - 145 binding site in PLCE1 3＇ untranslated region （ 3 ＇ UTR ）. Proliferation and invasion capacity were assessed by MTr and wound healing assay separately. Results The relative expression of miR - 145 in four types of ESCC cells were 0. 63± 0. 06, 0. 72 ± 0. 05, 0. 55± 0. 05 and 0. 79± 0. 04 separately, which were obviously lower than in normal esophageal epithilum （ P = 0. 000, P = 0. 001, P = 0. 000, P = 0. 003 ）. While, relative expression of miR - 145 in ESCC tissues was 0. 68±0. 03, which was 31.9% lower than in normal esophageal tissue （ P = 0. 000 ）. An inverse correlation between miR - 145 expression and tumor invasion depth and TNM stage were observed （X2 = 8. 380, P = 0. 039; X2 = 6. 810, P = 0. 033 ）. Luciferase reporter gene assay proved that PLCE1 was a direct target of miR - 145. PLCE1 was overexpressed and inversely correlated with miR - 145 expression in ESCC （r = -0. 828, P =0. 002）. The relative expression of PLCE1 in four types of ESCC cells were 1.69 ± 0. 04, 1.46± 0. 06, 1.31 ± 0. 06 and 1.60 ±0.08, which were obviously higher than in normal esophageal epithilum （ P = 0. 000, P = 0. 000, P = 0. 004, P = 0. 001 ）. And relative PLCE1 expression was 1.85 ±0. 04 in ESCC tissues, which was 1.85 times higher than in normal esopha- geal tissue （ P = 0. 000 ）. In addition, overexpression of miR - 145 suppressed Eeal09 cells proliferation （P= 0. 006） and decreased invasion capacity by 22. 1% （P= 0. 013 ）. Whereas, overexpression of PLCE1 induced proliferation （ P = 0. 003 ） and elevated invasion capacity by 24. 9% （ P = 0. 029 ）. En- forced expression of miR - 145 could partially reversed the promoting effect of PLCE1 （ P = 0. 003 ）. Conelusion MiR - 145 suppresses proliferation and invasion of ESCC by targeting PLCE1.
出处
《中华实验外科杂志》
CSCD
北大核心
2017年第9期1472-1475,共4页
Chinese Journal of Experimental Surgery
基金
中山大学“985”工程项目（82000-31101301）
广东省科技计划项目2014A020212429）
广东省医学科研基金（A2015118）
关键词
微小RNA-145
磷脂酶Cε1
食管癌
增殖
侵袭
MicroRNA - 145
Phospholipase C epsilon 1
Esophageal neoplasms
Prolifera- tion
Invasion
