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Cε1(PLCE1)mRNA表达;Western blot检测PLCE1蛋白水平;Targetscan软件预测PLCE1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PLCE1 mRNA的3’端非编码区域(3'UTR);噻唑蓝(MTI)和划痕实验检测细胞增殖和侵袭能力.结果 4种ESCC细胞miR-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=0.001,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PLCE1 mRNA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PLCE1相对表达量为1.85 ±0.04,为癌旁正常组织的1.85倍(P=0.000).PLCE1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Eca109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.

abstractsObjective To investigate the mechanism of microRNA (miRNA,miR)-145 in modulating the capacity of proliferation and invasion in esophageal squamous cell carcinoma (ESCC).Methods 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 software.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 MTT 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 esophageal tissue (P =0.000).In addition,overexpression of miR-145 suppressed Eca109 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).Enforced expression of miR-145 could partially reversed the promoting effect of PLCE1 (P =0.003).Conclusion MiR-145 suppresses proliferation and invasion of ESCC by targeting PLCE1.