食管鳞癌中微小核糖核酸-21的作用及对靶蛋白的影响

The role of microRNA-21 in esophageal squamous cell carcinoma and its effects on the PDCD4

目的探讨微小RNA（miRNA）-21对食管鳞癌细胞Ecal09和哈萨克族食管癌的促增殖作用及对程序性细胞死亡基因4（PDCD4）表达的影响。方法将食管鳞癌细胞系Ecal09分为miRNA-21Mimics组（转染序列5＇-UCAACAUCAGUCUGAUAAGCUA-3’）、miRNA-21抑制剂组（转染序列5＇-UAGCUUAUCAGACUGAUGUUGA-3＇）、阴性对照组（转染随机序列）和正常对照组（不予转染等任何处理）。细胞按4．5×10^5个／孔接种于6孔板，基于RNA干扰（RNAi）技术、使用脂质体2000试剂进行细胞转染。采用细胞计数法检测转染后各组细胞的增殖情况。使用实时荧光定量聚合酶链反应（qRT—PCR）检测各组细胞的miRNA-21转录水平。采用Western印迹技术检测转染后各组PDCD4蛋白表达情况。检测18对哈萨克族食管癌及其对应的癌旁正常食管组织中miRNA-21转录和PDCD4蛋白表达情况。结果转染48h后，与正常对照组比较，miRNA-21Mimics组Ecal09细胞数增加36％（P=0．002），miRNA-21抑制剂组细胞数减少28％（P=0．002），阴性对照组细胞数变化不明显（P=0．515）。转染48h后，miRNA-21抑制剂组、miRNA-21Mimics组、阴性对照组、正常对照组miRNA-21的相对表达量分别为0．37±0．10、9．17±1．08、0．74±0．23和1．04±0．34，PDCD4蛋白相对表达量分别为1．47±0．11、0．61±0．09、0．89±0．12、0．79±0．02。与正常对照组比较，miRNA-21抑制剂组miRNA-21相对表达量下降（P=0．031）、PDCD4蛋白相对表达量上调（P=0．001），miRNA-21Mimics组miRNA-21相对表达量上升（P=0．001）、PDCD4相对表达量下调（P=0．030），阴性对照组则差异无统计学意义（P值分别=0．272和0．541）。18对哈萨克族食管癌组织中16对的miRNA-21相对表达量（0．11±0．09）高于其对应的癌旁正常食管组织（0．03±0．03，P=0．001），PDCD4蛋白相对表达量（0．92±0．39）低于其对应的癌旁正常食管组织（1．57±0．80，P=0．004），且癌组织中miRNA-21相对表达水平越高，PDCD4蛋白相对表达水平越低（r=-0．538，P=O．046）。结论miRNA-21可能通过抑制PDCD4蛋白表达而促进食管鳞癌细胞Ecal09增殖，并且参与哈萨克族食管癌的发生。

Objective To investigate the role of microRNA-21 （miRNA-21） in promoting proliferation of Kazakh＇s esophageal squamous cell carcinoma （ESCC） cell line Eca109 and Kazakh＇s ESCC tissues, and its effects on the expression of programmed cell death 4（PDCD4）gene. Methods The Ecal09 ceils were divided into miRNA-21 Mimics group （ transfer sequence： 5＇- UCAACAUCAGUCUGAUAAGCUA-3＇）, miRNA-21 inhibitor group （ transfer sequence： 5＇- UAGCUUAUCAGACUGAUGUUGA-3＇）, negative control group （the transfer random sequence） and normal control group （no transfection）. The cells were seeded at 4. 5 × 10^5/well in 6-well cell culture plates. According to RNA interference technology, the cells were transfeeted with LipofectamineTM 2000. After transfection, the cell proliferation was determined by cell number counting. The expression of miRNA-21 was detected by real-time quantitative polymerase chain reaction （qRT-PCR）, and the expression of PDCD4 protein was measured by Western blot. Meanwhile, the expression of miRNA-21 and PDCD4 protein were performed in 18 pairs of Kazakh＇s ESCC and corresponding adjacent normal esophageal mucosa. Results At 48 hour after transfection, compared with normal control group, the cell number increased 36% （P= 0. 002） in miRNA-21 Mimics group, decreased 28% （P= 0. 002） in miRNA-21 inhibitor group, and did not change significantly in negative control group （P = 0. 515）. At 48 hour after transfection, the relative expression of miRNA-21 in miRNA-21 Mimics group, miRNA-21 inhibitor group, negative control group and normal controlgroup was 0. 37±0. 10, 9. 17±1. 08, 0. 74±0. 23 and 1. 04±0. 34, respectively, and the relative protein expression of PDCD4 was 1. 47± 0. 11, 0. 61 ±0.09, 0. 89 ± 0.12 and 0.79±0.02 accordingly. Compared with normal control group, the relative expression of miRNA-21 decreased （P= 0. 031） and the relative protein expression of PDCD4 up regulated （P= 0. 001） in miRNA-21 inhibitor group, the relative expression of miRNA-21 increased （P=0. 001） and the relative protein expression of PDCD4 down regulated （P= 0. 030） in miRNA-21 Mimics group, and there was no significant difference in negative control group （P=0. 272 and 0. 541）. In 16 pairs of Kazakhrs ESCC tissues, the expression of miRNA-21 was significantly higher in tumor tissues （0.11 ± 0.09） than that of corresponding adjacent esophageal normal tissues （0.03±0.03, P=0. 001）, while the relative protein expression of PDCD4 was significantly lower （0.92 ±0. 39） than that of corresponding adjacent normal esophageal tissues （1. 57 ± 0. 80, P = 0. 004）. And the higher expression of miRNA-21, the lower relative protein expression of PDCD4 （r=-0. 538, P= 0. 046）. Conclusion miRNA-21 may promoted the cell proliferation through inhibiting PDCD4 expression, which involved in the pathogenesis of Kazakh＇s ESCC.