miR-122调控PDK4表达对食管癌增殖和凋亡影响的实验研究

Experimental study of miR-122 regulating PDK4 on proliferation and apoptosis of esophageal cancer

目的探讨微小RNA122(miR-122)对食管癌细胞增殖、凋亡的影响及可能机制。方法向食管癌Eca-109细胞转染miR-122 mimics(miR-122 mimics组)和阴性对照质粒(miR-NC组),另设未处理的对照组。实时荧光定量(qPCR)法检测miR-122表达。MTT和流式细胞术检测细胞增殖、凋亡,Western blotting和qPCR检测PDK4表达。双荧光素酶报告实验评价miR-122与PDK4的靶向调控作用。结果 Eca-109、KYSE-30、TE-1细胞中miR-122表达水平分别为0. 46±0. 05、0. 85±0. 10、0. 83±0. 07,均低于正常食管上皮细胞HEEC的1. 00±0. 08,差异有统计学意义(P<0. 05)。miR-122 mimics组miR-122表达水平为16. 33±0. 88,均高于对照组和miR-NC组,差异有统计学意义(P<0. 05)。miR-122 mimics组24、48、72 h的A值分别为0. 389±0. 016、0. 590±0. 019、0. 692±0. 020,低于对照组和miR-NC组,差异有统计学意义(P<0. 05)。miR-122 mimics组转染48 h的凋亡率为(32. 11±5. 71)%,高于miR-NC组和对照组,差异有统计学意义(P<0. 05)。miR-122 mimics组PDK4mRNA表达水平为0. 132±0. 044,低于对照组的1. 0±0. 017和miR-NC组的1. 025±0. 058,差异有统计学意义(P<0. 05)。miR-122 mimics组PDK4蛋白表达水平为0. 217±0. 071,低于对照组的0. 853±0. 035和miR-NC组的0. 830±0. 015,差异有统计学意义(P<0. 05)。miR-122可抑制野生型PDK4 3’UTR报告基因载体的荧光素酶活性,而对突变型PDK4 3’UTR的荧光素酶活性无影响。结论上调miR-122表达能靶向抑制PDK4表达,进而抑制食管癌细胞增殖和促进凋亡。

Objective To investigate the effect of microRNA 122(miR-122)on the proliferation and apoptosis of esophageal cancer cells and its possible molecular mechanism.Methods The Eca-109 cells of esophageal cancer were transfected with miR-122 mimics(miR-122 mimics group)and the negative control plasmid(miR-NC group),and an untreated control group was set.The expression of miR-122 was detected by real-time fluorescence quantification(qPCR).Cell proliferation and apoptosis were detected by MTT and flow cytometry,and the expression of PDK4 was detected by Western blotting and qPCR.Dual luciferase reporter assay was used to evaluate the regulatory effect of miR-122 on PDK4.Results The expression levels of miR-122 in Eca-109,KYSE-30 and TE-1 cells were 0.46±0.05,0.85±0.10,and 0.83±0.07,respectively,which were all lower than 1.00±0.08 of HEEC,and the differences were statistically significant(P<0.05).The A values of 24,48 and 72 hours in miR-122 mimics group were 0.389±0.016,0.590±0.019 and 0.692±0.020,respectively,which were lower than those in the control group and miR-NC group(P<0.05).The apoptosis rate of miR-122 mimics group was(32.11±5.71)%,which was higher than that of miR-NC group and control group(P<0.05).The expression of PDK4 mRNA in miR-122 mimics group was 0.132±0.044,lower than that in control group and miR-NC group(P<0.05).The expression of PDK4 protein in miR-122 mimics group was 0.217±0.071,lower than that in the control group and miR-NC group(P<0.05).MiR-122 inhibited the luciferase activity of wild-type PDK43’UTR reporter gene vector,but had no effect on the luciferase activity of mutant-type PDK43’UTR.Conclusion Up-regulation of miR-122 expression can inhibit PDK4 in a targeted manner,thereby inhibiting proliferation and promoting apoptosis of esophageal cancer cells.