食管鳞状细胞癌中miR-888-3p表达及其对癌细胞表型影响

Expression of microRNA-888-3p in esophageal squamous cell carcinoma and its effect on cancer cell phenotype

目的探讨微小RNA(miRNA,miR)-888-3p在食管鳞状细胞癌(ESCC)中表达及对细胞恶性表型影响及其机制。方法选取2016年3月至2019年4月白求恩医院行手术切除治疗的68例ESCC和癌旁组织,采用定量聚合酶链反应(qPCR)检测ESCC和癌旁组织以及细胞中miR-888-3p表达,将抗miR-NC、抗miR-888-3p转入细胞,分别记为抑制miR-NC组、抑制miR-888-3p组,噻唑蓝(MTT)检测细胞增殖,流式细胞术检测细胞周期和凋亡;生物信息学预测miR-888-3p和程序性死亡蛋白5(PDCD5)靶向关系,荧光素酶报告实验验证两者靶向关系;蛋白质印迹法(Western blot)检测细胞中PDCD5及其下游蛋白p53、B细胞淋巴瘤/白血病-2相关X蛋白(bax)、半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3和B细胞淋巴瘤/白血病-2(bcl-2)表达。用SPSS 17.0软件进行统计学分析,两组间比较采用t检验,多组间比较采用LSD-t检验。结果食管鳞状细胞癌组织中miR-888-3p表达较癌旁组织显著增加(4.57±3.27、1.28±0.82,t=8.047,P<0.01);上皮细胞-18(EC-18)、上皮细胞-1(EC-1)、上皮细胞-109(EC-109)中miR-888-3p表达较正常食管上皮细胞NEEC显著增加(分别为1.74±0.19、2.56±0.27、2.85±0.29、1.06±0.11,t=8.047,F=38.524,P<0.01),差异有统计学意义;与抑制miR-NC组相比,抑制miR-888-3p后EC-1和EC109细胞增殖显著降低(分别为0.48±0.05、0.35±0.04,t=3.517,P<0.05;0.71±0.07、0.51±0.05,t=4.027,P<0.05;和0.49±0.05、0.33±0.03,t=4.753,P<0.05;0.74±0.07、0.47±0.05,t=5.436,P<0.05),细胞周期阻滞在G2/M期(EC-1分别为15.54±0.45、27.68±0.51和EC109分别为9.52±0.24、22.25±0.31),细胞凋亡率显著增加(EC-1分别为4.35±0.87、18.59±1.92和EC109分别为4.26±0.83、15.34±0.83,t=11.701、8.327,P<0.05);PDCD5是miR-888-3p靶基因,抑制miR-888-3p可显著升高细胞中PDCD5及其下游蛋白p53、bax、Caspase-3表达,降低bcl-2表达(PDCD5分别为0.44±0.04、0.97±0.09;p53分别为0.42±0.04、0.95±0.09;bax分别为0.40±0.04、0.92±0.10;Caspase-3分别为0.34±0.03、0.94±0.08;bcl-2分别为0.79±0.08、0.33±0.03,t=9.321、9.321、8.362、12.163、9.325,P<0.01),差异有统计学意义。结论miR-888-3p在食管鳞状细胞癌中高表达,抑制其表达可通过上调PDCD5激活p53依赖凋亡信号途径,抑制细胞增殖并诱导细胞凋亡。

Objective To investigate the expression of microRNA(miRNA,miR)-888-3p in esophageal squamous cell carcinoma(ESCC),its influence on malignant phenotype and its potential mechanism.Methods Sixty-eight cases of ESCC and adjacent tissues were selected from March 2016 to April 2019 at Bethune Hospital for surgical resection.Quantitative polymerase chain reaction(qPCR)was used to detect the expression of miR-888-3p in ESCC and adjacent tissues and cells by qPCR.Anti-miR-nc and anti-miR-888-3p were transferred into cells,which were recorded as miR-nc inhibition group and miR-888-3p inhibition group,respectively.Methyl thiazol tetrazolium(MTT)assay was used to detect cell proliferation,and flow cytometry was used to detect cell cycle and apoptosis.Bioinformatics predicted the targeting relationship between miR-888-3p and programmed cell death 5(PDCD5),and luciferase reports verified the targeting relationship between the two.The expression of PDCD5 and its downstream proteins p53,B cell lymphoma/leukemia-2 associated X protein(bax),cysteinyl aspartate-specific protease(Caspase)-3 and B cell lymphoma/leukemia-2(bcl-2)was detected by Western blotting.The SPSS 17.0 software was used for statistical analysis,t test was used for comparison between two groups,and LSD-t test was used for comparison between multiple groups.Results The expression levels of miR-888-3p in ESCC tissues were significantly higher than those in the adjacent tissues(4.57±3.27 and 1.28±0.82,t=8.047,P<0.01).and those in cells EC-18,EC-1 and EC109 were significantly higher than those in the normal esophageal epithelial cells(1.74±0.19,2.56±0.27,2.85±0.29 and 1.06±0.11,respectively,t=8.047,F=38.524,P<0.01).Compared with miR-nc group,the proliferation of EC-1 and EC109 cells was significantly decreased after miR-888-3p inhibition(0.48±0.05,0.35±0.04,t=3.517,P<0.05;0.71±0.07,0.51±0.05,t=4.027,P<0.05;and 0.49±0.05,0.33±0.03,t=4.753,P<0.05;0.74±0.07,0.47±0.05,t=5.436,P<0.05),and cell cycle arrest was observed in G2/M phase(EC-1:15.54±0.45,27.68±0.51 and EC109:9.52±0.24,22.25±0.31,t=30.916,56.241,P<0.05).Apoptosis was significantly increased(EC-1:4.35±0.87,18.59±1.92 and EC109:4.26±0.83,15.34±0.83,t=11.701,8.327,P<0.05).PDCD5 is the target gene of miR-888-3p.Inhibition of miR-888-3p significantly increased the expression of PDCD5 and its downstream proteins p53,bax and caspase-3,and decreased the expression of bcl-2(PDCD5:0.44±0.04,0.97±0.09;p53:0.42±0.04,0.95±0.09;bax:0.40±0.04,0.92±0.10;Caspase-3:0.34±0.03,0.94±0.08;bcl-2:0.79±0.08,0.33±0.03,t=9.321,9.321,8.362,12.163,9.325,P<0.01).Conclusion MiR-888-3p is highly expressed in ESCC,and inhibition of miR-888-3p expression can activate p53-dependent apoptotic signaling pathway by up-regulating PDCD5,inhibit cell proliferation and induce cell apoptosis.