环状蛋白质精氨酸甲基转移酶5在结直肠癌细胞增殖、迁移中的作用和机制

Role and mechanism of circular RNA protein arginine methyltransferase 5 in proliferation,migration of colorectal cancer cells

目的评估环状蛋白质精氨酸甲基转移酶5(circPRMT5)在结直肠癌发生、发展中的作用。方法收集2013年1月至2017年12月上海健康医学院附属嘉定区中心医院普外科行结直肠癌根治术患者96例。采用实时荧光定量PCR检测circPRMT5在结直肠癌组织中的表达水平;分析circPRMT5表达水平与结直肠癌患者年龄、性别、肿瘤最大径、肿瘤位置、病理分化、TNM分期、淋巴结转移等因素的相关性。将结直肠癌细胞株SW620和LOVO按不同的处理方式分为对照组、circPRMT5-lenti组和circPRMT5-shRNA-lenti组,检测circPRMT5表达水平对SW620和LOVO细胞活性、凋亡、线粒体膜电位、迁移的影响。采用蛋白质印迹法检测circPRMT5表达水平对上皮钙黏蛋白、Slug、神经钙黏蛋白和波形蛋白表达的影响。采用StarbaseV2.0软件预测circPRMT5的潜在靶miRNA。统计学分析采用Studentt检验、方差分析和卡方检验。结果实时荧光定量PCR结果显示,结直肠癌组织中circPRMT5表达水平高于癌旁组织(2.167±0.345比1.103±0.144),差异有统计学意义(t=26.847,P<0.01),其表达水平与肿瘤最大径、TNM分期、淋巴结转移和远处转移显著相关(χ^2=6.010、10.971、5.321、6.272,P均<0.05)。circPRMT5表达上调可以促进SW620和LOVO细胞的增殖和迁移;circPRMT5表达下调可以抑制细胞增殖,诱导细胞凋亡,且降低细胞线粒体膜电位。蛋白质印迹结果显示,与对照组相比,circPRMT5-lenti组Slug、神经钙黏蛋白和波形蛋白表达均升高(分别为1.023±0.038比2.105±0.042,1.051±0.309比2.277±0.111,1.055±0.040比2.002±0.537),上皮钙黏蛋白表达降低(2.074±0.214比0.627±0.023),差异均有统计学意义(t=31.817、22.065、14.536、9.148,P均<0.01);circPRMT5-shRNA-lenti组Slug、神经钙黏蛋白和波形蛋白表达均降低(1.023±0.038比0.585±0.023,1.051±0.309比0.616±0.043,1.055±0.040比0.537±0.022),上皮钙黏蛋白表达升高(2.074±0.214比2.756±0.148),差异均有统计学意义(t=-13.795、-14.252、-11.794、-13.116,P均<0.05)。StarbaseV2.0软件预测共有21个miRNA与circPRMT5存在潜在结合位点,并且实时荧光定量PCR检测证实SW620和LOVO细胞中miRNA4735-3p、miRNA202-3p、miRNA326、let-7i-5p、miRNA4500与circPRMT5表达水平呈负相关。结论circPRMT5是结直肠癌发生、发展中的重要促癌基因,在结直肠癌靶向药物研发中具有一定应用前景。

Objective To evaluate the role of circular RNA protein arginine methyltransferase 5 (circPRMT5) in the genesis and progression of colorectal cancer. Methods From January 2013 to December 2017, 96 patients with colorectal cancer who underwent radical resection in Department of General Surgery, Jiading District Central Hospital Affiliated to Shanghai Medical College of Health were collected. The expression of circPRMT5 in colorectal cancer tissues was examined by real-time polymerase chain reaction (RT-PCR). The correlation between circPRMT5 expression level and age, gender, tumor size, tumor location, pathological differentiation, TNM stage, lymph node metastasis of patients with colorectal cancer was analyzed. The SW620 and LOVO cells were divided into control group, circPRMT5-lenti group and circPRMT5-shRNA-lenti group according to different interventions. The effects of circPRMT5 expression level on viability, apoptosis, mitochondrial membrane potential and migration of SW620 and LOVO cells were detected. The influence of circPRMT5 expression level on E-cadherin, Slug, N-cadherin and vimentin was determined by Western blotting method. The potential target miRNA of circPRMT5 was predicted by Starbase V2.0. Student′s t test, analysis of variance and chi-square test were performed for statistical analysis. Results The results of RT-PCR showed that the expression of circPRMT5 in colorectal cancer tissues was higher than that of adjacent cancer tissues (2.167±0.345 vs. 1.103±0.144), and the difference was statistically significant (t=26.847, P<0.01). The circPRMT5 expression level was positively correlated with tumor size, TNM stage, lymph node metastasis and distant metastasis (χ^2=6.010, 10.971, 5.321 and 6.272, all P<0.05). The upregulation of circPRMT5 expression could promote proliferation and migration of SW620 and LOVO cells. The circPRMT5 downregulation could inhibit cell proliferation, induce apoptosis and decrease mitochondrial membrane potential. The results of Western blotting indicated that, compared with those of control group, the expression of Slug, N-cadherin and vimentin increased in circPRMT5-lenti group (1.023±0.038 vs. 2.105±0.042, 1.051±0.309 vs. 2.277±0.111, 1.055±0.040 vs. 2.002±0.537, respectively), however the expression of E-cadherin decreased (2.074±0.214 vs. 0.627±0.023), and the differences were statistically significant (t=31.817, 22.065, 14.536 and 9.148, all P<0.01). Compared with the control group, the expression of Slug, N-cadherin and vimentin decreased in circPRMT5-shRNA-lenti group (1.023±0.038 vs. 0.585±0.023, 1.051±0.309 vs. 0.616±0.043, 1.055±0.040 vs. 0.537±0.022), while the expression of E-cadherin increased (2.074±0.214 vs. 2.756±0.148), and the differences were statistically significant (t=-13.795,-14.252,-11.794 and -13.116, all P<0.05). A total of 21 miRNAs might have potential binding sites with circPRMT5 predicted by Starbase V2.0 software. The expression of miRNA4735-3p, miRNA202-3p, miRNA326, let-7i-5p and miRNA4500 was negatively correlated with circPRMT5 expression in both SW620 and LOVO cells confirmed by RT-PCR. Conclusion CircPRMT5 is an important oncogenic gene in the genesis and progress of colorectal cancer, and may have certain potential application prospect in the research and development for colorectal cancer.