CIZ1基因通过调控MEK-ERK1-2信号通路影响视网膜母细胞瘤细胞增殖和凋亡的实验研究

doi:10.3877/cma.j.issn.2095-2007.2020.04.002

目的

探讨锌指蛋白(CIZ)1基因影响视网膜母细胞瘤(RB)细胞增殖和凋亡的机制。

方法

RB组织芯片购自西安艾丽娜生物科技有限公司，人神经母细胞瘤细胞系Y79购自美国ATCC公司。应用免疫组化方法检测CIZ1基因在RB肿瘤组织和正常视网膜组织中的表达情况；应用荧光定量聚合酶链式反应(PCR)和Western blot方法检测CIZ1基因在RB细胞中信使核糖核酸(mRNA)和蛋白的表达水平。选取RB细胞系Y79细胞，采用数字表法随机分为对照组、干扰CIZ1基因组(shCIZ1)-1及shCIZ1-2等3组。采用不同的慢病毒分别感染阴性对照组和两组shCIZ1基因组细胞。应用荧光定量PCR及Western blot方法检测敲低效率；应用细胞增殖测量试剂盒(CCK8)方法检测细胞的生长、半胱氨酸天冬氨酸蛋白酶(caspase)3-7的活性和细胞凋亡相关蛋白表达的情况；通过Western blot方法检测丝裂原活化蛋白激酶激酶(MEK)-细胞外调节蛋白激酶(ERK)1-2信号通路相关蛋白的表达变化。CIZ1的基因表达量、蛋白表达水平、RB细胞的增殖倍数、Caspase3-7、MEK、p-MEK、ERK及p-ERK蛋白的表达水平，采用单样本K-S拟合优度法进行正态性检验，符合正态分布者以均数±标准差进行描述。多组样本间不同组间均数的比较采用重复测量资料的方差分析，采用LSD检验进行两两比较。

结果

与正常视网膜组织相比，CIZ1基因在RB肿瘤组织中的表达水平显著上调；在RB细胞中，CIZ1基因mRNA水平及蛋白水平均显著高于正常人视网膜色素上皮细胞(ARPE)，Y79细胞、人神经母细胞瘤细胞系(WERI-Rb)-1细胞中mRNA相对表达量分别为(0.061±0.001)、(0.041±0.001)，与ARPE19细胞中mRNA相对表达量(0.025±0.001)相比，差异均具有统计学意义(t＝41.58，18.68；P<0.05)；慢病毒干扰CIZ1基因的表达可以显著降低CIZ1的mRNA与蛋白表达水平，shCIZ1-1组和shCIZ1-2 CIZ1组mRNA的相对表达量分别为(0.015±0.008)和(0.008±0.003)，与对照组mRNA的相对表达量(0.032±0.003)相比，差异均具有统计学意义(t＝3.72，5.357；P<0.05)；敲低CIZ1基因可以显著抑制RB细胞的增殖，也可以抑制增殖相关基因即细胞周期蛋白(Cyclin)D1蛋白的表达，与对照组相比差异有统计学意义(t＝21.18，21.80；P<0.05)；同时还可以抑制增殖相关基因Cyclin E1蛋白的表达，与对照组相比差异有统计学意义(t＝17.26，16.41；P<0.05)。敲低CIZ1可以显著增强细胞凋亡相关蛋白Caspase3-7的活性，上调Caspase-3和Caspase-7蛋白的表达。Western blot检测敲低CIZ1基因对MEK-ERK1-2信号通路影响的结果显示，CIZ1下调可以显著抑制磷酸化的MEK的表达水平，与对照组相比差异具有统计学意义(t＝3.925，8.461；P<0.05)；CIZ1下调可抑制ERK1-2蛋白的磷酸化水平，与对照组相比差异具有统计学意义(t＝14.12，28.36；P<0.05)。

结论

敲低CIZ1基因可以通过抑制MEK-ERK1-2信号通路抑制RB的生长。

关键词: 视网膜母细胞瘤, CIZ1, 增殖, 凋亡, MEK-ERK1-2信号通路

Objective

To investigate the effect of CIZ1 gene on the proliferation of retinoblastoma cells and exploring the underlying mechanism.

Methods

Retinoblastoma tissue chip was purchased from Xi′an Alina Biotechnology Co., Ltd., and human neuroblastoma cell line Y79 was purchased from ATCC. Immunohistochemistry was used to detect the expression of CIZ1 gene in tumor tissue of retinoblastoma and normal retinal tissue. mRNA and protein expression levels of CIZ1 in retinoblastoma cells were detected by fluorescence quantitative PCR and Western blot. Retinoblastoma cell line Y79 cells were selected and infected with the negative control group and two lentiviruses interfering with CIZ1 gene, respectively. The experiment was divided into three groups: nine samples in total, control group, shCIZ1-1 group and shCIZ1-2 group. Fluorescence quantitative PCR and Western blot were used to detect knock down efficiency. Then, CCK8 assay kit was used to detect cell growth, and apoptosis was detected by Caspase-3 to 7 activity and expression of apoptosis-related proteins. Finally, Western blot was used to detect the expression of MEK-ERK1-2 signaling pathway related proteins.The gene and protein expression level CIZ1, RB cell proliferation ability, Caspase-3 to7, MEK, p-MEK, ERK, p-ERK protein expression levels were statistically described as mean±standard deviation. The normality test uses the one-sample K-S goodness-of-fit method. The comparison of the mean between different groups among multiple samples adopts repeated measures analysis of variance, and LSD test was used for pairwise comparison.

Results

Compared with normal retinal tissue, the expression level of CIZ1 gene in tumor tissue of retinoblastoma was significantly up-regulated. The mRNA level and protein level of CIZ1 were highly expressed in retinoblastoma cells, and the relative mRNA expression levels in Y79, WERI-Rb1 and ARPE19 cells were (0.061±0.001), (0.041±0.001), (0.025±0.001); the differences were statistically significant (t=41.58, 18.68; P<0.05). Compared with the control group, the mRNA and protein expression levels of CIZ1 were significantly decreased by interfering with CIZ1. The relative mRNA expression levels of CIZ1 in the control group, shCIZ1-1 group and shCIZ1-2 group were (0.032±0.002), (0.015±0.007), (0.008±0.003); the differences were statistically significant (t=3.72, 5.357; P<0.05). Knocking down CIZ1 significantly inhibited the proliferation of retinoblastoma cells, down-regulated the expression of Cyclin D1, compared with the control group; the difference was statistically significant (t=21.18, 21.8; P<0.05). And down-regulated the expression of Cyclin E1, compared with the control group; the difference was statistically significant (t=17.26, 16.41; P<0.05). Knockdown of CIZ1 significantly enhanced Caspase-3 to 7 activity and upregulated the expression of Caspase-3 and Caspase-7. Finally, Western blot was used to detect the effect of CIZ1 knockdown on the MEK-ERK1-2 signaling pathway. The results showed that, compared with the control group, down-regulation of CIZ1 significantly inhibited the expression levels of phosphorylated MEK (t=3.925, 8.461; P<0.05) and ERK1-2 proteins (t=14.12, 28.36; P<0.05); the difference was statistically significant.

Conclusions

Knockdown of CIZ1 inhibits the growth of retinoblastoma cells by inhibiting the MEK-ERK1-2 signaling pathway.

Key words: Retinoblastoma, CIZ1, Proliferation, Apoptosis, MEK-ERK1-2 signaling pathway

图1 慢病毒转染后细胞周期素依赖性激酶抑制因子1A相互作用的锌指蛋白(CIZ1)基因在视网膜母细胞瘤组织的免疫组化染色显微结构图和在视网膜母细胞瘤细胞中的基因表达量柱状图及蛋白条带图　图A和图B示免疫组化法检测CIZ1基因在视网膜母细胞瘤肿瘤组织和正常视网膜组织中的表达情况(CIZ1免疫组化染色，×20，标尺＝200 μm)；图C示荧光定量聚合酶链式反应检测CIZ1基因信使核糖核酸水平的相对表达量；图D示Western blot方法检测CIZ1蛋白水平的蛋白条带图。注：*与ARPE19细胞组比较，差异有统计学意义；CIZ1，细胞周期素依赖性激酶抑制因子1A相互作用的锌指蛋白

图2 慢病毒转染后细胞周期素依赖性激酶抑制因子1A相互作用的锌指蛋白干扰慢病毒感染细胞后的显微结构图　图A~图C示不同病毒载体干扰细胞后的荧光显微镜观察结果(×10，标尺＝100 μm)；图D~图F示不同病毒载体干扰细胞后的光学显微镜观察结果(×100，标尺＝100 μm)

图5 慢病毒转染后细胞周期素依赖性激酶抑制因子1A相互作用的CIZ1后Y79细胞凋亡相关蛋白的活性变化柱状图、蛋白条带图及差异柱状图　图5A示酶活性方法检测凋亡相关蛋白Caspase3-7活性；图5B示不同干扰组间Caspase3、Caspase7蛋白的条带灰度比较柱状图；图5C示Western blot方法检测凋亡相关蛋白Caspase3、Caspase7的蛋白条带图。注：*与对照组比较，差异有统计学意义；CIZ1，细胞周期素依赖性激酶抑制因子1A相互作用的锌指蛋白

图6 干扰CIZ1后MEK/ERK1/2信号通路相关蛋白的条带灰度比值柱状图及蛋白条带图　图6A示不同干扰组间丝裂原活化蛋白激酶激酶、p-丝裂原活化蛋白激酶激酶、细胞外调节蛋白激酶、p-细胞外调节蛋白激酶蛋白的条带灰度比值柱状图；图6B示Western blot方法检测丝裂原活化蛋白激酶激酶-细胞外调节蛋白激酶1-2信号通路相关蛋白的条带结果。注：*与对照组比较，差异有统计学意义；CIZ1，细胞周期素依赖性激酶抑制因子1A相互作用的锌指蛋白

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Effect of CIZ1 gene on the proliferation and apoptosis of Y79 cells through MEK-ERK1-2 signaling pathway

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Effect of CIZ1 gene on the proliferation and apoptosis of Y79 cells through MEK-ERK1-2 signaling pathway