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

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.

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.

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