| Objective:Small ubiquitin-like modifier(SUMO)was a class of peptides similar to ubiquitin,which participated in the important post-translational modification of proteins(SUMOylation)in cells.It has been demonstrated to play a crucial role on tumorigenesis.In this study,we preliminarily explored the role of small ubiquitin-related modified protein 2(SUMO2)in the development of liver cancer,and provided data as marker for the early diagnosis and prognosis of liver cancer.Methods:1.The public databases of TCGA(50 cases of liver cancer adjacent tissues and371 cases of liver cancer tissues)and GEO(52 cases of liver cancer adjacent tissues and 115 cases of liver cancer tissues)were applied for bioinformatics analysis of the differential expression of SUMO2 in liver cancer tissues and adjacent tissues.The clinical data from liver cancer patients was analized with Kaplan-Meier method(www.kmplot.com)in order to evaluate the relationship between SUMO2 expression and patient prognosis.2.The expression of SUMO2 in cancerous tissues and adjacent tumors was conducted by immunohistochemical staining(IHC)from 70 patients with liver cancer;the correlation between expression of SUMO2 protein and overall survival of liver cancer was evaluated by Kaplan-Meier method.The multi-factor including gender,age,degree of differentiation,tumor size,T stage,and the presence of lymph node metastasis and vascular invasion were analized by Multivariate Cox risk regression model and Chi-square test(Fisher test),in which may affect the survival and prognosis of patients with liver cancer,relationship between pathological parameters and SUMO2 protein expression.Finally,ROC curve was used to detect the diagnostic efficiency of SUMO2.3.Two hepatocellular carcinoma cell lines,SMMC-7721 and Bel-7404 were constructed with si-SUMO2 for cytology experiments in vitro.4.The cell proliferation was checked by CCK-8 assay and the ability of colony formation was assessed by soft agar assays in SUMO2 knockdown cell lines respectively.5.Scratch and transwell invasion assay were performed to detect the ability of migration and invasion from SUMO2 knockdown cell lines,respectively.6.Western bolt was performed to detect the inhibition of SUMO2 protein expression,and its effect on the expression of downstream gene Ki67 protein.7.The expression of SUMO2 protein,vascular endothelial growth factor(VEGF)and matrix metalloproteinase 9(MMP-9)were detected by RT-qPCR and Western bolt respectively.8.Cell experiments in vitro were carried out for three times independently,experiments data were expressed as means ± standard deviation.One-way analysis of variance(ANOVA)was used for the comparison between different experimental groups,and p<0.05 indicated a statistically significant difference.Results:1.Bioinformatics analysis from public databases TCGA and GEO showed that the expression level of SUMO2 m RNA in liver cancer tissues was significantly higher than that in adjacent tissues(p<0.001 and p<0.001);the results from clinical data of364 patients with liver cancer revealed that the overall survival time of patients with high expression of SUMO2 was shorter than that of the group with low expression(p<0.05).2.45 samples of SUMO2 protein were highly expressed in 70 hepatocellular carcinoma tissue samples,which were mainly localized in the nucleus with a high rate of 64.3%(45/70);Meanwhile,the high rate of SUMO2 protein in the matched adjacent tissue was only 20.0%(14/70),indicating that the expression level of SUMO2 protein in liver cancer tissues was significantly higher than that in adjacent tissues(p<0.05);Compared with the overall survival time of liver cancer patients with low expression of SUMO2,the high expression group was shorter(23.0 months vs 61.0 months,p <0.001).3.The results from multi-factor and expression of SUMO2 protein in the 70 patients suggested that the presence of lymph node metastasis and vascular invasion and SUMO2 protein expression were independent prognostic factors of liver cancer patients.Moreover,the high expression of SUMO2 protein was closely related to lymph node metastasis(85.3%)and vascular invasion(87.5%),and there was a high mortality rate(82.0%)with no significant correlation with other clinicopathological parameters.4.The expression of SUMO2 in cancer tissues provided medium diagnostic value and statistically significant by ROC curve.However,the expression in paracancer showed low diagnostic value and no statistical significance.5.The proliferation ability of SUMO2 knockdown cell lines was significantly inhibited with time-dependent of 24 h,48h,and 72 h compared with the control group(Con)and negative control group(NC).6.The results of the plate clone experiment showed that the down-regulation of SUMO2 expression could lead to the decrease of the clonal formation ability of liver cancer cells after two weeks.7.The results of Western Bolt indicated that down-regulation of SUMO2 expression could lead to significant decrease of Ki67 expression level in liver cancer cells.8.The results of the scratch test showed that down-regulation of SUMO2 expression could lead to a decrease in the wound healing rate of liver cancer cells from 0 to 24 hours,that is,a decrease in the migration ability of liver cancer cells.The results of Transwell invasion showed that down-regulation of SUMO2 expression could lead to a decrease in the number of HCC cells invading and penetrating the membrane,namely,the invasion ability of HCC cells decreased.9.Down-regulating the expression of SUMO2 could lead to a decrease in the expression of VEGF and MMP-9 in liver cancer cells.Conclusions:1.The expression of SUMO2 in liver cancer tissues is significantly higher than that in adjacent tissues.2.The overall survival time of liver cancer patients with high expression of SUMO2 was lower than that of liver cancer patients with low expression of SUMO2.3.The high expression of SUMO2 protein in liver cancer tissues was closely related to lymph node metastasis and vascular invasion with a high mortality rate.4.Down-regulating the expression of SUMO2 protein significantly inhibited the proliferation,migration and invasion of liver cancer cells,which might be related to the decrease expression of VEGF and MMP-9. |
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