Bioinformatics Analysis Of RAD18 In Colorectal Cancer And Its Molecular Mechanism For Promoting Invasion And Metastasis

Background and objective: Colorectal cancer is one of the common malignant tumors in China.It ranks third and second in morbidity and mortality among all tumors in world,respectively.At present,the effects of various treatments are limited,and distant metastasis is still the root cause of treatment failure.With the advent of the big data era of biological information,data mining has provided a very powerful and convenient tool for life sciences,especially tumor research.RAD18 is an E3 ubiquitin protein ligase,and because of its involvement in error-prone replication,it plays a key role in the occurrence and progression of many tumors.Despite the significance of RAD18,its function has not been reported in colorectal cancer.In this study,we investigated the role of RAD18 in colorectal cancer through three levels of in silicon,in vivo,and in vitro,and sought new potential targets for the diagnosis and treatment of colorectal cancer.Materials and methods:(1)In silicon: Data mining is performed using databases such as GEO,TCGA,Oncomine,UALCAN,HPA,STRING,and DAVID,and R language software is used to study the RAD18 gene in colorectal cancer.(2)In vivo: A total of 93 patients with colorectal cancer were collected,of which 87 samples were obtained from adjacent normal tissues,and the expression level of RAD18 was detected by immunohistochemistry.The Chi-square test was used to analyze the correlation between RAD18 and clinicopathological factors.Follow-up data were used for survival analysis and Kaplan-Meier survival curve was drawn.Log-rank test compares the differences in overall survival between high and low RAD18 expression groups.Univariate and multivariate COX regression models were used to analyze the independent prognostic factors that affect the survival of colorectal cancer.(3)In vitro: Three colorectal cancer cells HCT116,DLD-1 and SW480 were selected and stable cell lines were established using lentiviral RNA interference and c DNA plasmids.RTPCR and Western blotting experiments were used to verify the transfected cell lines,and cell proliferation CCK8,scratch,and Transwell experiments were used to verify the effects of RAD18 on cell proliferation,migration,and invasion in vitro.The possible molecular mechanism was initially explored by Immunohistochemistry and western blotting.Results:(1)In silicon: Using bioinformatics analysis,RAD18 is highly expressed in cancerous tissues in colorectal cancer and lowly expressed in normal tissues adjacent to the cancer.The difference is statistically significant.The high expression of RAD18 in colorectal cancer is associated with poor prognosis,and the difference is statistically significant.No statistically significant correlation was found between RAD18 and the clinical characteristics of colorectal cancer.RAD18 may affect its biological behavior through microtubule-associated proteins and its related signaling pathways.(2)In vivo: Through clinical cases and samples,it was verified that the expression of RAD18 in tissue samples of colorectal cancer patients was significantly higher than that in normal tissues adjacent to the cancer.Correlation analysis confirmed that RAD18 expression was positively correlated with tumor differentiation,lymph node metastasis,tumor stage,and MSH2.Kaplan-Meier survival analysis suggested that patients with high expression of RAD18 had low overall survival.Univariate and multivariate COX regression revealed that RAD18 is an independent prognostic factor for the survival of colorectal cancer patients.(3)In vitro: In cell experiments,we established RAD18 stably transfected over-and low-expressing cell lines that were verified by RT-PCR and Western Blotting experiments.CCK-8 cell proliferation experiments have not found that RAD18 has an effect on the proliferation of colorectal cancer cells,but the scratch test and Transwell test have clearly shown that RAD18 promotes the migration and invasion of colorectal cancer cells.EMT-related proteins have been validated in cells by Western Blotting experiments and have been validated by RT-PCR in patient tissues.The above conclusions were further verified in rescue experiments.Conclusion: Through three levels of In silicon,In vivo and In vitro,we confirm that RAD18 promotes the migration and invasion of colorectal cancer cells through the EMT signaling pathway,thus leading to the metastasis of colorectal cancer.Our study suggests that RAD18 is a novel prognostic biomarker that may be a potential target for future treatment of colorectal cancer.