Study On The Clinical Significance And Related Oncogenic Mechanisms Of RAP1A In Colorectal Cancer

Purpose To investigate the clinical significance of RAP1A(Ras-related protein1,RAP1A)in colorectal cancer(CRC)and its related oncogenic mechanism in CRC development.Materials and Method 1.144 cases of stage II / III colorectal cancer samples were performed by immunohistochemistry to detect RAP1 A protein expression,and its clinical significance was analyzed.2.QRT-PCR was performed to detect RAP1 A expression in different CRC cell lines.Stable knock-down of RAP1 A was constructed in HT29 and SW620.The biological functions of CRC cells after knockdown of RAP1 A were detected by MTT assay,clone formation assay,flow cytometry,transwell invasion and metastasis assay.To investigate the effect of RAP1 A on the proliferation of CRC cells in vivo,we constructed the xenograft model in nude mice.3.The high-throughput transcriptome microarray and related bioinformatics analysis were used to study the downstream molecules regulated by RAP1 A and the involved signaling pathways.The downstream proteins in CRC cells was detected by QRT-PCR and western blot.4.Functional assays including MTT assay,clonogenic assay and flow cytometry were performed to verify the dominant downstream of RAP1 A in CRC cells.5.The expression of RAP1 A downstream molecular vimentin was detected by immunohistochemistry in stage II CRC tissues and its clinical significance was analyzed statistically based on the pathological data.Results 1.The expression of RAP1 A in colorectal cancer tissues was significantly correlated with the degree of tumor invasion(P <0.05).The patients with high RAP1 A expression had worse prognosis than those with low RAP1 A expression,and RAP1 A expression was an independent prognostic factor in colorectal cancer patients.RAP1 A combined with CEA testing is more helpful in the prognosis of patients with colorectal stratification.2.After RAP1 A was down-regulated by lentivirus,the capacity of proliferation,invasion,migration,anti-apoptosis and colony formation were inhibited(P <0.05).In vivo animal experiments also showed that silencing RAP1 A can inhibit the tumorigenic ability of CRC cell lines HT29 and SW620 in nude mice(P <0.05).3.The 30 genes related to PTEN signal were selected by Patharray biochip.Further confirmation assay showed that FOXO3 was significantly up-regulated after RAP1 A knockdown(P <0.05),confirming that FOXO3 might be the target protein in downstream of RAP1 A regulation.QRT-PCR and WB results showed that the expression of E-cadherin was upregulated while vimentin and N-cadherin was downregulated after knockdown of RAP1 A.4.Downstream gene validation experiments showed that after overexpression of FOXO3,biological changes were similar to those after silencing RAP1 A.The proliferation,anti-apoptosis and colony formation of HT29 and SW620 were inhibited(P <0.05).5.The expression of vimentin was significantly correlated with the degree of tumor infiltration(p <0.05).The expression of vimentin was an independent prognostic factor influencing cancer-specific overall survival and disease-free survival in stage II patients with high risk(p <0.05).Conclusion RAP1 A promotes the development of CRC through the PTEN / FOXO3 / CCND1 signaling pathway and the regulation of EMT.Targeting RAP1 A to inhibit tumor growth is expected to be a new strategy for the treatment of CRC.