The Mechanism Of RBBP6 On Modulating The Invasion And Metastasis Of Colorectal Cancer By Regulating Epithelial-mesenchymal Transition

Colorectal cancer(CRC)is one common malignant tumor with high incidence and mortality.The recurrence and metastasis of colorectal cancer is multi-step process of various factors and genes.Epithelial-mesenchymal transition(EMT)is a key step in cancer metastasis.EMT is a biological process that epithelial cells transited to cells with mesenchymal characters.E-cadherin is an important epithelia marker,and its loss is a significant symbol during the process of EMT.RBBP6(Retinoblastoma Binding Protein 6)is involved in the development of lung cancer,breast cancer and cervical cancer,but the role in colorectal cancer has not yet been reported.In our previous study,the expression of RBBP6 was gradually up-regulated in the normal colorectal epithelial-adenoma-adenocarcinoma of the colorectal.The loss of E-cadherin was observed in colorectal cancer tissues with high expression of RBBP6,suggesting that RBBP6 may promote the occurrence of EMT and participate in the metastasis process of cancer cells,but the specific mechanism is not yet clear.Part 1.Expression of RBBP6 in colorectal cancer and its correlation with clinicopathological featuresObjective: To detect the expression of RBBP6 in colorectal cancer tissue,and to explore the correlation between the expression level and clinicopathological features.Methods: Immunohistochemical staining was used to detect the expression of RBBP6 in colorectal cancer tissues,and the correlation between the expression level and clinicopathological characteristics of colorectal cancer was analyzed statistically.Results: Compared with adjacent tissues,the positive expression rate of RBBP6 in colorectal cancer tissues was significantly higher than that in adjacent tissues(57.2% vs.34.4%),which was statistically significant(P < 0.01).The expression of RBBP6 was related with tumor stage(P = 0.006),histological grade(P = 0.036),distant metastasis(P = 0.018).The COX regression model showed that RBBP6 was an independent prognostic factor(P = 0.012).Conclusion: The expression of RBBP6 in colorectal cancer tissue is elevated and may be associated with the invasion and metastasis of colorectal cancer.Part 2.The effect of RBBP6 on the invasion and metastasis of colorectal cancerObjective: To investigate the effect of RBBP6 on the invasion and metastasis of colorectal cancer.Methods: Western blot were used to detect the difference in expression of RBBP6 in colorectal cancer cell lines.RBBP6 knockdown and overexpressed viral vectors were constructed to infect HT29 and RKO cells respectively,and stable cell lines were obtained.The effects of RBBP6 expression on the invasion and migration of HT29 and RKO cells were detected by cell scratch and transwell test.Results: RBBP6 was highly expressed in high-invasive cell lines,such as HT29,SW620,Caco2 and LoVo,while it was low in RKO,HT15,SW480 and other low-invasive cell lines.After overexpression of RBBP6,the invasion and migration ability of RKO was enhanced.On the contrary,the invasion and migration of HT29 was weakened after RBBP6 knockdown.Conclusion: RBBP6 can enhance the invasion and migration of colorectal cancer cells.Part 3.The mechanism of RBBP6 to promote the invasion and metastasis of colorectal cancerObjective: To investigate the effect of RBBP6 on the invasion and metastasis of colorectal cancer.Methods: Immunohistochemical staining and Western blot assays were used to detect and analyze the correlation between the expression of Ecadherin,N-cadherin and p-p65 and RBBP6 in colorectal cancer tissues.The effects of RBBP6 on the activation of the NF-κB pathway were detected.Results: In colorectal cancer tissues,the expression of E-cadherin was negatively correlated with the expression of RBBP6,while the expression of N-cadherin and NF-κB signaling pathway was positively correlated with the expression of RBBP6.RBBP6 enhanced p65 nuclear translocation and N-cadherin expression,decreased E-cadherin expression and promoted cell EMT and cell metastasis.Either exogenous IκBα-M or NF-κB inhibitor BAY11-7082 treatment dramatically abolished the effects of RBBP6-overexpression on CRC cell migration and invasion,whereas the overexpression of p65 restored the effects of RBBP6-sh on CRC cell migration and invasion.Conclusion: RBBP6 induces EMT and promotes the invasion and metastasis of colorectal cancer by activating NF-κB signaling pathway..