CtBP2 Facilitated By CCNH/CDK7 Promoting Escc Invasion And Metastasis

ObjectiveTo investigate the expression of Cyclin H/Cyclin-dependent kinase 7(CCNH/CDK7) and CtBP2 in human esophageal squamous cell carcinoma(ESCC), and analyze the correlation of CtBP2, CCNH/CDK7 and invasion and metastasis of ESCC.MethodsParting One1. Immunohistochemistry was used to detect the expression of CCNH/CDK7、CtBP2 and EMT marker E-cadherin in 105 samples of ESCC tissue. To analyze the connection between CtBP2, CCNH/CDK7 and clinicopathologic data of ESCC patients, such as age, gender, tumor diameter, histological grade, metastasis and so on.2. 105 cases of ESCC patients were followed up, survival curve was calculated using the Kaplan-Meier method, and the prognosis was analyzed with multivariate Cox proportional hazards regression analysis.Parting Two1. In ESCC cells, immunoprecipitation analysis were performed to verify the interaction of CtBP2 and CCNH/CDK7.2.Western Blot analysis were used to detect the protein levels of CtBP2 in EAC109 transfected with sh-CCNH or FLAG-tagged CCNH respectively.3. The change of E-cadherin, Vimentin and N-cadherin expression as markers of EMT was evaluated by Western blot and immunofluorescence, wound-healing assays and transwell assays were performed to show change of ESCC cell migration, after ECA109 cells and TE1 cells were transfected with Flag-Ct BP2 or sh-CtBP2 respectively.4. The change of E-cadherin, Vimentin and N-cadherin expression as markers of EMT was evaluated by Western blot and immunofluorescence, the change of ESCC cell migration were showed by wound-healing assays and transwell assays, after ECA109 cells and TE1 cells were transfected with sh-CCNH、sh-CCNH and flag-CtBP2、flag-CCNH 、flag-CCNHand sh-CtBP2 respectively.Results1. Immunohistochemistry analysis showed that the expression of CtBP2 and CCNH/CDK7 in tumor tissues with metastasis were higher than that in tumor tissues without metastasis, opposite to E-cadherin expression. CtBP2 and CCNH/CDK7 expression was correlated with histological grade(P=0.000), lymph nodes metastases(P=0.005) and E-cadherin expression(P=0.004). The Kaplan-Meier survival curves showed that high CtBP2(P<0.01), CCNH/CDK7 expression related to a poor survival with statistical significance. Unvaried analysis showed that CtBP2 and CCNH/CDK7 expression was associated with poor prognosis of ESCC(P=0.010). Multivariate Cox proportional hazards regression analysis indicated that CtBP2 and CCNH/CDK7 expression was an independent prognostic marker for ESCC(P=0.005).2. Immunoprecipitation analysis were performed to verify that CtBP2 interacts with CCNH/CDK7 in ESCC tissues. Western blot analysis were used to confirm that CCNH/CDK7 could positively impact the expression of Ct BP2 in ESCC cells.3. Western blot analysis and immunofluorescence analysis showed that the silence of CtBP2 was accompanied by the upregulation of epithelial marker E-cadherin and downregulation of mesenchymal marker N-cadherin while the expression of E-cadherin and N-cadherin were in a reverse tendency in ESCC cells transfected with FLAG-tagged CtBP2. Wound-healing assays and transwell assays were performed to show the silence of CtBP2 could interfere the migration of ESCC cells.4. The silence of CCNH resulted in the upregulation of epithelial marker E-cadherin and downregulation of mesenchymal marker N-cadherin in accord with CtBP2-treated cells. The ability of CtBP2 to promote ESCC cell migration is impaired by the lack of CCNH by using wound-healing assays and transwell assays.Conclusions1. The expression of CtBP2, CCNH/CDK7 was upregulated in ESCC and CtBP2 was an independent prognostic indicator of overall survival. These data revealed that CtBP2 and CCNH/CDK7 may play a role in the oncogenesis and development of ESCC and may be a new prognostic marker for ESCC.2. It is confirmed that CCNH/CDK7 interacts with CtBP2 in ESCC cells and CCNH/CDK7 could positively impact the expression of Ct BP2 in ESCC cells.3. High expression of CtBP2 promotes ESCC cell migration and CCNH/CDK7 facilitates CtBP2 induced ESCC cell migration. These results indicate that CCNH/CDK7-CtBP2-EMT may be a potential target for treating ESCC.