The Research On Survivin Regulating Bad In Esophageal Squamous Cell Carcinoma

Objective: Survivin is a member of the inhibitor of apoptosis protein family,which involved in regulation of cell apoptosis.Survivin also takes part in tumor occurrence,development and has related to the clinical features of patients.Bad is one of the members of Bcl-2 protein family and is a pro-apoptotic regulator of apoptosis.Our previous results showed that Survivin is the most important proto-oncogene involved in the carcinogenesis and progression of esophageal cancer;By screening results in small samples of esophageal cancer,we found that the expression of survivin in the ESCC tumors was significantly lower than that of in adjacent normal tissues(P <0.05).Comparing with the expression in normal tissues,the mRNA expression of Bad in tumor tissues was low,suggestin that there was a negative correlation between the expression of Bad and survivin,but the specific mechanism and role of survivin on Bad expression in esophageal squamous cell carcinoma was not fully understood.In this study,by using various methods to regulate the expression of survivin to see whether surviving could downregulate the expression of Bad.Finally,we investigated the effect of survivin on the expression of surviving on cell biological behavior of the study.Methods: 1.Comparing with the expression of survivin ESCC tissue and in normal tissues.2.Survivin overexpression and shRNA vector,and transfection of esophageal squamous cell carcinoma cell line by electroporation method.Cells cultured in complete culture medium were used as blank control group.The total RNA and protein were collected after 48 hours.The expression of survivin was detected by RT-qPCR and Western-blotting,and the transfection efficiency was verified.And detect the expression of Survivin,Bad and Caspase-3,and verifying whether Bad was regulated by Survivin.The cell cycle and apoptosis were detected by flow cytometry.The effects of over-expression of Bad and up-regulation or down-regulation of Survivin on cell cycle and apoptosis were evaluated.3.Cell viability was measured after 48 h of treatment with YM155,Dox,VP-16,or a combination of their designated concentrations.And 48 hours after the protein collection,protein immunoblotting detection of Caspase-3,PARP to see whether YM155 and traditional chemotherapy drugs Dox and VP-16 combined effect could cause tumor cell apoptosis and increase the sensitivity of esophageal cancer cells to chemotherapy and reduce the drug resistance of these cells to Dox and VP-16.2.Colony forming ability is an important index to evaluate the malignancy degree of tumor cells.The colony formation ability of colorectal cancer cells is a kind of method to detect the survival and migration ability of tumor cells.Cultures of esophageal squamous cell carcinoma KYSE450 and KYSE510 cells were seeded in appropriate amounts in a soft agar/complete medium containing cell mixture and 24 hours later the YM155 was added to the soft agar gel top layer followed by a cell incubator For 2 to 3 weeks.When the colonies showed microscopic size of soybeans,they were stained with crystal violet.After 4 hours,the photographs were taken with the Bio-Rad gel imaging system and the number of colonies per well was counted using "Quantity One" to analyze whether YM155 inhibits Survivin activity and inhibits tumor cell growth.Results: 1.Positive expression of survivin in cancer tissues was 88% higher than the distal normal tissues,47.5%,the diffefence was statically significant(P<0.05);the expression level of Bad in cancer tissues was lower than normal tisses,with statical significance(P<0.05);There was a positive correlation between the expression of survivin and poorly differentiated in esophageal carcinoma(r = 0.412,P = 0.009),but there was no correlation between Bad and tumor differentiation.There was no relevance between surviving,Bad mRNA and other clinicopathological markers.2.Survivin overexpression vector was constructed.Survivin was up-regulated in KYSE450 and ECA109 cells after transfection with survivin overexpression vector.The expression of Bad mRNA and protein was also down-regulated.3.The expression of survivin mRNA and protein was down-regulated in KYSE450 and ECA109 cells after transfection with Survivin shRNA,while the mRNA and protein levels of Bad were also up-regulated in KYSE450 and ECA109 cells.The change of cleavedCaspase3 was also detected.Flow cytometry showed that cell cycle arrest and cell apoptosis induced by survivin.4.Here we report that YM155,a specific inhibitor of Survivin,could induce Bad-mediated apoptosis in esophageal squamous cell carcinoma cells.The combination of YM155 with Dox and VP-16 significantly sensitized KYSE450 and KYSE510 cells to chemotherapy,thereby reducing drug resistance of these cells to Dox and VP-16.In addition,YM155 inhibited the colony-forming potential of esophageal cancer cells.Conclusions: 1.In esophageal squamous cell carcinoma KYSE450 and ECA109 cells,survivin may promote the apoptosis of tumor cells through directly or indirectly binding to Bad promoter region to downregulate the expression of Bad.2.Overexpression of survivin could block the activity of Bad,inhibit apoptosis and promote cell proliferation.Survivin could be involve in oncogenesis and development of esophageal squamous cell carcinoma.3.In this study,the molecular mechanism of negative regulation of survivin overexpression in esophageal squamous cell carcinoma established a new way of thinking;The discovery of molecular regulation mechanism between survivin and Bad will provide new targets for clinical drug intervention.They can be used as new indicators of esophageal squamous cell carcinoma diagnosis and treatment.