The Mechanism Of SOX2 Reduces The Chemosensitivity Of Irinotecan In Colorectal Cancer Cells Via Regulating ABCC2

Background and ObjectionColorectal cancer is one of the most common malignancies.It is the fourth most deadly cancer worldwide.The main treatment is surgery-plus chemotherapy-based comprehensive therapy.Adjuvant treatment before surgery or after surgery or patients without surgical conditions can use chemotherapy,radiotherapy treatment,but the biggest obstacle during the chemotherapy is the chemoresistance.Studies have shown that the main reason for the decreased chemosensitivity is the presence of cancer stem cells.SOX2,as a stem cell related transcription factor,promotes chemoresistance in a variety of tumors.Previous studies have found that the silenced of SOX2 can increase the chemosensitivity of colorectal cancer cells to irinotecan,indicating that the presence of cancer stem cells is related to the chemosensitivity.Chemoresistance of cancer stem cells is mainly induced through the high expression of ATP-binding cassette transporters and anti-apoptotic gene,etc.In the preliminary experiment,ABCC2 was screened as the downstream protein of SOX2.Therefore,our study hypothesized that SOX2 affect the chemosensitivity of colorectal cancer cells to irinotecan via regulating ABCC2.Materials and methodsImmunohistochemical staining of SOX2 and ABCC2 was performed in tissue arrays and the related clinical data of 90 CRC patients were analyzed to study the expression of SOX2 and ABCC2 in colorectal cancer and its adjacent tissues,also the effect of SOX2 and ABCC2 on survival and prognosis and the correlation between SOX2 and ABCC2 expression of patients were analyzed.The background expressions of SOX2 and ABCC2 in SW480 and SW620 cells were detected by RT-qPCR and Western blot.The overexpression and silencing of SOX2 and ABCC2 were constructed in SW480 and SW620 cells,then the proliferation-toxicity test,cell apoptosis test,colony formation assay,the establishment of tumor-bearing nude mouse model and rescue experiments were carried out to study the effect of SOX2 and its downstream protein ABCC2 on the chemosensitivity of colorectal cancer cells to irinotecan.Dual luciferase reporter assay was carried out to confirmed that SOX2 binds to the promoter of ABCC2.The binding range of SOX2 and the promoter of ABCC2 was determined by constructing truncated promoter plasmid.The binding sequence of SOX2 and the promoter of ABCC2 was determined by CHIP and the dual luciferase reporter assay of mutant ABCC2 promoter,in order to study the molecular mechanism of how SOX2 regulating the expression of ABCC2.Results1.The expression of SOX2 in colorectal cancer was significantly higher than that in its adjacent tissues;The group with high expression of SOX2 in Colorectal cancer had lower differentiation,larger tumor volume,more lymph node metastasis,and advanced AJCC staging.Patients with high expression of SOX2 had worse prognosis and lower survival rate.Silenced SOX2 in colorectal cancer cells showed decreased cell viability,increased cell apoptosis rate,and the number of colony formation decreased.The survival time of nude mice treated with silenced SOX2 and irinotecan was the longest.2.The expression of ABCC2 in colorectal cancer was significantly higher than that in its adjacent tissues.The group with high expression of ABCC2 had more lymph node metastasis and advanced AJCC staging.Patients with high expression of ABCC2 had worse prognosis and lower survival rate.The expression of SOX2 and ABCC2 in colorectal cancer tissues were weakly positively correlated.ABCC2 is the downstream protein of SOX2.Silenced ABCC2 in colorectal cancer cells showed decreased cell viability,increased cell apoptosis rate,and the number of colony formation decreased.The survival time of nude mice treated with silenced ABCC2 and irinotecan was the longest.ABCC2 can rescue the phenotypic changes in cell viability,cell apoptosis rate and number of colony formation induced by SOX2.3.Luciferase activity of the upregulated SOX2 group was significantly increased;Luciferase activity of the group that truncated the site-2000/-1518 was significantly decreased.Luciferase activity of the group with mutant promoter was significantly decreased.Conclusion1.Silenced SOX2 increased the chemosensitivity to irinotecan in colorectal cancer cells.2.Silenced SOX2 increased the chemosensitivity to irinotecan in colorectal cancer cells via the down-regulation of ABCC2.3.SOX2 can bind to the promoter of ABCC2 and positively regulate the expression of ABCC2,and its binding sequence is-1593/-1583(TTACAATGCCT).