GOLPH3 Overexpression Promotes The Resistance Of HT29 Colon Cancer Cell To Cisplatin By Activating Cell Signaling

Objective: The treatment of colon cancer includes important method—chemotherapy,which often consist of platinum drugs.However,chemotherapy resistance can affect the efficacy and prognosis of colon cancer,and its mechanism is related to abnormal gene expression.Golgi phosphorylated protein 3(GOLPH3)is overexpressed in colon cancer tissue,which can promote the proliferation of colon cancer cells and associate with poor prognosis.At present,the relationship between the GOLPH3 gene overexpression and the resistance to platinum drugs in colon cancer is still unknown.In this study,to explore the effect of GOLPH3 gene overexpression on cisplatin resistance and its mechanism in colon cancer cells,the GOLPH3 expression was silenced with si RNA interference technology in colon cancer cell experiment in vitro and animal model by cisplatin treatment.Methods: 1.The cell groups: 1.1 Selection of Cisplatin concentration: HT29 colon cancer cell was divided into four groups according to different cisplatin concentrations of 0,2.5,5,10 ?M for follow-up experiments of cisplatin concentration,which is on the basis of the related protein expressions by Western blot detection.1.2 Experimental grouping:(1)control group: HT29 human colon cancer cell;(2)transfection group: GOLPH3 gene silencing of HT29 cells by si RNA;(3)experimental group 1: HT29 cells treated by Cisplatin;(4)the experimental group 2: HT29 si RNA-GOLPH3 transfected cells treated by Cisplatin;(5)experimental group 3: HT29 cells treated with Cisplatin and ERK1/2 inhibitor PD98059.2.Cell transfection:The expression of GOLPH3 gene in HT29 colon cancer cellwas silenced by si-RNA interference technique.RT-PCR and Western blot were used to detect the silencing effect of HT29 cell after transfected with si RNA-GOLPH3.3.The colon cancer cells were cultured in the medium for 24 h,which containing the corresponding concentration of cisplatin respectively.4.Cell proliferation and tumor formation were detected by MTT assay,Plate Clone Formation assay and Tumorsphere Formation Assays.Annexin V-FITC/PI flow cytometry was used to detect the apoptosis of each group.5.Western blot was used to detect the expression of GOLPH3,P-gp,?-catenin,ERK1/2 and p ERK1/2 protein in each group.6.16 nude mice were randomly divided into 4 groups,4 mice in each group,control group,transfection group,experimental group 1 and experimental group 2 respectively.The corresponding colon cancer cells were injected subcutaneously into axillary tissue.The tumor formation in nude mice was observed within 30 days.7.To compare the differences of proliferation,tumorigenesis,apoptosis and expression of corresponding proteins in each cell group in vitro,and calculate the difference of the volume of axillary metastatic tumor in nude mice,the correlation and mechanism of GOLPH3 gene overpression and the resistance to platinum drugs in colon cancer cell were explored.Results: 1.Transfection effect of HT29 colon cancer cell.1.1 Detection of the GOLPH3 expression: compared with the control group,the relative expression of GOLPH3 m RNA in the transfected group was significantly lower,the difference was statistically significant(P<0.01).Moreover,the results of Western Blot showed that the expression of GOLPH3 protein in the transfected group was significantly lower than that in the control group(P<0.01).1.2 MTT assay was used to detect the proliferation of HT29 cells: the OD490 value of the transfection group was significantly lower than control group(P<0.01).Moreover,the colony number of the transfected group was significantly lower than the control group in plate colony formation assay(P<0.05).1.3 Annexin V-FITC/PI flow cytometry was used to detect the apoptosis of HT29 cells: the cell apoptosis rate of the transfection group was significantly higher than the control group(P<0.01).2.GOLPH3 gene involved in the regulation of resistance to cisplatin chemotherapy in HT29 colon cancer cell 2.1 With the increase of the cisplatin concentration,the protein expression of P-gp,GOLPH3,p ERK1/2,?-catenin up-regulated synchronously in HT29 colon cancer cell.Compared with the control group(0?M cisplatin),the expression of the related protein was most significantly different in the group of 10?M cisplatin concentration(P<0.01),therefore,10?M cisplatin intervention concentration was selected in the next tests.2.2 Under cisplatin treatment,the results of MTT,Clone Formation and Tumorsphere Formation Assays showed that the OD490 value,the number of colony count and tumorsphere number in experimental group 2 were significantly lower than those in experimental group 1 respectively,all the difference were statistically significant(P<0.001).2.3 Under cisplatin treatment,Annexin V-FITC/PI flow cytometry showed that the apoptosis rate of the experimental group 2 was significantly higher than the experimental group 1,and the difference was statistically significant(P<0.001).3.The mechanism of GOLPH3 gene involved in HT29 cell resistance to cisplatin chemotherapy 3.1 The expression of GOLPH3,P-gp,p ERK1/2,?-catenin in experimental group 2 was significantly lower than that in experimental group 1,the difference was statistically significant(P<0.01).3.2 The expression of ?-catenin and P-gp in experimental group 3 was significantly lower than that in experimental group 1,the difference was statistically significant(P<0.01).4.Silencing GOLPH3 expression increases the sensitivity of HT29 cells to cisplatin in animal tumorigenesis experiment.Termination of the transplanted tumor in nude mice,the volume of the axillarysubcutaneous tumor of the si RNA group,the experimental group 1 and the experimental group 2 were significantly lower than the control group,the difference was statistically significant(P<0.05).In the experimental group 2,the tumor volume of the nude mice was lower than that of the experimental group 1 in the same period after 15 days,the difference was statistically significant(P<0.001).Conclusion: 1.Overexpression of GOLPH3 may be involved in the resistance of HT29 colon cancer cell to cisplatin chemotherapy;2.The overexpression of GOLPH3 can promote the resistance of HT29 colon cancer cell to cisplatin by activating Wnt/?-catenin and MAPK/ERK signaling pathway.3.The expression of silencing GOLPH3 can reverse the resistance of HT29 colon cancer cell to cisplatin to some extent.