The Role Of IL-8 On Growth Of Xenograft Tumor And Sensitivity To Cisplatin In Small Cell Lung Cancer

Purpose1.To screen the metabolism related genes differentially expressed in small cell lung cancer stem cells(SCLC)and the differentiated cells.2.To research the effect of IL-8 on the energy metabolism and anti-cancer sensitivity in SCLC.3.To explore the effect of IL-8 on the growth of subcutaneous xenograft tumor in SCLC.Methods1.Our previous work identified that the uPAR~+cells enriched with stem cell-like cells in SCLC cell line H446.In our study,uPAR~+and uPAR~-cells were selected as the stem cells and the differentiated cells by flow cytometry in SCLC cell line H446 cell.2.we used tanscriptome sequencing analysis to screen the genes differentially expressed in SCLC stem cells and the differentiated cells.3.According to the metabolic correlation through the literature search and the significance of fold change,we screened the energy metabolism related genes differentially expressed in SCLC stem cells and the differentiated cells through GO analysis and KEGG analysis.4.The other member of the project team verified and further screened the desired gene through the fluorescence quantitative PCR experiment and the ELISA experiment.5.we knocked out the target gene using RNA interference technique in SCLC cell line H446 tumor sphere cells.ATP levels in tumor sphere cells were detected by ATP assays before and after gene knockout.The cell viability of the sphere cells was observed by MTS assays,and the effect of the target gene on cisplatin sensitivity of tumor sphere cells was analyzed.The recombinant protein of the target gene was used to treat H446 cells,ATP levels in H446 cells were detected before and after the treatment by ATP assays.The cell viability of H446 cells was observed by MTS assays,and the effect of the recombinant protein on cisplatin sensitivity of H446 cells was analyzed6.The model of xenograft tumor was established by subcutaneous injection of tumor sphere cells.The targeted IL-8 small interfering RNA was imported into the xenograft tumor by local injection in the tumor.Then observe the tumor volume and analyse the effect of IL-8 on the growth of xenograft tumor.Results1.The differentially expressed genes associated with energy metabolism in SCLC stem cells and the differentiated cells(1)The transcriptome sequencing analysis showed that 13 genes differentially expressed in uPAR~+cells and uPAR~-cells.There were 10 genes up-regulated,and three genes down-regulated.(2)According to the correlation with metabolism and the significance of fold change,Combining GO analysis and KEGG analysis.Five differentially expressed genes(IL-8,IL-2,SAT1,SPINK6 and IGFBP3)were selected from the above10up-regulated genes.(3)The other member of the project group verified and further screened IL-8 as the target gene through fluorescence quantitative PCR and ELISA assays.2.The effect of IL-8 on the energy metabolism in SCLC(1)we knocked out the target gene using RNA interference technique in SCLC cell line H446 tumor sphere cells.ATP assays showed that the ATP level in tumor spheres with treatment of anti-IL-8 siRNA was decreased relative to the control group.(2)The recombinant protein of the target gene was used to treat H446 cells,ATP assays showed that the ATP level in H446 cells with treatment of rh-IL-8 was increased relative to the control group.3.The effect of IL-8 on the sensitivity to cisplatin in SCLC(1)we knocked out the target gene using RNA interference technique in SCLC cell line H446 tumor sphere cells.MTS assay showed that the cell viability of tumor spheres with treatment of anti-IL-8 siRNA was lower relative to the control group.It was suggested that inhibition of IL-8 could reduce the sensitivity of tumor cells to cisplatin.(2)The recombinant protein of the target gene was used to treat H446 cells,MTS assays showed that the cell viability of H446 cells with treatment of rh-IL-8 was higher relative to the control group.It was suggested that rh-IL-8 can improve the sensitivity of H446 cells to cisplatin.4.IL-8 promote the growth of subcutaneous xenograft tumor in SCLC.(1)The results showed that the growth rate of subcutaneous xenograft tumor with treatment of anti-IL-8 siRNA was lower relative to the control group.(2)On the 20th day since xenograft tumor formation,the average volume of the xenograft tumor with treatment of anti-IL-8 siRNA decreased by about 20%compared with the control group.Conclusion1.IL-8 is the key gene differentially expressed in SCLC stem cells and the differentiated cells.2.IL-8 promote cell energy metabolism of cancer stem cells in SCLC.3.IL-8 may reduce the anticancer drugs sensitivity In SCLC.4.The inhibition of IL-8 may suppress the tumor growth in SCLC.In conclusion,The inhibition of IL-8 may suppress the tumor growth in SCLC,and enhance the anticancer drugs sensitivity in SCLC.