| Purpose 1.To identify the differentially expressed genes screened between CSCs and their differentiated cells in small cell lung cancer cell line H446 from our previous work.2.To test the effect of IL-8 on cancer stem cells in small cell lung cancer cell line H446.Methods 1.To identify differentially expressed gene in CSCs and differentiated cells in small cell lung cancer cell line H446:(1)q RT-PCR demonstrated the presence of m RNA levels in differentially expressed genes.(2)Elisa validated the protein levels of the genes that had differences in the above m RNA levels.2.To test the effect of IL-8 on CSCs in small cell lung cancer cell line H446:(1)We used the RNA interference technology to silence the expression of IL-8 in the H446 spheroidal cells and confirmed that IL-8 in the tumor spheres was inhibited successfully by q RT-PCR and Elisa assay,then we separately analyzed the tumor sphere forming rate and differencial expression level of the stemness genes before and after RNA interference by tumor sphere formation and q RT-PCR experiments.(2)We put human recombinant protein IL-8(rh-IL-8)in H446 cells and analyzed the effects of IL-8 on the expression of stemness genes such as u PAR,CD44,SOX2 and Nanog by q RT-PCR assay,then we analyzed the effects of IL-8 on the tumor sphere forming rate and cell mobility of H446 cells by tumor sphere formation and migration experiments.(3)We put the human recombinant protein IL-8(rh-IL-8)in H446 cells and analyzed the survival rate of small cell lung cancer cells by MTS assay.(4)We analyzed the effect of reparixin on the tumorsphere-forming ability of H446 cells by using IL-8 receptor inhibitor reparixin.Results 1.Differentially expressed genes in CSCs and differentiated cells in small cell lung cancer cell line H446:(1)We verified the differentially expressed five up-regulated genes(IL-8,IL-2,SAT1,SPINK6 and IGFBP3)screened on the whole transcription sequencing from our previous work:q RT-PCR showed that the m RNA expression level of IL-8 and IL-2 in the tumor sphere is higher than that of adherent cells(n=5,P<0.05),but there was no significant difference in SAT1,SPINK6 and IGFBP3 in the two groups in the expression level of m RNA(n=5,P>0.05).(2)We verified the differences in the level of protein expression of IL8 and IL2 and the Elisa result showed that the secretion of IL-8 in tumorspheres were higher than that of adherent cells(n=4,P<0.05),but the secretion of IL2 detected in the two groups had no significant difference(n=4,P>0.05).2.The effect of IL-8 on CSCs in small cell lung cancer cell line H446:(1)The q RT-PCR and Elisa experiments results showed that the m RNA and protein level expression of IL-8 after si IL-8 treated tumorspheres were significantly inhibited(n=5,p<0.01),indicating that expression of IL-8 was silenced successfully in tumorspheres.The relative expression levels of stemness genes of the tumorspheres treated by si IL-8,such as OCT4,CD133 and Nanog,were significantly lower than that of tumorspheres in si NC treatment(n=5,P<0.01),and the expression of genes CD44 in the two groups had no significant difference(n=5,P=0.05).Tumor sphere formation assay showed that the tumorspheres treated by si IL-8 was lower than that of tumorspheres in si NC treatment(n=5,P<0.05).(2)The q RT-PCR experiments showed that the stemness genes expression after human recombinant IL-8 protein(rh-IL-8)treatment of adherent cells,such as u PAR,CD44,SOX2 and Nanog,were higher than the untreated group(n=5,P<0.01),but the expression of CD133,ABCG2 and OCT4 in the two groups had no significant difference(n=5,P=0.05).The spheres formation ability after human recombinant protein IL-8(rh-IL8)treatment of adherent cells was higher than that of untreated group(n=5,P<0.05).The migration experiment results showed that the tumor cell migration rate after treated by 5ng/ml and 10ng/ml rh-IL-8 was higher than that in untreated group(n=5,P<0.001).(3)The results of MTS showed that the survival rate of H446 adherent cells treated with human recombinant protein IL-8 was higher than that in untreated group(n=5,P<0.01).(4)The tumor sphere formation assay showed that the tumorsphere formation rate after 100 n M reparixin treated H446 adherent cells was slightly lower than the control group(n=5,P<0.05),the tumor sphere formation rate afer 500 n M Reparixin treatment of adherent cells was significantly lower than that in the control group(n=5,P<0.001).Conclusion 1.The expression of IL-8 in the cancer stem cells of small cell lung cancer cell line H446 was significantly higher than that in differentiated cells.2.IL-8 may promote the stemness of cancer stem cells in small cell lung cancer cell line H446. |
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