Effect Of Up-regulated MicroRNA-145 On Differentiation And IL-9 Expression In Th9 Cells

Background&Aims:To investigate the effect of up-regulated micro RNA145 on differentiation and IL-9 expression in mouse Th9 cells.Methods:Naive CD4~+T cells were isolated from the spleen of mouse with a Mouse Naive CD4~+T Cell Isolation Kit,and the purity of Naive CD4~+T cells were detected by Flow cytometry.Naive CD4~+T cells were randomly allocated into 2 groups:differentiation group and control group,cells were stimulated with anti-mouse CD3e and anti-mouse CD28,differentiation group was treated with Interleukin-4(IL-4)and transforming growth factorβ(TGF-β),and flow cytometry was used to detect the cell markers of the th9 cells after 3 days of differentiation culture.Naive CD4~+T cells were randomly allocated into 3groups:miR-145 mimic transfected group,miR-145 mimic NC transfected group and blank control group,the cells were transfected with Trans IT-TKO transfection reagent.After transfection,the expression level of miR-145 and NFATc1 mRNA was detected by real-time PCR.Transfected cells were stimulated via anti-CD3 and anti-CD28,together with TGF-βand IL-4 for 3days,the Th9 markers were detected by flow cytometry.IL-9 secretions were detected by ELISA.The mRNA expression levels of IL-9 and Smad3 were detected by RT-PCR.Results:After separation,the pure rate of Naive CD4~+T cells was 91.4±2.7%,and high-purity Naive CD4~+T cells were successfully obtained.After differentiation culture 3 days,the proportion of CD4~+IL9~+cells in differentiation group was 10.9±2.0%,which was higher than that in control group 2.22±0.2%(P<0.05),the difference is statistically significant,Th9 cells were successfully induced.After transfection 24h,compared with the miR-145 mimic NC transfected group and blank control group,the miR-145 expression level of the miR-145 mimic transfected group was up-regulated significantly(P<0.01),and the difference was statistically significant,cells were transfected successfully;the NFATc1 mRNA expression level of the miR-145 mimic transfected group was decreased compared with the miR-145 mimic NC transfected group(P<0.01),the difference was statistically significant.After transfection,the cells were cultured under the Th9 cells induced condition for 3 days,the results of flow cytometry showed that the proportion of CD4~+IL9~+cells was reduced in the miR-145 mimic transfected group compared with the miR-145 mimic NC transfected group and blank control group(P<0.05),the difference was statistically significant;Compared with the miR-145 mimic NC transfected group and blank control group,ELISA showed that the IL-9 secretion levels of the miR-145 mimic transfected group was reduced in the cell supernatant(P<0.01),the difference was statistically significant;The IL-9 mRNA expression level was decreased in the miR-145 mimic transfected group compared with the miR-145 mimic NC transfected group and blank control group(P<0.05),the difference was statistically significant;Compared with the blank control group,the Smad3 mRNA expression level of the miR-145 mimic transfected group was decreased(P<0.05),the difference was statistically significant;Compared with the miR-145 mimic NC transfected group,the Smad3 mRNA expression level of the miR-145 mimic transfected group was decreased(P>0.05),the difference was not statistically significant.Conclusion:IL-4 and TGF-βinduced Naive CD4~+T cells to Th9 cells,up-regulated miR-145 of Naive CD4~+T cells can inhibit the differentiation of Th9 cells,inhibit the mRNA expression levels of IL-9,NFATc1 and Smad3,and inhibit the secretion level of IL-9.The results of this study demonstrated the inhibitory effect of mirna-145 on Th9 cells,providing a solid experimental basis for the development of drugs to reduce the inflammatory response in the microenvironment of malignant ascites and inhibit the development of malignant ascites in the future.