| ObjectiveTo investigate the effect of MSCs overexpressive Sirt1(MSCs-Sirt1)on the growth of 4T1 breast cancer cells and its potential molecular mechanism.Methods1.BALB/ C mice were treated with subcutaneous xenograft model to observe the effect of MSCs-Sirt1 on the growth of experimental cells-4T1 breast cancer cells in vivo.2.The effects of MSCs-Sirt1 on the proliferation and apoptosis of 4T1 breast cancer cells were detected byReal-time quantitative PCR,Western-blot,immunohistochemical stain,TUNEL stain and Flow cytometry.3.The levels of IL-6,IL-8,IL-10,IFN-γ and TNF-α in serum of tumor-bearing mice were detected by enzyme-linked immunosorbent assay(ELISA).4.The quantityofNK cells infiltrated in tumor tissuewas detected by Flow cytometry.The cytotoxicity of NK cells in tumor-bearing mice was detected by isotope release and labeling.5.The expression levels of CCL3,CCL4 and CXCL10 in the serum of tumor-bearing mice were measured by ELISA.The expression of CXCL10 in tumor tissues of tumor-bearing mice was detected by Real-time quantitative PCR and Western blot.6.The chemotaxis of CXCL10 on NK cells was detected by Transwell assay in vitro.And the effect of CXCL10 on the growth of 4T1 cells induced by MSCs-Sirt1 was further studied by using CXCL10 reduced tumor bearing mice model.7.To construct a CXCL10-reducing tumor-bearing mouse model to further study the role of CXCL10.Results1.Compared with the 4T1 group,the volume and weight of the transplanted tumor in MSCs group were significantly increased(P <0.05).The volume and weight of the transplanted tumor of MSCs-Sirt1 group were significantly decreased(P<0.01).2.(1)Real-time PCR and Western-blot showed that the expression of PCNA was increased(P<0.001)and the expression of Caspase-3 was decreased(P < 0.01)in MSCs group.While the expression of PCNA in MSCs-Sirt1groupwasdecreased(P<0.01)and the expression of Caspase-3 was increased(P<0.001).(2)Both immunohistochemical stain and TUNEL stain showed thatthe number of Ki-67 positive cells in MSCs group was significantly increased while compared with 4T1 group(P <0.001),and the number of TUNEL positive cells was significantly decreased(P<0.01).On the contrary,the number of TUNEL positive cells in MSCs-Sirt1 group was significantly increased(P < 0.001),Ki-67 positive tumor cells decreased significantly(P<0.01).(3)Flow cytometry showed that the apoptosis rate of MSCs-Sirt1 groupwas significantly higher than that of 4T1 group(P <0.001).3.Detection of serum inflammatory cytokines in mice by ELISA showed that the levels of IFN-γ in serum of MSCs-Sirt1 mice were higher than those of other groups(P <0.001),and other inflammatory cytokines(IL-6,IL-8,IL-10,TNF-α)were not significantly different(P> 0.5).4.The number of NK cells detected by flow cytometry showed that the number of NK cells in MSCs-Sirt1 group was significantly higher than that in the control group(P <0.001).The cytotoxicity of NK cells was detected by isotope release and labeling.The results showed that NK cells in MSCs-Sirt1 group Cell viability was significantly enhanced compared to other groups.5.(1)Detection of serumNK cell aggregation related chemokines in mice by ELISA showed that CXCL10 levels were significantly higher in MSCs-Sirt1 group than in other groups(P <0.01),while CCL3 and CCL4 were not statistically significant(P> 0.5).(2)Real-time PCR and Western-blot were used to detect the expression of CXCL10 in each group.The result showed that the expression of CXCL10 mRNA and protein in MSCs-Sirt1 group weresignificantly higher than that in other groups(P <0.001).6.Transwell results show that,the number of NK cells in the MSCs-Sirt1 group was significantly higher than that in the control group(P <0.001).The number of NK cells in the MSCs-Sirt1 group was significantly decreased after adding the rabbit anti-mouse CXCL10 antibody(P < 0.001).7.Compared with MSCs-Sirt1 group,the tumor volume and weight of mice in anti-CXCL10 group were significantly higher than those of the control group(P <0.05).Conclusions1.MSCs-Sirt1 significantly inhibited the growth of 4T1 breast cancer cells.2.MSCs-Sirt1 may raise NK cells by CXCL10 to enhance local inflammatory response to inhibit the growth of 4T1 breast cancer cell. |
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