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Objective To investigate the role of miR-10a in CD4+CD25+Treg-mediated immunosuppression during sepsis and its potential role in immunotherapy for sepsis.Methods Sepsis mouse model was established by cecal ligation and puncture(CLP).Balb/c mice of clean grade were sacrificed 1,3,5,and 7 days after operation.Blood as well as spleen samples were harvested at given intervals.The splenic CD4+CD25+Treg cells and CD4+T cells were isolated by MACS microbeads.Cells were cultured,and phenotypes were analyzed by flow cytometry.The miR-10a expressed in Treg cells were detected by Real-time PCR.After administration of LV-mmu-miR-10a-5p-inhibition,the immunosuppressive function have been detected.Statistical analyses were performed using one-way analysis of variance (SPSS 19.0,Chicago,USA) test followed by Dunnett-t test to compare among three or more groups or by Student's t-test to compare between two groups.Results The percentages of splenic Tregs (CD4+CD25+/CD4+T) was (7.34±1.2)％ in normal group,and the increase in percentage of Tregs in spleen has been observed in septic mice (P＜0.05).The mean fluorescence intensity (MFI) of Foxp3+Treg was increased in septic mice compared with sham group (P＜0.05).The expression of miR-10a was significantly elevated on CLP 1-7 day (P＜0.05).After down-regulation of miR-10a in septic mice,the percentages of Tregs (CD4+CD25+/CD4+T) was significantly increased in septic mice (P＜0.05),the MFI of Foxp3+Treg was increased in septic mice compared with control group (P＜0.05).The CD4+T cell proliferative activity in CLP-induced mice was significantly suppressed on CLP 3 day compared with sham group (P＜0.05).After down-regulation of miR-10a in septic mice,the CD4+T cell proliferative activity was significantly suppressed compared with control group (P＜0.05).Conclusions Treg plays a critical role in immunosuppression in septic mice.Inhibition of miR-10a in vivo could enhence immunesuppression of CD4+CD25+Treg.Therefore miR-10a may participate in the regulation of CD4+CD25+Treg immunosuppression in sepsis and become the target for immunotherapy.
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OBJECTIVE To investigate the effect of microRNA-10a on the development of granulosa cells tumor (GCT). METHODS FISH was used to detect the miR-10a expression in tissues from GCT patients. Several functional assays were performed to investigate the effect of miR-10a on proliferation,migration, invasion, spheroid formation and repressed anticancer drug-induced apoptosis of GCT in vitro.CRISPR- Cas9 system mediated miR- 10a knockout in cancer GC and two mice GCT models wereconstructed to show the knockdown effect of miR-10a on cancer GC both in vitro and in vivo. RNA-seq,Western blot, luciferase reporter assay and FISH were used to identify potential direct functional targets and related pathways of miR-10a in cancer GC. RESULTS Strong miR-10a signal was detected in tissues from malignant GCT patients. And amplification of miR- 10a negatively correlated with overall survival rate of ovarian cancer patients. In addition, ectopic expression of miR- 10a significantly promoted cell proliferation, migration, invasion, spheroid formation and repressed anticancer drug-induced apoptosis in vitro. CRISPR-Cas9 system mediated miR-10a knockout in cancer GC showed opposite phenotype compared to miR-10a overexpressed cancer GC. By using xenograft and orthotropic models, the onco?genic role of miR-10a was further confirmed in vivo. RNA-seq, Western blot, luciferase reporter assay and FISH were used to identified PTEN/TET2 as direct functional targets of miR-10a in cancer GC; Akt and Wnt were found as two associated signaling pathways of miR- 10a in cancer GC. CONCLUSION Taken together, our results demonstrate that the miR-10a is positively involved in development of GCT.