The Effects And Mechanism Of MicroRNA-7 Overexpression On The Development Of Murine CD8 Single Positive Thymocyte

ObjectiveTo observe the effects of miR-7 overexpression on the development of murine CD8single positive（SP）thymocyte and preliminarily expore its molecular mechanism.Methods1.MiR-7 eukaryotic expression vector was constructed by molecular biology technology,and miR-7 OE mice were successfully constructed by microembryo injection.Then,genomic DNA extracted by DNA kit instructions from the tail of seven F0 generation miR-7 OE mice and one wild-type（WT）mice as a negative control.Furthermore,the DNA fragment was amplified by PCR with specific primers.2.Total RNAs were extracted from eleven different kinds of organs and tissues by Trizol from WT mice and miR-7 OE mice.Then,the relative expression level of miR-7 in those organs and tissues was detected by Real-time PCR.3.The body weight changes of WT and miR-7 OE mice from 2 to 9 weeks old were observed and recorded.The histological changes of heart,lung,liver and so on were observed in 4-week-old and 8-week-old WT and miR-7 OE mice by HE staining respectively.4.The thymus size,weight and total cell numbers of WT and miR-7 OE mice at 4 and 8weeks old were detected respectively.The histological changes of thymus in each group of mice were observed by HE staining.5.The percentage of thymocyte subsets and the percentage of activation related molecules CD44,CD62L and CD69 in CD8⁺SP cells in each group of mice were analyzed by Fluorescence-activated cell sorter（FACS）.Moreover,the proliferation and apoptosis changes of CD8⁺SP cells in each group of mice were detected by Ki67 and PI staining through FACS.6.The proliferation,activation and apoptosis of CD8⁺SP cells in 8-week-old WT and miR-7 OE mice in vitro were stimulated by anti-CD3/CD28.After 72 hours,the percentage of CD44,CD62L and CD69 as well as the percentage of Ki67 and PI in CD8⁺SP cells were analyzed by FACS.7.Adoptive cell transfer（ACT）experiment was performed:Bone marrow cells of8-week-old CD45.1^+/+mice or CD45.1⁺miR-7 OE mice were transferred into Rag1^-/-mice through tail vein.After 7 days,their thymus was collected,then the percentage and absolute number of CD45.1⁺T cell subsets were analysed by FACS.8.The whole gene expression in the thymus of 8-week-old WT and miR-7 OE mice were detected by cDNA microarray technique.According to the results of gene chip,combined with some target molecule prediction software such as miRbase,TargetScan,mirWalk,the possible target molecules of miR-7 were screened out by Venn analysis.Then,the mRNA expression level of these possible target genes was detected by Real-time PCR and finally to determine the only one.9.The expression level of final target molecule of miR-7 in CD8⁺SP cells was detected by FACS.Moreover,the changes of related signal pathways in CD8⁺SP cells including AKT,ERK,p-AKT and p-ERK were detected by FACS.Results1.The results of enzyme digestion and vector sequencing showed that the miR-7eukaryotic expression vector was successfully constructed;then,the vector was sent to Cyagen Biosciences Inc.to generate the miR-7 OE mice.Finally,seven F0 generation miR-7 OE mice were obtained.Furthermore,PCR result showed that the DNA fragment of 492bp could be amplified from the mice tail DNA,suggesting the successful construction of miR-7 OE mice.2.The Real-time PCR result showed that miR-7 OE mice lymphoid organs and some important organs had significantly higher expression levels of miR-7 than WT mice（P﹤0.05）,suggesting that miR-7 OE mice was successfully constructed and can be passaged stably.3.It was found that the body weight of miR-7 OE mice was significantly higher than that of WT mice,and the body size was slightly larger likewise（P﹤0.05）.HE staining results further showed that there were no significant histological changes in various important organs and tissues.4.In 4-week-old mice,the thymus size,weight and total cell number of miR-7 OE mice were not significantly changed compared with WT mice.Meanwhile,in 8-week-old mice,the thymus size,weight and total cell number of miR-7 OE mice increased significantly compared with WT mice（P﹤0.05）.Besides,HE staining results showed that there were no significant histological changes in the thymic structure of 4-week-old and 8-week-old miR-7 OE mice compared with corresponding week-old WT mice.5.FACS results showed that compared with corresponding week-old WT mice,there was no significant change in the proportion and total cell number of thymocyte subsets in4-week-old miR-7 OE mice.The changes of thymocyte subsets in the 8-week-old miR-7OE mice were as follows:the percentage of CD4⁺CD8⁺DP cell,CD4⁺SP and CD8⁺SP cells were not altered obviously,but their cell counts increased significantly（P﹤0.05）;meanwhile,the percentage of CD4^-CD8^-DN celsl decreased significantly,but its cell numbers did not change significantly（P﹤0.05）.6.FACS results showed that compared with WT mice,the proliferation,activation and apoptosis of CD8⁺SP cells in miR-7 OE mice（8 weeks）were as follows:the percentage of CD62L was significantly reduced,while the percentage of CD69 and CD44 was significantly increased（P﹤0.05）.In addition,the percentage of apoptosis decreased significantly,while the percentage of Ki67 increased significantly（P﹤0.05）.7.In vitro experiments showed that compared with WT mice,thymocyte clones in miR-7 OE mice increased significantly.And FACS results were similar to corresponding in vivo experiments.8.The results of ACT experiment showed that compared with the control group,the percentage of thymocyte subsets in the CD45.1⁺miR-7 OE group were as follows:the percentage and cell number of CD8⁺SP cell increased significantly（P﹤0.05）;meanwhile,the percentage of CD4^-CD8^-DN,CD4⁺CD8⁺DP and CD4⁺SP cells had no obvious change,although their cell number changes were not statistically significant,there was an increasing trend on them.9.cDNA gene chip results showed that there were a large number of differentially expressed genes in thymus tissue of miR-7 OE mice compared with WT mice.Combined with the analysis of target gene prediction software such as miRbase,TargetScan,miRWalk,etc.,24 candidate target genes were screened out.At the same time,based on some literature reports,7 candidate target genes such as PIK3R1,POU2F1,SLC38A2,BBX,RAB7,ARF4,and PIK3CA were finally selected.Furthermore,Real-time PCR results showed that the expression levels of these seven candidate target genes were all down-regulated（P﹤0.05）in the thymus tissue of miR-7 OE mice compared with WT mice,and the expression levels of PIK3R1 and PIK3CA were down-regulated most obviously（P﹤0.05）.Furthermore,combined with literature analysis,PIK3R1 was finally selected as the target gene of miR-7.10.Furthermore,FACS results showed that the expression level of PIK3R1 in CD8⁺SP cells in miR-7 OE mice was downregulated compared with WT mice（P﹤0.05）,which is further clarified that PIK3R1 is the targtet molecular of miR-7.11.FACS results showed that compared with WT mice,the expression levels of AKT and ERK in CD8⁺SP cell of miR-7 OE mice did not change significantly;while in CD8⁺SP cells,the expression level of p-AKT increased significantly,the expression level of p-ERK decreased significantly（P﹤0.05）.ConclusionsMiR-7 overexpression can significantly affect murine CD8⁺SP cell development,which is related to the down-regulation of PIK3R1,a novel target of miR-7,and altered transduction of AKT and ERK signaling pathways.