| BackgroundMalignant tumors (especially lung cancer) are serious and lethal diseases which aredifficult to cure. Surgery, radiotherapy and chemotherapy are traditional treatment of tumor,but these therapeutic methods have great harm or adverse effect to body. At present, toimprove the quality of life and survival time has become the primary consideration oftumor treatment, so, immunotherapy of T lymphocytes is gradually attached greatimportance by clinician.CD8~+T cells are key members of adaptive immunity against tumorigenesis. It is ofgreat value to explore and clarify the regulatory mechanism of CD8~+T lymphocytes. Assubset of CD8~+T cells, effector T cells (Te) and memory T cells (Tm) have differentbiological activities. The former can kill tumor cells but come into apoptosis in a certainperiod and the latter is static with the ability of self-renewal. Thereby, is there anyregulatory difference which exists between Te and Tm cells? Are there any potential targetmolecules that can enhance anti-tumor ability of T cells?More recently, a large number of studies showed that microRNAs (miRNA) playedcritical roles in regulating adaptive immunity and miRNAs were important for subtle oraccurate regulation of T cells, therefore, to explore and analyze the target miRNA which isinvolved in regulating differentiation and function of CD8~+T cells may provide effectivestrategies to enhance the anti-tumor activity of T lymphocytes.ObjectiveThis study aimed to sort out the target miRNAs which differentially expressedbetween CD8~+Te and Tm cells in mice with Lewis lung carcinoma and to analyze theregulatory role and target genes of miRNA which participated in regulating CD8~+T cells. MethodsThe model of C57BL/6tumor-bearing mice was established with Lewis lungcarcinoma cells, CD8~+Te and Tm of spleen from tumor-bearing mice were separated andpurified. miRNA expression profiling was performed between CD8~+Te and Tm cells.Differentially expressed miRNA as target miRNA (miRNA-15b) was chosen and analyzedby qRT-PCR. The expression of miRNA-15b was observed during the activation of CD8~+Tcells.Synthetic miRNA-15b mimic was transfected into CD8~+T lymphocytes to realize theectopic expression of miRNA-15b in vitro. Then, apoptosis detection kit, ELISA, flowcytometry, and CFSE kit were used to evaluate the biological effects of miRNA-15b onapoptosis, cytokine secretion, phenotype, and proliferation of CD8~+T cell. The relevantdownstream target genes of miRNA-15b were also analyzed and confirmed.Results1. Analysis of miRNA microarray and qRT-PCR showed that the level of miRNA-15bwas higher in CD8~+Tm cells than in Te cells.2. miRNA-15b was more highly expressed in CD8~+T cells from tumor-bearing micethan those from healthy ones.3. The expression of miRNA-15b was negatively correlated with activation of CD8~+Tlymphocytes.4. Transfection of CD8~+T cells with miRNA-15b mimics could prevent T cells fromapoptosis.5. Ectopic miRNA-15b could inhibit the activation of CD8~+T cells (via repressing theproduction of IL-2and IFN-γ and expression of CD69) and promote expression of CD44through unknown pathways.6. Ectopic miRNA-15b does not affect proliferation of T cell.7. DEDD (Death Effector Domain-containing DNA binding protein) is a target geneof miRNA-15b and ectopic miRNA-15b can inhibit the translation of DEDD in CD8~+Tcells.ConclusionUp-regulation of miRNA-15b in tumor environment might negatively regulateanti-tumor immunity through inhibiting function of CD8~+T cells (such as secretion of IFN-γ and IL-2). On the other hand, it could enhance anti-apoptotic ability of CD8~+T cellsand improve the expression of CD44.miRNA-15b may has multiple effects in regulating anti-tumor function of CD8~+Tlymphocytes. It played a negative regulatory role in activation of T lymphocytes, but at thesame time, it mought induce CD8~+T cells differentiate into Tm and contributes toestablishment of immune memory. miRNA-15b might be a potential therapeutic target forimmunotherapy. |
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