The Regulation Of Microrna On CD4⁺CD25⁺CD127^dim/-Treg Cells In The Microenvironment Of Hepatocellular Carcinoma

[Objective] In tumor microenvironment,CD4⁺CD25⁺CD127^dim/-Regulatory Tcells （Treg cells） are a group of negative regulatory cells,which playan important role in the mechanism of immune inhibition and immune escapeof liver cancers.The increased number of tumor antigen and cytokine intumor microenvironment could induce the accumulation and expansion oftumor specific Treg cells.However, The cause of increased cytokines IL-2,IL-10and TGFβ1in tumor microenvironment is not clear.In our study,wecarried out our research in function of microRNA associated with thedysregulated Treg cells in Hepatocellular carcinoma.[Methods] Microarray were utilized to detect dysregulated microRNAs inthe differential hepatocellular carcinoma classified by the level ofCD4⁺CD25⁺CD127^dim/-Regulatory T cells. The results were further confirmedby Real－time qPCR. We looked for the target genes of miR-608with theapplication of bioinformatics tools.Potential target genes expressionwere assessed by westernblot for proteins,real-time PCR and Luciferaserepot gene assays.[Results]1. According to the microarray,7microRNAs expressions includingmiR-1224-3p,miR-181b,miR-21,miR-221,miR-23b,miR-4259,miR-92a weresignificantly increased,and13microRNAs expressions includingmiR-1258-p,miR-1973,miR-210-p,miR-3178-p,miR-323-5p,miR-375-p,miR-4301,miR-494,miR-608,miR-708,miR-934,miR-133a-2-p,miR-134-p weresignificantly decreased in the tumor high-expressedCD4⁺CD25⁺CD127^dim/-Treg．2. Confirmed by Real-time qPCR,we demonstrated both up-regulatedmiR-21-5p,miR-221-3p and down-regulated miR-608were consistent with theresults of Microarray.3. Through westernblot technique,we discovered that in the HepG2,SMMC7721,293T cell lines transfected with miR-608mimics,theincreased accumulation of miR-608resulted in decrease of TGFβ1proteinlevels.However,the HepG2,SMMC7721transfected with miR-608inhibitor,the decreased accumulation of miR-608had no effect on levelof TGFβ1protein,and in293T transfected with miR-608inhibitor,theprotein expression of TGFβ1was significantly higher than transfectedwith miR-608inhibitor control.4. Through Real－time qPCR experiment, we found that both of miR-608mimics or miR-608inhibitor in the HepG2,SMMC7721,293T cell lines had noeffect on level of TGFβ1mRNA.5. The expression vector was constructed containing the target gene3’UTR region of TGF β1and FoxP3. Detected by luciferase report geneassays,the interaction between miR-608and target gene3’ UTR region wasconfirmed. We hypothesized that TGFβ1may be the target gene miR-608.[Conclusions] In tumor microenvironment, Treg play an important role inthe mechanism of immune inhibition.miR-608can negatively regulate TGFβ1gene, which could be used as a tumor suppressor function. Its abnormalexpression also indirectly regulate the expression of Treg cells.