Inhibition Of Mir-21Expression Of Tumor Microenvironment To Promote The Antitumor Activity Of STAT3-deleted DC

Objective: Dendritic cells (DCs) are professional antigen presenting cellswith the ability to effectively induce Naive T cell activation, as well asdifferentiation of cytotoxic T lymphocytes (CTL) and T helper cells (Th).Therefore, more and more researchers are trying to use DC vaccines to treat avariety of diseases including cancer. However, the immunogenic capacity ofthe tumor DC vaccines may be limited by the microenvironment. The releaseof IL-10, IL-6, VEGF and M-CSF as well as other cytokines by tumor cells,could regulate stat3phosphorylation levels of DC and consequently inhibit theactivity of DC vaccines. miR-21has been identified as one of the most highlyexpressed miRNAs in various tumors which involved in the regulation oftumor microenvironment by regulation of stat3/IL-6, TGF-β, and other targetgenes in tumor cell.In this study, the stat3-deleted tumor DC vaccine was prepared and theanti-tumor effect of inhibition of tumor miR-21combined with stat3-deletedDC vaccine was observed with the tumor-bearing mouse model. It is providean experimental basis for the preparation and application of clinical DCvaccine.Methods:1Preparation of stat3-deleted DC vaccine(1) Preparation of mouse dendritic cells (BMDCs): The mouse bonemarrow cells were prepared according to conventional method and DCsdifferentiation was induced by mGM-CSF and mIL-4.(2) Packaging Lentiviral: The Lentiviral carrying silencing stat3genewas packaged with the method of transfecting293T cell lines in vitro. Then,the BMDCs were infected at the fourth day with the fresh virus according tokit instructions. (3) DCs were loaded with tumor antigen and DC maturation was induced:DCs were loaded with proteins from X-ray-irradiated4T1cellsLysates(50μg/ml) at the sixth day for12h, then LPS combined with IFN-γ(100ng/ml and10ng/ml, respectively) was used to induce DC maturation forstimulating further36h. The stat3-deleted DCs were obtained.2stat3expressions in stat3-silenced DCs were detected: Total protein andRNA were extracted respectively from the stat3-silenced DCs, then, stat3level was analyzed by real-time PCR and Western blot.3Detecting the activity of stat3-silenced DC vaccine:(1) The expressions of CD86, MHC II and ICAM-1on the surface ofstat3-silenced DCs. Collecting stat3-depleted DCs, APC-anti-mouse CD11cand PE-anti-mouse CD86, MHC II and ICAM-1antibody were added,respectively. Then, the expressions of CD86, MHC II and ICAM-1weredetected by flow cytometry.(2) Cytokine level in the culture supernatant of the stat3-deleted DCs:Collecting the culture supernatant of stat3-depleted DCs, IL-12p40/p70levelwas assessed by ELISA according to the kit construction.4The anti-tumor effect of inhibiting miR-21in tumor combining withstat3-silenced DC vaccine(1)preparing the tumor-bearing mouse model:4T1cells was implanted onthe chest Subcutaneous of6-to7-wk-old BABL/c mice.When the tumorvolume reached200mm3, anti-miR-21plasmid (100μg/mouse) were injectedinto the tumor twice at3days interval. Then three days later after the finalinjection, tumor-bearing mice were injected with stat3-deleted DC vaccine(106/mouse), a week later the DC vaccine was injected again.(2)Observing the tumor growth: The tumor diameters were measuredwith a vernier caliper every3d after tumor inocalation.Tumor volume wascalculated according to the formula (Long diameter×short diameter2)/2, thetumor growth curve was graphed in lines.(3)The effect of inhibiting tumor miR-21combining with stat3-deletedDC vaccine on spleen lymphocyte: The7day of final vaccination with DC vaccines, tumor-bearing mice were killed, the production of IFN-γ in CD8+T,as well as CD4+CD25+of spleen lymphocytes were analyzed by FCM,respectively; Cytolytic activity of cytotoxic T lymphocyte cells was detectedby LDH release assay.Results:1The plasmid with highest inhibition efficiency on stat3expression wasselected from four plasmids which carrys silencing stat3gene by the methodof cell transfection.2Compared with the control group, stat3expression levels ofstat3-depleted DC decreased. The stat3protein decrease70%and that ofmRNA declined30%.3Compared with the control group, ICAM-1, CD86and MHC IIexpressions of stat3-depleted DC were significantly increased, and silence ofstat3could promote the secretion of IL-12.4Inhibition of tumor miR-21combined with stat3-deleted DC vaccinesignificantly slowed tumor growth; the production of IFN-γ and CTL activityof tumor-bearing mouse spleen lymphocytes were increased; But had noobvious regulatory role on the distribution of Treg cells.Conclusion:1stat3-depleted DC vaccine was obtained by lentiviral transductionmethod.2stat3-depletion in DCs increased the expression of MHC II, CD86andICAM-1, as well as promoted IL-12secretion.3The anti-tumor effect of stat3-silenced DC vaccine enhanced afterinhibiting miR-21in tumors.4The production of IFN-γ and CTL activity of spleen lymphocytes fromtumor-bearing mouse were enhanced by inhibiting tumor miR-21combiningwith stat3-deleted DC vaccine; But had no obvious regulatory role on thedistribution of Treg cells.