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Objective:To investigate the key mechanism of transforming growth factor-β (TGF-β) inducing the expression of interleukin-17D (IL-17D) in lung cancer-associated fibroblast (CAF) and promoting the recruitment of myeloid-derived suppressor cells (MDSCs).Methods:C57BL/6 mice were established for B16 lung melanoma metastasis model (tumor model group), and control group was set up, 6 mice in each group. Flow cytometry (FACS) was used to detect the lung CAF and the changes of its ability to secrete IL-17D and the proportion of MDSCs in tumor mice. The changes of TGF-β level in lung tumor were examined by ELISA and quantitative real-time PCR (RT-qPCR). Lung fibroblasts were screened by FACS, and the effects of TGF-β on the secretion of IL-17D, C-C motif chemokine ligand (CCL)2 and CCL7 in fibroblasts were detected by RT-PCR. The migration of MDSCs under the condition of TGF-β stimulating fibroblasts was detected by Transwell.Results:The proportion of CAF (CD45 -CD326 -CD31 -) in the tumor model group was higher than that in the control group [(28.02±2.23)% vs. (7.35±2.14)%, t=9.956, P<0.001]. The ability of CAF to secrete IL-17D in the tumor model group was significantly higher than that in the control group [(38.27±2.93)% vs. (19.04±3.16)%, t=5.995, P=0.001]. The proportion of MDSCs in the tumor model group was significantly higher than that in the control group [(12.93±1.27)% vs. (8.21±1.40)%, t=4.804, P=0.009]. Compared with the control group, the protein and transcription levels of TGF-β in lung of the tumor model group were significantly increased [(1 685.07±135.61) ng/L vs. (1 047.98±68.50) ng/L, t=5.051, P=0.002; 2.17±0.03 vs. 1.00±0.05, t=51.237, P<0.001]. In vitro, lung fibroblasts were stimulated with different concentrations of TGF-β (0, 5 and 10 μg/L) for 24 hours, the relative expressions of IL-17D mRNA secreted by stimulated fibroblasts were 0.42±0.01, 0.67±0.01 and 0.84±0.04 respectively, the relative expressions of CCL2 mRNA in each group were 0.89±0.08, 1.08±0.04, 1.19±0.01 and CCL7 were 0.53±0.05, 0.65±0.04, 0.74±0.03 respectively. With the increase of TGF-β concentration, the expression levels of IL-17D, CCL2 and CCL7 in fibroblasts were significantly increased ( F=57.384, P<0.001; F=15.802, P=0.004; F=14.544, P=0.005). In addition, compared with the control group (0 μg/L TGF-β), fibroblasts treated with 10 μg/L TGF-β for 24 hours could promote the migration of MDSCs in spleen of tumor mice [(9.59±0.21)% vs. (2.14±0.24)%, t=6.585, P<0.001]. Conclusion:TGF-β can induce high expression of IL-17D in lung CAF, which is an important factor in promoting the expressions of CCL2 and CCL7 and the migration of MDSCs in tumor microenvironment.
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Objective To investigate the function of tripartite motif protein 22 (TRIM22) and the interaction with eukaryotic translation initiation factor-4E (eIF4E) in the differentiation of NB4 cells, one kind of acute promyelocytic leukemia cells, which elucidates the mechanism of TRIM22 targeting to regulate eIF4E.Methods The model of NB4 cells inducing differentiation was established in vitro.The expression changes of gene and protein of TRIM22 and eIF4E were detected by using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting.In addition, the effect on cell function and protein expression level of eIF4E after adopting electroporation technology to depress or over-express TRIM22 was detected by CCK-8 and flow cytometry.Finally, the interaction of TRIM22 and eIF4E was verified by using co-immunoprecipitation (CO-IP).Results The mRNA relative expression level of TRIM22 was gradually increasing from 1.01±0.15 to 30.98±2.79 (F=280.700, P=0.000), and the protein relative expression level was gradually increasing from 0.22±0.03 to 0.51±0.05 (F=51.430, P=0.000) after the all-trans-retinoic acid (ATRA) induction for NB4 cell.However, the mRNA relative expression level of eIF4E was gradually decreasing from 1.01±0.09 to 0.47±0.06 (F=20.520, P=0.000), with the same trend, the protein relative expression level was gradually decreasing from 0.97±0.02 to 0.64±0.09 (F=14.700, P=0.001).The expression level of PE-CD11b in the TRIM22 over-expression group with ATRA detected by flow cytometry [(78.80±2.00)%] was higher than that in the transfection group of empty vetor with ATRA [(58.70±2.70)%] (t=9.535, P=0.000) and the cotransfection group with ATRA [(61.60±3.80)%] (t=8.187, P=0.000).Meanwhile, the protein level of eIF4E changed reversely after over-expressing the gene level of TRIM22 (t=4.985, P=0.007).The CO-IP experiment was used to verify the interaction of TRIM22 and eIF4E.ConclusionTRIM22 is able to promote the cell differentiation during the process of NB4 cells differentiation.Furthermore, eIF4E is a target of TRIM22 for binding with, which plays an important role in depressing the expression of eIF4E.
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Objective To study the molecular regulation mechanism of VEGF in the model of ATRA induced differentiation in HL-60 cells,and to provide new targets for leukemia anti-angiogenic therapy.Methods The morphology was observed by Wright-Gimesa staining; HL-60 cells differentiation was detected by NBT reduction experiment.VEGF,STAT3,c-myc mRNAs were measured by reverse transcription-PCR;VEGF,STAT3 and c-myc proteins were determined by Western blot.Results The proliferation of HL-60 cells was inhibited obviously by ATRA(1 μmol/L) with the induction of differentiation,NBT positive rate was 82.59％ (t =-24.157,P ＜ 0.01) ; VEGF mRNA (t =7.339,P ＜ 0.05),STAT3 mRNA (t =3.667,P ＜0.05) and c-myc mRNA (t =6.858,P ＜ 0.05) were all down-regulated.VEGF protein (t =3.386,P ＜0.05),STAT3 protein(t =4.074,P ＜ 0.05) and c-myc protein (t =3.333,P ＜ 0.05) were all down-regulated.Conclusion VEGF expression level is reduced with the procession of differentiation of HL-60 cells,which may be largely correlated with the down regulation of STAT3 and c-myc.