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Objective:To investigate the effect of tripartite motif-containing 23 (Trim23) on the differentiation and maturation of dendritic cells and the possible mechanism.Methods:Mouse bone marrow-derived dendritic cells (BMDCs) were prepared from bone marrow cells of C57BL/6 mice with the presence of Flt3L. Real-time quantitative PCR and Western blot were used to detect the expression of Trim23 in BMDCs after LPS stimulation. An overexpression vector for full-length Trim23 (Trim23 OE) was constructed and transfected into BMDCs, and the pcDNA3.1 empty vector was used as control. Flow cytometry was used to detect the expression of CD80, CD86, CD40 and MHCⅡ on the surface of vector-transfected BMDCs after LPS stimulation and ELISA was used to detect the secretion of IL-12p40, TNF-α, IL-6 and IL-10 by these cells. CD8 + and CD4 + T cells were isolated from spleen and lymph nodes of OT-Ⅰ and OT-Ⅱ mice by magnetic beads and co-cultured with LPS-treated BMDCs in the presence of ovalbumin (OVA). Flow cytometry was used to detect the proliferation and differentiation of CD8 + and CD4 + T cells. Western blot was performed to analyze the phosphorylation of p38, ERK1/2 and AKT in BMDCs. Two overexpression vectors for Trim23 mutants lacking RING or ARF domain (Trim23 ΔRING and Trim23 ΔARF) were constructed and transfected into BMDCs. Then flow cytometry and ELISA were used to detect the expression of surface molecules and cytokines. Results:The expression of Trim23 in BMDCs was significantly down-regulated after LPS stimulation. The expression of MHCⅡ, CD86 and CD80 and the secretion of TNF-α and IL-6 decreased significantly in BMDCs overexpressing Trim23. Furthermore, overexpression of Trim23 inhibited the ability of BMDCs to induce the proliferation and differentiation of CD4 + T cells and the proliferation of CD8 + T cells. Western blot showed that the phosphorylation of p38 and ERK1/2 decreased significantly in Trim23-overexpressing BMDCs. Compared with wildtype Trim23, overexpression of Trim23 ΔRING had no significant influence on the expression of surface molecules (MHCⅡ and CD86) and the secretion of cytokines (TNF-α and IL-6) in BMDCs stimulated by LPS. Conclusions:Trim23 overexpression inhibited the maturation and immune activation of BMDCs via MAPK signal pathway and its RING domain. This study provided reference for targeting Trim23 to improve the immune response of dendritic cell-based tumor vaccines.
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Objective:To detect the differential expression of microRNA(miRNA) in abdominal fat of mice fed with high fat and to explore the role of highly expressed miR-335 in lipid metabolism.Methods:MicroRNA microarray was used to detect the differential expression of miRNAs in abdominal fat of mice fed with high fat. The target genes of miR-335 were predicted by Targetscan, the target genes of ectonucleotide pyrophosphatase/phosphordiester-ase 4(ENPP4) and fatty acid desaturase1(FADS1) were verified with double luciferase reporter system and point mutation test. miR-335 mimics was transfected into 3T3L1 cells to detect the expression of target gene mRNA and protein; Realtime PCR was used to detect the expression levels of miR-335 and target genes in different tissues of mice.Results:After high-fat feeding, the size of mice in the model group was significantly larger than the control group, and the serum total cholesterol and triglyceride levels of mice were significantly increased( P=0.016, P=0.014). miRNAs were differentially expressed in adipose tissues of mice fed with high fat, and the expression of miR-335 increased significantly( P=0.024). Double luciferase reporter system showed that miR-335 combined with the predicted target sites of ENPP4 and FADS1 through the " seed region" to inhibit the expression of ENPP4 and FADS1 genes, and the point mutation test confirmed the binding target sites between miR-335 and ENPP4/FADS1. MiR-335 mimics were transfected into 3T3L1 cells, the expression level of FADS1 mRNA decreased significantly( P=0.038), and the protein levels of ENPP4 and FADS1 decreased significantly( P=0.033, P=0.007). Realtime PCR showed that, miR-335 was significantly higher expressed in liver, brown adipose tissue, and brain after high-fat feeding, especially in white adipose tissue( P=0.002). The expression of ENPP4 and FADS1 in brain( P=0.006, P=0.034) and brown adipose tissue( P=0.014, P=0.037) decreased significantly, and the expression of ENPP4 in liver also decreased significantly after high-fat diet( P<0.001). Conclusion:miR-335 is a miRNA related to lipid metabolism and fat deposition. ENPP4 and FADS1 are the target genes of miR-335.
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Objective To investigate the effects of lncRNA FENDRR on the proliferation, migration, invasion and apoptosis of LUSC H226 cells and its molecular mechanism. Methods The expression levels of FENDRR in normal lung epithelial cells BEAS, lung adenocarcinoma A549 and H1299 cells and LUSC H226 cells were detected by qRT-PCR. H226 cells were transfected with FENDRR-siRNA as the experimental group, or with FENDRR-siNC as a negative control group. Cell proliferation was detected by CCK-8 assay. Cell migration and invasion were detected by Transwell assay. Cell apoptosis was detected by flow cytometry. The protein expression levels of MEK, p-MEK, ERK and p-ERK were determined by Western blot. Results FENDRR levels in H226 cells were significantly lower than those in normal lung epithelial cells. Compared with the negative control group, the knockdown of FENDRR could significantly promote the proliferation, migration and invasion of H226 cells, inhibit the cell apoptosis, and increase the protein levels of p-MEK and p-ERK. The addition of ERK inhibitor U0126 rescued the effect of FENDRR knockdown on H226 cells. Conclusions The knockdown of lncRNA FENDRR can promote the proliferation, migration and inhibit the apoptosis of H226 cells through ERK/MAPK pathway.
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Objective:To investigate the effects of β-glucan combined with agonistic anti-CD40 monoclonal antibody (5C11) on the immune functions of dendritic cells (DCs) and the possible molecular mechanism.Methods:Mononuclear cells were separated from fresh concentrated white cells (granulocytes) of healthy subjects using Ficoll density gradient centrifugation, and induced by GM-CSF and IL-4 to differentiate into immature DCs. Following various stimulation (5C11 alone, β-glucan alone, 5C11 combined with β-glucan), flow cytometry was used to detect the expression of CD40, CD80, CD83, CD86 and MHC-Ⅱ molecule HLA-DR on the surface of DCs. ELISA was used to detect the secretion of cytokines including IL-12, IL-6, TNF-α and IL-10. Western blot was used to detect the phosphorylation of proteins related to MAPK signaling pathway.Results:Flow cytometry suggested that β-glucan significantly induced the expression of co-stimulatory molecule CD40 on the surface of DCs ( P<0.05). After the DCs were co-stimulated with β-glucan and 5C11, CD80, CD83 and CD86 expression were further significantly increased, and a strong synergistic effect on CD83 expression, a key marker of DC maturation, was observed ( P<0.01). ELISA showed that β-glucan combined with 5C11 could significantly promote the secretion of cytokines such as IL-12, IL-6 and TNF-α by DCs, and have a synergistic effect on the secretion of IL-12, a critical cytokine in regulating DC functions ( P<0.01). Western blot indicated that the phosphorylation of p38 MAPK and p44/42 MAPK in DCs was increased significantly after combined treatment, and the phosphorylation started earlier and lasted longer compared to that in DCs stimulated with 5C11 or β-glucan alone ( P<0.01). Conclusions:This study suggested that β-glucan combined with 5C11 had a synergistic effect on promoting the maturation and improving the immune functions of DCs, providing a new strategy for the preparation of anti-tumor DC vaccines.
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Objective The inhibitory effect of the PARP inhibitor olaparib on human acute myeloid leukemia HL-60 cells was studied. Methods The HL-60 cells in logarithmic growth phase were treated with different concentrations(1. 25,2. 5,5 and 10 μmol/L) of olaparib for different time. The CCK-8 assay was used to detect the inhibitory effect of olaparib on HL-60 cells. The apoptotic level of HL-60 cells was detected by Annexin-V/PI double staining method,and the expression of related signal proteins ( PARP-1 and caspase-3)in HL-60 cells was detected by Western blot. Results HL-60 cells were inhibited by olaparib at dif-ferent concentrations(1. 25,2. 5,5 and 10 μmol/L) for 48 h,and the inhibition rate gradually increased with the prolongation of the action time;at the same time,the apoptotic rate was increased in HL-60 cells after olaparib treatment for 48 h,showing a dose-de-pendent manner;the PARP activity was inhibited and caspase-3 was activated in HL-60 cells treated with olaparib. Conclusion The PARP inhibitor olaparib not only inhibits proliferation of HL-60 cells,but it also promotes apoptosis of HL-60 cells by inhibi-ting PARP activity and activating caspase-3.
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MicroRNAs are non-coding small RNAs that participate in the regulation of post transcriptional gene expression.MicroRNAs play the roles of inhibiting cancer or promoting cancer in different tumors.MicroRNA-373 is one of the members of the microRNA-520/373 family,which is involved in cancer anoxic response and DNA damage repair.MicroRNA-373 is abnormal expressed in breast cancer,glioma,lung cancer,prostate cancer and other tumors,which may play an important role in tumor development process,especially in tumor invasion and migration.
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Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is an inflammatory lung injury induced by a variety of factors, and these diseases are associated with high rates of mortality due to the lack of effective treatments. Based on the latest research in ALI/ARDS, it is widely accepted that generalized inflammatory responses play a critical role in initiating and developing process of ALI/ARDS. We make a brief review on the immune-pathogenesis and the signaling pathways of ALI/ARDS from the perspective of inflammation, thereby helping develop novel therapeutic strategy for treatment of patients with ALI/ARDS.
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Objective Our study aims to investigate the inhibitive effects of miR -124 on the growth of breast cancer cell MCF -7 induced by paclitaxel .Methods MTT was used to detect the growth inhibition of MCF-7 induced by paclitaxel .Flow cytometry was used to detect the effect of paclitaxel on cell cycle .Real-time quantitative PCR(qRT-PCR) was used to detect the expressive level of miR -124,while MCF -7 cells were treated with paclitaxel .MiR-124 inhibitor was transfected into MCF -7 breast cancer cells ,and growth in-hibition was detected by MTT .Results The results showed that paclitaxel could significantly inhibit the growth of breast cancer cell line MCF -7 by blocking the G2 phase.The results from qRT-PCR showed that the relative expression of miR-124 was increased when the dosage of paclitaxel was increased .When the expression of miR-124 was inhibited ,the cell growth inhibition caused by paclitaxel was also prominently decreased .Conclusion The higher expression of miR -124 in MCF-7 induced by paclitaxel was dose dependent .And miR-124 in-hibitor can significantly influence the cell growth inhibition caused by paclitaxel .These results indicat that miR -124 plays an important role in paclitaxel -induced chemotherapy drug resistance ,and provides a new direction to solve the problem .
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Objective:To observe and identify the inhibitory effect of oncostatin M (OSM) combined with dacarbazine (DTIC) on mouse melanoma cells B16 in vitro and in vivo. Methods:The inhibitory effect of OSM combined with DTIC on the proliferation and apoptosis of B16 melanoma cell line B16 were determined through MTT assay and flow cytometry, respectively. The change in nu-cleus morphology of B16 cells was observed under a fluorescence microscope by Hoechst staining method. The effects of single agents OSM and DTIC, as well as OSM-DTIC joint treatments, on tumor in mice in vivo were observed by inoculating B16 cells into C57 BL of six mice. Results:The OSM, DTIC, and combined OSM-DTIC treatments inhibited the proliferation of B16 cells by (11.2±2.3)%, (25.3±4.6)%, and (32.5±3.8)%, respectively (P<0.05). Apoptosis of B16 occurred at (1.32±0.42)%, (10.64±2.13)%, and (15.86±2.76)%, respectively (P<0.05). Cell morphology showed a significant increase in nuclear fragmentation, as proven by OSM-DTIC combined treatment. In the in vivo experiment, DTIC caused an apparent inhibition on the growth of mouse melanoma compared with the control group, and the joint treatment showed that the addition of OSM enhanced the tumor suppression effect of DTIC. Conclusion: OSM combined with DTIC has a synergistic effect that inhibits proliferation and apoptosis of B16 in vitro. This approach suggests a new po-tential treatment for melanoma.
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Objective: This work aimed to investigate the relationships of the serum levels of IL-17 and TGF-β with the carcinogenesis and progression of colorectal cancer (CRC), as well as the clinical significance of these serum levels. Methods: Data of 30 healthy subjects, 59 patients with simple CRC and 44 CRC patients with postoperative (post-op) metastasis were recruited in this study. The patients were respectively divided into group A (30 healthy subjects as the control group), group B (59 CRC patients without distant metastasis after surgery), and group C (44 CRC patients with post-op metastasis). The patients in each group had a mean age of 53.8 ± 20.8, 62.0 ± 11.8, and 64.0 ± 15.7 years, respectively. All patients were confirmed by pathological diagnosis. The serum levels of IL-17 and TGF-β were measured by enzyme-linked immunosorbent assay. All quantitative data were analyzed using SPSS 13.0. Results: The IL-17 serum level was significantly higher in groups B and C than in group A. The preoperative (pre-op) serum level of IL-17 was significantly higher than the post-op serum level in group B (P0.05). However, the TGF-β serum level in group C was significantly higher than that in groups A and B (P<0.05). No significant correlation was observed in the serum level IL-17 or TGF-β between colon and rectum cancers in groups B and C. Conclusion: The serum level of IL-17 is significantly correlated with that of CRC. The serum level IL-17 increases with the aggravation of CRC and increased tumor burden. A strong correlation exists between the serum level of TGF-β and metastasis of CRC. Cytokine IL-17 and TGF-β may play an important role in the progression and metastasis of CRC.
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<p><b>OBJECTIVE</b>To explore the effect of recombinant human soluble CD(40) ligand (rhsCD(40)L) and CD(40)L cDNA transfected cell (CD(40)L-TC) on the behavior of malignant B lymphocytes, and investigate the possibility of using rhsCD(40)L as a new bio-factor in tumor immunotherapy.</p><p><b>METHOD</b>rhsCD(40)L and CD(40)L-TC were obtained by gene recombinant techniques. Multiple myeloma cell lines, XG2, XG7, U266 and 8226, B-lymphoma cell lines, Raji and Daudi were selected to detect responses to rhsCD(40)L and CD(40)L-TC stimulation. Cell growth curve, cell cycle, early apoptosis as well as membrane surface molecules on these cell lines were analyzed.</p><p><b>RESULTS</b>(1) The expression levels of CD(40) molecule on malignant B lymphocytes showed heterogeneity. High level of CD(40) on XG2, moderate on 8266, Raji, and Daudi, and no expression on U266 and XG7 were detected. The rhsCD(40)L stimulation gave rise to a typical homo-type cell aggregation of XG2 and Daudi. Meanwhile, at least 10 to 20 of CD(40)(+) XG2 or CD(40)(+) Daudi cells were found adherent to one pre-treat ed CD(40)L-TC. (2) Co-incubation with rhsCD(40)L (5 micro g/ml), or CD(40)L-TC (tumor cell: CD(40) = 5:1) resulted in a significant inhibition of in vitro cell growth of XG2, Raji and Daudi, with G(1)-phase arrest for XG2 and G(2)-phase for Raji and Daudi. These two kinds of CD(40) stimulators induced XG2, Raji and Daudi cells to apoptosis in vitro. The apoptotic rate for XG2 was 23.3% (rhsCD(40)L) and 18.8% (CD(40)L-TC), for Daudi 14.2% and 15.9%, and for Raji 11.6% and 8.9% respectively. (3) Phenotype analysis showed that CD(95) expression levels were significantly up-regulated on XG2, Raji and Daudi after stimulation with rhsCD(40)L or CD(40)L-TC, and CD(80) and CD(18) expression levels on Raji were respectively enhanced and decreased.</p><p><b>CONCLUSION</b>The abilities to directly inhibit XG2, Daudi and Raji cell proliferation, to induce themapoptosis, as well as to up-regulate immune co-stimulator molecule CD(80) expression on Raji cells would make rhsCD(40)L a potential bio-factor for tumor immuno-therapy.</p>