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Objective:To investigate the role and mechanism of miR-146b in the proliferation, metastasis and apoptosis of thyroid papillary carcinoma cells.Methods:qRT-PCR was used to detect the expression of miR-146b in thyroid papillary carcinoma cells (NPA, GLAG-66, ONCNO-DG1 and B-CPAP) and normal human thyroid cell line HTori3. After B-CPAP cells were transfected with miR-146b inhibitor, the inhibition efficiency was detected by qRT-PCR, the effect of miR-146b on PTC cells proliferation was detected by MTT assay, the effect of miR-146b on PTC cells invasion was studied by Transwell assay, and the effect of miR-146b on tumor cell apoptosis was detected by flow cytometry. SiRNA-IRAK1 was transfected into B-CPAP cell line. The cell proliferation rate, migration ability and apoptosis rate were detected by MTT, cell scratch test and flow cytometry respectively. The target gene of miR-146b, interleukin-1 associated receptor kinase 1 (IRAK1) , was predicted by bioinformatics software, and the regulatory effect of miR-146b on IRAK1 was verified by double fluorescein reporter gene experiment.Results:QRT-PCR showed that the expression of miR-146b in NPA87, KAT-5, FTC-133 and B-CPAP cell lines was significantly higher than that in normal cell HTori3, especially B-CPAP ( P<0.05) . MiR-146b inhibitor transfection could significantly reduce the expression level of miR-146b in B-CPAP cells ( P<0.01) . MTT results showed that miR-146b inhibitor could inhibit the proliferation of B-CPAP cells ( P<0.05) . Flow cytometry showed that miR-146b inhibitor could promote the apoptosis of B-CPAP cells ( P<0.05) . Transwell results showed that miR-146b inhibitor could reduce the invasive ability of B-CPAP cells ( P<0.05) . After transfection with siRNA-IRAK1, the proliferation rate of B-CPAP cells increased significantly (MTT test) , the migration ability increased (cell scratch test) , and the apoptosis rate decreased significantly (flow cytometry) ( P<0.05) . The results of double luciferase reporter gene showed that irak1 was the target gene of miR-146b, and miR-146b inhibitor could significantly up regulate the expression level of irak1 protein in B-CPAP cells. Conclusion:miR-146b may play a role in promoting the proliferation and metastasis and inhibiting cell apoptosis of PTC cells by inhibiting the downstream target protein IRAK1.
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BACKGROUND:Interleukin-1 receptor-associated kinase 4 activity-induced inflammations and infection have been extensively accepted. However, there was no report concerning its effects on flap ischemia-reperfusion injury. OBJECTIVE:To explore the significance of interleukin-1 receptor-associated kinase 4 activity in flap ischemia-reperfusion injury. METHODS:A total of 36 adult male Sprague-Dawley rats were randomized into sham-operated group (n=12), ischemia-reperfusion group (n=12) and interleukin-1 receptor-associated kinase 4 group (n=12). The models of right lower abdominal island flap ischemia-reperfusion injury were set up. Interleukin-1 receptor-associated kinase 4 group was intraperitoneal y injected with 1 mL of interleukin-1 receptor-associated kinase 4 (100μmol/L) before reperfusion. The flaps were col ected at 1, 2, 4, and 6 hours after ischemia-reperfusion injury for histopathhological observation. At 1 hour after ischemia-reperfusion, protein expression of interleukin-1 receptor-associated kinase 4 was detected in flaps. The proportion of flap survival was calculated at 7 days after surgery.RESULTS AND CONCLUSION:Histopathological observation demonstrated that compared with the ischemia-reperfusion injury group, neutrophil infiltration and edema was evidently improved, and the protein expression of interleukin-1 receptor-associated kinase 4 was gradual y reduced in the interleukin-1 receptor-associated kinase 4 group. Flap survival proportions were respectively (51.70 ±7.62)%and (86.56±12.23)%in the ischemia-reperfusion injury group and interleukin-1 receptor-associated kinase 4 group at 7 days after surgery. There were significant differences in the flap survival proportion between the two groups (P<0.01). These results showed that after flap ischemia-reperfusion injury, the inhibition of interleukin-1 receptor-associated kinase 4 activities could elevate the survival rate of transplanted flap.
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Objective To explore the dynamic expressions of interleukin-1 (IL-1)receptor associated kinase (IRAK)-M and IRAK-4 in rats with or without endotoxin tolerance (ETT)in acute liver failure (ALF).Methods Sixty-six male SD rats were divided into three groups:ALF group,ETT group and control group.The rats in ETT group received daily lipopolysaccharide (LPS)intraperitoneal injection for 5 days,while the rats in ALF group received daily injection with same volume of 0.75% NaCl solution.Both ETT group and ALF group received intraperitoneal injection with D-galactosamine (D-GalN)and LPS at 24 hours after the 5th injection of LPS or NaCl solution.At 2,6,12,24 and 48 h after D-GalN and LPS injection,the serum levels of tumor necrosis factor-α (TNF-α)and interleukin-6 (IL-6)were detected by enzyme-linked immunosorbent assay (ELISA).The liver pathologic changes were observed with HE staining by microscope.The mRNA expressions of IRAK-4,IRAK-M and NF-κB (p65)were detected by reverse transcriptase polymerase chain reaction (RT-PCR).The pairwise comparison between groups was done by lease significant difference (LSD)and Dunnet's t test.Results The liver pathologic changes in ETT group were much milder than those of ALF group.In ALF group,IRAK-4 mRNA/β-actin absorbance ratios at 2,6,12,24and 48 h after D-GalN and LPS challenge were 0.711 ±0.074,0.904±0.118,1.012 ±0.098,1.534±0.279 and 1.451±0.290,respectively,while the IRAK-M mRNA/β-actin absorbance ratios were 0.496±0.018,0.516±0.089,0.503±0.023,0.503±0.057 and 0.469±0.142,respectively.In ETT group,the IRAK-4 mRNA/β-actin absorbance ratios at 2,6,12,24 and 48 h after D-GalN and LPS challenge were 0.619±0.083,0.587±0.033,0.623±0.034,0.720±0.044 and 0.654±0.041,respectively,while the IRA'K-M mRNA/β-actin absorbance ratios were 0.929 ± 0.064,1.111±0.138,1.113±0.027,1.891±0.315 and 1.710±0.303,respectively.The IRAK-M mRNA expression level was significantly increased and IRAK-4 mRNA expression level was relatively decreased in ETT group compared to ALF group.The differences were all statistically significant at 2,6,12,24 and 48 h after D-GalN and LPS challenge (F= 17.305,54.921,121.031,67.607,55.279,respectively; F=19.506,43.777,110.823,302.681,202.822,respectively; F=172.003,59.519,987.055,68.463,96.601,respectively; all P＜0.05).Conclusion LPS pretreatment may induce ETT in rat model by downregulating the expression of IRAK-4 and upregulating the expression of IRAK-M.
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Objective To investigate the effects of curcumin on mRNA expression of cytokines related to Toll-like receptor (TLR) 4 signaling in THP-1 cells.Methods After pretreatment with different concentrations (50,25,12.5 mg/L) of curcumin or dexamethasone for 12 hours,THP-1 cells were stimulated by lipopolysaccharide (LPS.1 mg/L) for 4 hours followed by the collection of cells.Then total RNA was isolated from these cells and subjected to reverse transcription-polymerase chain reaction (RT-PCR) for the detection of mRNA expression of tumor necrosis factor receptor-associated factor 6 (TRAF6),interleukin-1 receptorassociated kinase (IRAK1) and nuclear factor (NF)-κB.THP-1 cells without pretreatment or stimulation served as negative control,and those only stimulated with LPS served as LPS group.Results After stimulation with LPS (1 mg/L) for 4 hours,the mRNA expressions of TRAF6,IRAK1 and NF-κB were significantly upregulated in THP-1 cells compared with negative control cells (f=38.69,39.13,23.99,all P<0.01).Curcumin of 50 mg/L and 25 mg/L significantly inhibited the mRNA expressions of TRAF6.IRAK 1 and NF-κB upregulated by LPS with an inhibition rate of more than 50% (all P<0.0 1).Conclusions Certain concentrations of curcumin can inhibit the mRNA expressions of TRAF6.IRAK1 and NF-κB.which demonstrates the regulatory effect of curcumin on the mRNA expressions of TLR4 signaling pathway-associated cytokines.