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Abstract Objectives Melanoma is one of the leading causes of cancer death. Kinesin Family member 22 (KIF22) is essential for the invasion of melanoma cells, but the role and mechanism of KIF22 in the proliferation and glycolysis in melanoma remains unknown. Methods KIF22 expression in melanoma tissues and the relationship between KIF22 high expression and overall survival rate in patients with melanoma were analyzed using the Tnmplot database. KIF22 expression in melanoma cells was examined by western blot. Then, KIF22 was silenced and CCK-8 assay, EDU staining and flow cytometry analysis were adopted for assessing cell proliferation and apoptosis. In addition, the glycolysis metabolism of melanoma cells was reflected by detecting Extracellular Acidification Rates (ECAR) and Oxygen Consumption Rates (OCR). The expression of proteins related to apoptosis, glycolysis and EGFR/STAT3 signaling was tested by western blot. Subsequently, melanoma cells were treated with EGF or Colivelin to further elucidate the regulatory effect of KIF22 on EGFR/STAT3 signaling. Results KIF22 expression was notably upregulated in melanoma tissues and cells, and KIF22 high expression was associated with a poor prognosis. Moreover, KIF22 insufficiency suppressed proliferation and accelerated apoptosis of melanoma cells. Additionally, glycolysis was reduced by KIF22 depletion, evidenced by the decreased ECAR and increased OCR, accompanied by the downregulated expression of HK2, PKM2 and LDHA. Importantly, the impacts of KIF22 depletion on the progression of melanoma were partially attenuated after EGF or Colivelin treatment. Conclusion Collectively, KIF22 knockdown suppressed the proliferation and glycolysis and facilitated the apoptosis of melanoma cells by inactivating EGFR/STAT3 signaling.
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Aim To investigate the anti-neuropathic pain effect of DXL-A-22 and further to explore the potential mechanisms. Methods The anti-neuropathic pain effect was evaluated by chronic constriction injury (CCI) model. The potential anti-neuropathic pain mechanisms of DXL-A-22 was studied by Western blot and qPCR. The acute toxicity was evaluated by ultimate test. Results DXL-A-22 (2,1,0. 5 mg . kg-1 ,i. g.) dose-dependently elevated the mechanical withdrawal threshold (MWT) and the paw withdrawal latency (PWL) in CCI rats (P < 0. 05, P < 0. 01), the percentage of pain threshold elevation (PTE%) and the percentage of Maximal Possible Effect (MPE%) was 108%,86%,71% and 77%,56%,43% respectively on day 7 post-operation. DXL-A-22 (2 mg . kg-1 ,i. g.) significantly reduced the expression of p-CaMK II α, p-CREB, p-JAK2, p-STAT3 proteins and TNF-α mRNA, c-Fos mRNA in DRG (P < 0. 05, P < 0.01), and the percent inhibition was 37%, 48%, 35%,58%, 39% and 32% respectively. The expression of TNF-α mRNA and c-Fos mRNA in spinal pord was reduced by 47% and 72% respectively in CCI rats (P <0. 01). Acute toxicity test showed that DXL-A-22 had no obvious toxicity reaction. Conclusions Spirocyclopiperazinium salt compound DXL-A-22 exerts significant antinociceptive effect on CCI model. The anti-neuropathic pain effect of DXL-A-22 may be related to the inhibition of CaMK II α/CREB and JAK2/STAT3 signaling pathways, and the inhibition of the mRNA expression of TNF-α and c-Fos.
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AIM:To determine the effects and mechanisms of interleukin-22 (IL-22) on the fibroblast-like sy-noviocytes ( FLSs) from rheumatoid arthritis ( RA) patients.METHODS:RA-FLSs were cultured by tissue culture meth-od.RA-FLSs were incubated with different concentrations of IL-22 (0,1,10,100μg/L) for 24 h, 48 h and 72 h.The cell viability was examined by CCK-8 assay.IL-22 at concentration of 10 μg/L was used to stimulate RA-FLSs for 24 h, and the change of cell cycle distribution was identified by flow cytometry.The effects of IL-22 at concentrations of 0, 1, 10, 100μg/L and/or STA-21 (a STAT3 inhibitor at concentrations of 0, 25, 50μmol/L) on the protein levels of Bcl-2 and p-STAT3 in the RA-FLSs were determined by Western blot.RESULTS:Compared with control group, stimulation of rhIL-22 at different concentrations for 24 h, 48 h and 72 h, the cells viabilityof RA-FLSs were obviously increased ( P<0.05 ) . After co-cultured with 10 μg/L rhIL-22 for 24 h, the percentages of RA-FLSs were obviously increased in the G2/M+S phase and decreased in the G0/G1 phase.At the same time, rhIL-22 increased, but STA-21 decreased the protein levels of Bcl-2 but p-STAT3 in the RA-FLSs obviously (P<0.05).Treatment with STAT3 inhibitor STA-21 reversed the effect of IL-22-induced Bcl-2 upregulation in the RA-FLSs ( P<0.01 ) .CONCLUSION: STAT3 is critical in the process of IL-22-induced Bcl-2 upregulation in RA-FLSs, indicating that IL-22 may play a role in the apoptosis of RA-FLSs.
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Objective To investigate the mechanisms underlying intedeukin-22 (IL-22)-induced expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in HaCaT cells.Methods Some HaCaT cells were divided into several inverention groups treated with IL-22 at concentrations of 12.5,25,50,100 μg/L,respectively and a control group treated with phosphate buffer saline (PBS).After 24-hour culture,total proteins were extracted from the HaCaT cells,and Western blot was performed to measure the expression of phosphorylated extracellular signalregulated kinase 1/2 (P-ERK1/2) in the mitogen-activated protein kinase (MAPK)-ERK1/2 pathway,as well as phosphorylated-JAK2 (P-JAK2) and phosphorylated-signal transducer and activator of transcription 3 (P-STAT3) in the JAK2/STAT3 pathway.In a blocking experiment,some HaCaT cells were divided into 4 groups to be treated with PBS,IL-22,PD98059 (an inhibitor of MAPK-ERK1/2) combined with IL-22 (PD98059 group),AG490 (an inhibitor of JAK2/STAT3) combined with IL-22 (AG490 group),respectively.After 24-hour treatment,total proteins and mRNAs were extracted from the HaCaT cells followed by Western blot and real-time quantitative reverse transcription-PCR for the measurement of protein and mRNA expressions of HB-EGF respectively.Statistical analysis was carried out with the software SPSS 16.0 by one-way analysis of variance (ANOVA) for intergroup comparisons and by Bonferroni's test for multiple comparisons.Results After treatment with IL-22 at the above 4 concentrations,the expressions of P-ERK1/2,P-JAK2 and P-STAT3 in HaCaT cells were all increased compared with the control group (all P ＜ 0.05).The protein and mRNA expression levels (expressed as the HB-EGF/β-actin ratio and 2-△△Cr respectively) of HB-EGF were both significantly decreased in the PD98059 group and AG490 group than in the IL-22 group (protein:0.183 ± 0.020 and 0.199 ± 0.011 vs.0.924 ± 0.032,F =37.700,36.400,respectively,both P ＜ 0.05; mRNA:1.034 ± 0.072 and 0.989 ± 0.038 vs.1.844 ± 0.135,F =11.271,13.429,respectively,both P ＜ 0.05).Conclusions IL-22 can activate the MAPK-ERK1/2 and JAK2/STAT3 signaling pathways in HaCaT cells,which may contribute to IL-22-induced expression of HB-EGF in HaCaT cells.
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Objective To evaluate the effects of interleukin-22(IL-22)on the expression of tazarotene-induced gene 3(TIG3)in HaCaT cells. Methods Cultured HaCaT cells were randomly divided into several groups to be treated with different concentrations (12.5, 25, 50, 100 μg/L)of IL-22 alone, or the combination of 50 μg/L IL-22 with the MAPK-ERK1/2 inhibitor PD98059 or the JAK/STAT inhibitor AG490 for 24 hours. Those HaCaT cells treated with phosphate buffered saline served as the control group. Subsequently, total proteins and mRNAs were extracted from the HaCaT cells. An immunofluorescence assay, Western blot and enzyme-linked immunosorbent assay (ELISA) were performed to determine the protein expression level of TIG3, and real-time fluorescence-based quantitative PCR to quantify the mRNA expression of TIG3 in HaCaT cells. Results The immunofluorescence assay showed that TIG3 protein was mainly expressed in the cytoplasm of HaCaT cells. As Western blot revealed, the protein expression level of TIG3 was 0.743 ± 0.035, 0.678 ± 0.040, 0.582 ± 0.041 and 0.328 ± 0.032 in HaCaT cells treated with IL-22 of 12.5, 25, 50 and 100μg/L, respectively, significantly lower than that in the control group (0.839 ± 0.045, all P<0.05). ELISA also showed a decrease in the protein expression of TIG3 in IL-22-treated HaCaT cells, which was consistent with Western blot results. Further more, the mRNA expression level (2-△△Ct)of TIG3 was significantly weaker in HaCaT cells treated with IL-22 of 12.5, 25, 50 and 100μg/L than in the control group (0.838 ± 0.036, 0.686 ± 0.061, 0.565 ± 0.047 and 0.457 ± 0.033 vs. 1.000, all P< 0.05). The decrease in TIG3 mRNA and protein expressions was significantly attenuated in HaCaT cells treated with the combination of 50 μg/L IL-22 with PD98059 or AG490 compared with those treated with 50 μg/L IL-22 alone. Conclusion IL-22 can dose-dependently inhibit the expression of TIG3 in HaCaT cells, likely through the MAPK-ERK1/2 and JAK2/STAT3 signaling pathways.