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Objective:To observe the effects of p21 activated kinase 4 (PAK4) on the mitochondrial function and biological behavior in retinal vascular endothelial cells.Methods:The experimental study was divided into two parts: in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 12 healthy C57BL/6J male mice were randomly divided into normal control group and diabetes group, with 6 mice in each group. Diabetes mice were induced by streptozotocin to establish diabetes model. Eight weeks after modeling, quantitative real-time polymerase chain reaction and Western blots were performed to detect the expression of PAK4 in diabetic retinas. In vitro cell experiments: the human retinal microvascular endothelial cells (hRMEC) were divided into three groups: conventional cultured cells group (N group), empty vector transfected (Vector group); pcDNA-PAK4 eukaryotic expression plasmid transfected group (PAK4 group). WB and qPCR were used to detect transfection efficiency, while scratching assay, cell scratch test was used to detect cell migration in hRMEC of each group. In vitro white blood cell adhesion experiment combined with 4 ', 6-diamino-2-phenylindole staining was used to detect the number of white blood cells adhering to hRMEC in each group. The Seahorse XFe96 cell energy metabolism analyzer measures intracellular mitochondrial basal respiration, adenosine triphosphate (ATP) production, maximum respiration, and reserve respiration capacity. The t-test was used for comparison between the two groups. Single factor analysis of variance was used for comparison among the three groups. Results:In vivo animal experiments: compared with normal control group, the relative expression levels of PAK4 mRNA and protein in retina of diabetic mice were significantly increased, with statistical significance ( t=25.372, 22.419, 25.372; P<0.05). In vitro cell experiment: compared with the N group and Vector group, the PAK4 protein, mRNA relative expression and cell mobility in the hRMEC of PAK4 group were significantly increased, with statistical significance ( F=36.821, 38.692, 29.421; P<0.05). Flow cytometry showed that the adhesion number of leukocytes on hRMEC in PAK4 group was significantly increased, and the difference was statistically significant ( F=39.649, P<0.01). Mitochondrial pressure measurement results showed that the capacity of mitochondrial basic respiration, ATP production, maximum respiration and reserve respiration in hRMEC in PAK4 group was significantly decreased, with statistical significance ( F=27.472, 22.315, 31.147, 27.472; P<0.05). Conclusion:Over-expression of PAK4 impairs mitochondrial function and significantly promotes leukocyte adhesion and migration in retinal vascular endothelial cells.
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Immunotherapy has revolutionized the landscape of cancer treatment. However, single immunotherapy only works well in a small subset of patients. Combined immunotherapy with antitumor synergism holds considerable potential to boost the therapeutic outcome. Nevertheless, the synergistic, additive or antagonistic antitumor effects of combined immunotherapies have been rarely explored. Herein, we established a novel combined cancer treatment modality by synergizing p21-activated kinase 4 (PAK4) silencing with immunogenic phototherapy in engineered extracellular vesicles (EVs) that were fabricated by coating M1 macrophage-derived EVs on the surface of the nano-complex cores assembled with siRNA against PAK4 and a photoactivatable polyethyleneimine. The engineered EVs induced potent PAK4 silencing and robust immunogenic phototherapy, thus contributing to effective antitumor effects in vitro and in vivo. Moreover, the antitumor synergism of the combined treatment was quantitatively determined by the CompuSyn method. The combination index (CI) and isobologram results confirmed that there was an antitumor synergism for the combined treatment. Furthermore, the dose reduction index (DRI) showed favorable dose reduction, revealing lower toxicity and higher biocompatibility of the engineered EVs. Collectively, the study presents a synergistically potentiated cancer treatment modality by combining PAK4 silencing with immunogenic phototherapy in engineered EVs, which is promising for boosting the therapeutic outcome of cancer immunotherapy.
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AIM:To explore the mechanism of p21-activated kinase 4 (PAK4) on non-small-cell lung cancer (NSCLC) migration and invasion.METHODS:After A549 and NCI-H520 cell lines were transfected with PAK4-siRNA or negative control , the expression of PAK4 at mRNA and protein levels was detected by real-time PCR and Western blot , re-spectively .The invasion and migration of A 549 cells and NCI-H520 cells were measured by Matrigel invasion assay and Transwell migration assay.LIMK1, cofilin, and their respective phosphorylation were examined by Western blot .The inter-action of PAK4 and LIMK1 was investigated by co-immunoprecipitation assay .The relationship between PAK4 and LIMK1 phosphorylation was examined by a protein kinase assay in the A 549 cells and NCI-H520 cells.The expression of PAK4 and p-LIMK1 in 10 human NSCLC tissues was examined by Western blot .A549 cells and NCI-H520 cells were individually or commonly transfected with PAK4-siRNA or LIMK1 plasmid in order to observe the cell migration and invasion .RE-SULTS:After A549 cells and NCI-H520 cells were transfected with PAK4-siRNA for 48 h, the expression of PAK4 at mR-NA and protein levels , and the numbers of invasion and migration cells in PAK 4-siRNA group were lower than those in con-trol group.Compared with control group , the phosphorylation of LIMK1 and cofilin was lower in PAK4-siRNA group, whereas the total expression levels of LIMK 1 and cofilin did not change .The results of co-immunoprecipitation assays showed that PAK4 specifically interacted with LIMK1 in A549 and NCI-H520 cells.LIMK1 phosphorylation in the presence of PAK4 (K350M) was significantly lower than that in the presence of PAK 4 (WT) or PAK4 (S445N) in the protein ki-nase assay.The PAK4 upregulation was positively correlated with the level of p-LIMK1 (P<0.05).After A549 cells and NCI-H520 cells were co-transfected with PAK4-siRNA and LIMK1 plasmid, the migration and invasion cell numbers in co-transfection group were higher than those in PAK 4-siRNA transfection group .CONCLUSION: PAK4 promotes the inva-sive and migratory abilities of NSCLC , which is mediated by LIMK 1 phosphorylation .
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AIM:To investigate the expression and clinical significance of PAK4 in the cell lines and tissues of non-small cell lung cancer (NSCLC).METHODS:PAK4 expression in human bronchial epithelial (HBE) cells, NSCLC cell lines, NSCLC tissues and adjacent non-tumor tissues were assessed by immunohistochemistry, real-time PCR and Western blot.Prognostic value of PAK4 expression was evaluated by Kaplan-Meier analysis and Cox regression.RE-SULTS:PAK4 was over-expressed in the NSCLC cell lines at both mRNA and protein levels compared with HBE cells ( P<0.05).PAK4 was over-expressed in the NSCLC tissues at both mRNA and protein levels compared with adjacent non-tumor tissues (P<0.05).PAK4 was over-expressed in the metastatic NSCLC tissues compared with the primary NSCLC tissues (P<0.05).Higher PAK4 staining scores were positively correlated with differentiation, lymph node metastasis, distant metastasis, and clinical stage.Kaplan-Meier analysis and log-rank test showed that overall survival was significantly different between the patients with up-regulated PAK4 and the patients with down-regulated PAK4 ( P<0.05) .PAK4 over-expression was associated with NSCLC progression.CONCLUSION:Increased PAK4 expression was associated with tumor invasion, metastasis and prognosis in the patients with NSCLC.PAK4 is an important prognostic marker and potential ther-apeutic target in NSCLC.
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p21 activated kinase 4 (PAK4) is a member of a family of serine/threonine kinase.PAK4plays an important role in a variety of cellular functions including cell cycle,cell proliferation,apoptosis,and cytoskeletal reorganization.Recently,PAK4 has been shown to overexpress in a variety of malignancies,and contribute to cancer cell migration and invasion through multiple signalling pathways.