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Purpose: Napabucasin (NP) is a natural compound that can suppress cancer cell proliferation and cell cycle by inhibition of the signal transducer and activator of transcription 3 (STAT3) gene. We examined the effects of NP on the proliferation and invasion of neuroblastoma cells (SH-SY5Y). Methods: Human neuroblastoma SH-SY5Y cell line was used in this study. NP was administered to groups at the doses of 0.3-1 µM. Cell viability was analyzed by MTT assay. Real-time quantitative reverse transcription polymerase chain reaction and western blot analysis assessed the expressions of interleukin (IL)-6 dependent Jak2/Stat3 signaling pathway. The MTT cell viability method was applied to determine the antagonistic-synergistic effects and inhibitory concentration (IC50) doses of doxorubicin (DX) and NP. Results: It was determined that 0.3-1 µM doses of NP killed the cells almost completely after 48 hours, and also that Jak2/Stat3 expressions decreased dose-dependently via IL-6. At the protein level, NP and DX were found to reduce Jak2 and Stat3 levels. Conclusions: NP showed that it suppresses the proliferation of neuroblastoma cells. Due to its inhibitory effect on Jak2 and Stat3, it can be used to prevent invasion of SH-SY5Y cells. NP, which can inactivate Jak2/Stat3, can be used as a treatment agent by combining with DX in proliferation pathway in neuroblastoma.
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Doxorubicin , Cell Culture Techniques , STAT3 Transcription Factor , Cerebrum , NeuroblastomaABSTRACT
Objective To investigate the effects of chronic starvation stress on the proliferation and migration of colorectal cancer cells, as well as the underlying mechanisms. Methods By using prolonged serum starvation to simulate chronic starvation stress in tumor cells, we established enduring serum-deprived models of SW480 and DLD-1 cells and observed cellular morphological change. Effects of prolonged serum starvation on SW480 and DLD-1 proliferative and migratory capabilities were assessed using CCK-8 and Transwell assays. Differential gene-expression analysis on SW480 cultured with 1% FBS or 10% FBS medium was followed by GO and KEGG pathway assessments. Migration-related protein interactions were explored using String database and Metascape software, leading to 16 genes being selected for RT-qPCR validation. Protein levels of ITGB1 and key molecules in the relevant pathways were measured. Mobility changes in SW480 were observed through Transwell assay after ITGB1 knockdown or STAT3 inhibition. Results Prolonged serum starvation significantly inhibited the proliferation of SW480 and DLD-1 cells, and DLD-1 mobility, while enhanced SW480 migration. Transcriptome analysis revealed that prolonged serum deprivation caused the upregulation of 3016 genes, among which 283 were involved in cell migration. Metascape analysis identified the correlations among potential core genes ITGB1, CD44, TNS1, STAT3, etc. Prolonged serum deprivation increased the mRNA levels of VTN, TNS1, VEGFA, STAT3, and ITGB1 while also increasing the protein levels of ITGB1 and MMP2 and the phosphorylation levels of JAK2 and STAT3. Mobility reduction in prolonged serum-starved SW480 cells was achieved through ITGB1 knockdown or a STAT3 inhibitor. Conclusion Colorectal cancer cells can endure chronic starvation stress which enhances migration capability by upregulating ITGB1 expression.
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Objective:To This study was to investigate the expression and possible clinical significance of microRNA-146b (miR-146b) and signal transducer and transcriptional activator 1 and 3 (STAT1/3) in peripheral blood mononuclear cells (PBMCs) of patients with primary gouty arthritis (GA).Methods:The peripheral blood samples, clinical data and laboratory indexes of 120 male cases of GA [including 57 cases of acute (AG group) and 63 cases of intermittent (IG group)]and 66 healthy subjects (HC group) were collected. The expression levels of miR-146b and STAT1/3 in PBMCs were detected by real-time fluorescence quantitative PCR (RT-qPCR). The differences among the three groups were compared and the correlation between them and clinical indexes was analyzed. The receiver operating characteristic curve (ROC) was constructed to evaluate its diagnostic value in GA. After the PBMCs of 18 healthy subjects were stimulated by 100 μg/ml MSU for 3 hours to simulate acute gout inflammatory environment, the transcriptional changes of IL-1β, miR-146b and STAT1/3 were detected by RT-qPCR, and the expressions of IL-1β, STAT1/3 protein and phosphorylated protein were detected by Western blotting. T test or one-way ANOVA and LSD- t test were used in accordance with the normal measurement data, Kruskal-Wallis H and Mann-Whitney U test were used in the non-normal data, Spearman correlation analysis was used in the correlation between variables, and the diagnostic value was evaluated by the receiver working characteristic curve ROC. Results:①There were statistical differences in the expression of miR-146b, STAT1 and STAT3 among the three groups ( F=7.02、19.52、17.07, all P<0.001). The expression of miR-146b in gout group [(0.32±0.28)] was significantly higher than that in HC group (0.19±0.18)( t=2.96, P=0.003), while STAT1(0.019±0.012) and STAT3(0.014±0.010) were significantly lower than those in HC group (0.038±0.029),(0.025±0.016)( t=6.26, 5.56, both P<0.001). Further subgroup analysis showed that the expression of miR-146b in AG and IG groups was higher than that in HC group[(0.27±0.17), (0.38±0.35), (0.19±0.18), t=2.09, 3.30, both P<0.05], but that in AG group was lower than that in IG group ( t=2.02, P<0.05). The expression of STAT1 mRNA in AG and IG groups was lower than that in HC group [(0.020±0.012), (0.019±0.012), (0.038±0.029), t=4.89, 4.56, both P<0.001], but there was no statistical significance between AG and IG groups ( t=0.24, P>0.05). The expression of STAT3 mRNA in AG and IG groups was lower than that in HC group [(0.016±0.012), (0.012±0.008), (0.025±0.016), t=5.64, 3.33, both P<0.01], and the expression of STAT3 mRNA in AG group was higher than that in IG group ( t=2.12, P<0.05). ② Spearman correlation analysis showed that the expression of miR-146b in GA was negatively correlated with HCY ( r=-0.37, P=0.014), STAT1 was negatively correlated with Crea ( r=-0.29, P=0.019), positively correlated with eGFR ( r=0.25, P=0.047), and STAT3 was negatively correlated with HDL-C ( r=-0.27, P=0.033). ③ROC curve showed that the AUC (95% CI) of miR-146b, STAT1 and STAT3 were 0.679(0.582, 0.776), 0.710(0.629, 0.791) and 0.705(0.626, 0.783), and the combined AUC(95% CI) of the three was 0.836 (0.765, 0.907). ④Compared with blank control group and negative control group, the expression of miR-146b in PBMCs of 18 cases of HC was significantly decreased ( H=14.44, P=0.003), while the expression of IL-1β, STAT1 and STAT3 mRNA was significantly increased after 3 h of MSU stimulation ( H=26.44、27.26、15.90, all P<0.001). The expression of IL-1β, STAT1 and STAT3 protein and phosphorylated protein in the model group were significantly higher than those in the blank control group, and the differences were statistically significant ( t=9.97、6.63、7.48、11.25、6.28, all P<0.01). Conclusion:The abnormal expression of miR-146b and STAT1/3 in GA is related to some clinical indicators, suggesting that it may be involved in the regulation of gout immune inflammatory response and metabolism, and the specific mechanism is worth further study.
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Objective:To evaluate the role of signal transducer and activator of transcription 3 (STAT3)/nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy in salidroside-induced attenuation of intestinal ischemia-reperfusion (I/R) injury in mice and the relationship with ferroptosis.Methods:Thirty-six SPF-grade healthy male C57BL mice, aged 6-8 weeks, weighing 20-25 g, were divided into 6 groups ( n=6 each) by a random number table method: sham operation group (S group), sham operation+ salidroside group (SS group), intestinal I/R group (IR group), intestinal I/R+ salidroside group (IS group), intestinal I/R+ salidroside+ autophagy activator rapamycin group (ISR group) and intestinal I/R+ salidroside+ STAT3 activator colivelin group (ISC group). The intestinal I/R injury model was established by clamping the superior mesenteric artery for 45 min followed by 30-min reperfusion in IR, IS, ISR and ISC groups, while the superior mesenteric artery was only isolated without clipping in S and SS groups. At 1 week before developing the model, salidroside 40 mg/kg was intraperitoneally injected once a day for 7 consecutive days in SS, IS, ISC and ISR groups, rapamycin 4 mg/kg was intraperitoneally injected once a day for 7 consecutive days in group ISR, colivelin 1 mg/kg was intraperitoneally injected once a day for 7 consecutive days in group ISC, while the equal volume of normal saline was given instead in the rest two groups. The mice were sacrificed at 30 min of reperfusion, and intestinal tissues were obtained for examination of the pathological changes after HE staining (with a optical microscope) which were scored according to Chiu and for determination of contents of malondialdehyde (MDA), Fe 2+, glutathione (GSH) and reactive oxygen species (ROS), activity of superoxide dismutase (SOD) and expression of p-STAT3, STAT3, glutathione peroxidase 4 (GPX4), NCOA4 and ferritin heavy chain 1 (FTH1) in intestinal tissues (by Western blot). Results:Compared with group S, the Chiu′s score and contents of MDA, Fe 2+ and ROS were significantly increased, the content of GSH was decreased, the activity of SOD was decreased, the expression of p-STAT3 and NCOA4 was up-regulated, the expression of GPX4 and FTH1 was down-regulated, the p-STAT3/STAT3 ratio was increased ( P<0.05), pathological injury was found in intestinal tissues, and no significant change was found in the aforementioned indexes in group IR( P>0.05). Compared with group IR, the Chiu′s score and contents of MDA, Fe 2+ and ROS were significantly decreased, GSH content was increased, SOD activity was increased, the expression of p-STAT3 and NCOA4 was down-regulated, the expression of GPX4 and FTH1 was up-regulated, p-STAT3/STAT3 ratio was decreased ( P<0.05), and the pathological injury was significantly alleviated in intestinal tissues in group IS. Compared with group IS, the Chiu′s score and contents of MDA, Fe 2+ and ROS were significantly increased, GSH content was decreased, SOD activity was decreased, the expression of p-STAT3 and NCOA4 was up-regulated, the expression of GPX4 and FTH1 was down-regulated, p-STAT3/STAT3 ratio was increased ( P<0.05), and the pathological injury was aggravated in intestinal tissues in ISR and ISC groups. There was no statistically significant difference in the expression of STAT3 among the five groups ( P>0.05). Conclusions:STAT3/NCOA4-mediated ferritinophagy is involved in the process of salidroside-induced reduction of intestinal I/R injury in mice, which may be related to inhibiting ferroptosis.
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Objective:To determine S100A10 protein expression in psoriatic lesions, and to investigate its effect on psoriasis-like skin inflammation in imiquimod (IMQ) -induced mouse models.Methods:From January 2020 to June 2022, skin lesions were surgically collected from 28 patients with psoriasis in the Department of Dermatology, the First Affiliated Hospital of Xinxiang Medical University; normal skin tissues were collected from 18 healthy subjects in the Department of General Surgery and Department of Ophthalmology during the same period, and served as a control group. Immunohistochemical staining was performed to determine the S100A10 protein expression in skin lesions from psoriasis patients and normal skin tissues from healthy controls. Ten wild-type (WT) C57BL/6J female mice and 10 S100A10 -/- C57BL/6J female mice were selected to establish the IMQ-induced mouse models of psoriasis-like skin inflammation. Then, the mice were randomly divided into gene knock-out (KO) /IMQ group, WT/IMQ group, KO/vaseline (VAS) group, and WT/VAS group by using a random number table, and there were 5 mice in each group. The mice in the KO/IMQ group and WT/IMQ group were topically treated with IMQ cream (62.5 mg) on the shaved back daily to establish the mouse models of psoriasis-like skin inflammation, while the mice in the KO/VAS group and WT/VAS group were topically treated with vaseline cream (62.5 mg) daily, and both treatments lasted 7 consecutive days. Skin lesions on the back were observed daily. On day 7, the mice were sacrificed by cervical dislocation, and their dorsal skin tissues were excised. The IMQ-induced psoriasis-like skin inflammation was evaluated by the psoriasis area and severity index (PASI), pathological manifestations of skin lesions were observed by hematoxylin and eosin staining, the expression of S100A10 and Ki-67 in skin lesions was determined by immunohistochemical staining, the expression of STAT3, IL-17A and other cytokines was determined by Western blot analysis, and IL-17 mRNA expression was determined by real-time fluorescence-based quantitative PCR (qPCR). Statistical analysis was carried out by using the independent sample t-test, nonparametric U test, and chi-square test. Results:Immunohistochemical staining showed that the S100A10 protein expression was significantly lower in the psoriasis vulgaris lesions than in the normal control skin tissues ( Z = -3.47, P < 0.001). In the mouse models, the S100A10 protein expression was significantly lower in the skin lesions of mice in the WT/IMQ group than in the skin tissues of mice in the WT/VAS group ( t = 3.64, P = 0.007), and the Ki-67 expression was significantly higher in the KO/IMQ group than in the WT/IMQ group ( t = 2.97, P = 0.041). Additionally, the mice in the KO/IMQ group presented with more severe clinical manifestations such as scales and infiltration compared with those in the WT/IMQ group. Western blot analysis showed that the phosphorylated STAT3/STAT3 expression and IL-17A protein expression was significantly higher in the KO/IMQ group than in the WT/IMQ group ( t = 3.27, 3.48, P = 0.031, 0.025, respectively), and qPCR revealed that the IL-17A mRNA expression was also significantly higher in the KO/IMQ group than in the WT/IMQ group ( t = 2.73, P = 0.029) . Conclusion:S100A10 protein was underexpressed in the skin lesions of psoriasis patients, and the deletion of S100A10 protein aggravated IMQ-induced psoriasis-like skin inflammation in mice, possibly by upregulating STAT3 phosphorylation in the epidermis.
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Objective:To explore the effects of breast cancer mesenchymal stem cells (BC-MSC) on the proliferation and migration of breast cancer MCF-7 cells and the related mechanisms.Methods:The resected cancer tissues and paracancerous tissues were taken from breast cancer patients after surgery, and the bone marrow samples of healthy people were selected. BC-MSC, breast cancer paracancerous mesenchymal stem cells (BCN-MSC) and bone marrow mesenchymal stem cells (BM-MSC) of healthy people were isolated and cultured by tissue adhesion method, and their differentiation ability was induced by the addition of osteogenic and lipogenic induction, and their surface markers were detected by flow cytometry. The supernatants of BC-MSC, BCN-MSC and BM-MSC of healthy people cultured for 48 h were collected and used for the culture of MCF-7 cells as BC-MSC group, BCN-MSC group and BM-MSC group, respectively, and the control group was the conventional cultured MCF-7 cells. The proliferation ability of MCF-7 cells in each group was detected by methyl thiazol tetrazolium (MTT) assay, the clone formation ability of MCF-7 cells was detected by plate cloning assay, the migration ability of MCF-7 cells was detected by Transwell assay, and the mRNA relative expressions of interleukin (IL)-6 and epithelial mesenchymal transition (EMT)-related genes (E-cadherin, vimentin, snail) were detected by quantitative real-time fluorescence polymerase chain reaction (qRT-PCR) in MCF-7 cells. Western blotting was used to detect expressions of p-STAT3, E-cadherin, vimentin and snail proteins in MCF-7 cells. Luminex liquid microarray technology was used to detect cytokine levels in culture supernatants of different mesenchymal stem cells (MSC). IL-6 neutralizing antibody was added into the supernatant of BC-MSC, MCF-7 cells were cultured with the supernatant (BC-MSC+IL-6 neutralizing antibody group), and then the proliferation and migration abilities of MCF-7 cells were tested, as well as the expression changes of related genes and proteins.Results:BC-MSC, BCN-MSC and BM-MSC were successfully isolated; BC-MSC had positive expressions of CD29, CD44 and CD90 and negative expressions of CD14, CD34 and CD45, which were in line with the characteristics of MSC. MTT assay showed that the absorbance values of MCF-7 cells cultured for 48 h in the control group, BC-MSC group, BCN-MSC group and BM-MSC group were 0.31±0.02, 0.54±0.03, 0.43±0.02 and 0.42±0.02, respectively, and the difference was statistically significant ( F = 56.52, P < 0.05); the results of plate cloning experiments showed that the number of clones in each petri dish of the four groups were 180±9, 439±17, 319±16 and 306±19, respectively, and the difference was statistically significant ( F = 222.70, P < 0.05); Transwell assay showed that the numbers of membrane-penetrating cells in the four groups were 6.5±1.0, 23.2±2.4, 16.0±1.3 and 14.8±2.0, respectively, with the statistically significant difference ( F = 49.44, P < 0.05); qRT-PCR assay showed that the relative expressions of IL-6 mRNA in the control group, BC-MSC group, BCN-MSC group and BM-MSC group were 1.07±0.11, 13.79±3.80, 6.68±1.66 and 6.12±1.52, respectively, and the difference was statistically significant ( F = 107.60, P < 0.05), and the relative expression of E-cadherin mRNA in MCF-7 cells of BC-MSC, BCN-MSC and BM-MSC groups was lower than that of the control group, while the relative expressions of vimentin and snail mRNA were higher than those of the control group, and the differences were statistically significant (all P < 0.05). Western blotting assay showed that the relative expression of E-cadherin mRNA in MCF-7 cells of BC-MSC, BCN-MSC and BM-MSC groups was lower than that of the control group. Western blotting showed that the level of E-cadherin protein in BC-MSC, BCN-MSC and BM-MSC groups was lower than that in the control group, and the levels of vimentin and snail proteins were higher than those in the control group; Luminex liquid microarray technology showed that the content of IL-6 cytokine in the supernatants of BC-MSC, BCN-MSC and BM -MSC cultures were higher, and the relative expressions were 1.75±0.21, 1.00±0.10 and 0.96±0.08, respectively, and the difference was statistically significant ( F = 43.22, P < 0.05). The results of MTT assay showed that the absorbance values of MCF-7 cells in BC-MSC group and BC-MSC+IL-6 neutralizing antibody group were 0.56±0.05 and 0.42±0.04, respectively, and the difference was statistically significant ( t = -3.11, P < 0.05); the results of Transwell assay showed that the numbers of membrane-penetrating cells in the two groups were 30.3±1.5 and 17.3±2.1, respectively, and the difference was statistically significant ( t = -7.12, P < 0.05); qRT-PCR assay showed that the relative expressions of E-cadherin mRNA were 0.44±0.05 and 0.76±0.05 ( t = 6.40, P < 0.01), the relative expressions of vimentin mRNA were 2.90±0.21 and 1.79±0.21 ( t = 5.29, P < 0.01), and the relative expressions of snail mRNA were 3.20±0.20 and 1.91±0.30 ( t = 2.16, P < 0.01); Western blotting assay showed that the degrees to down-regulate the expression of E-cadherin protein and up-regulate the expressions of vimentin and snail proteins in the BC-MSC+IL-6 neutralizing antibody group were weakened compared with the BC-MSC group. Conclusions:BC-MSC can promote the proliferation and migration of breast cancer MCF-7 cells probably through activating IL-6-STAT3 signaling pathway-induced EMT by its secretion of IL-6.
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Objective:To investigate the effects of mild hypothermia on microglia polarization and janus kinase 2/signal transduction and transcriptional activation factor 3 (JAK2/STAT3) signaling pathway during cerebral ischemia-reperfusion (I/R) in rats.Methods:Forty-five clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 260-280 g, were divided into 3 groups ( n=15 each) by the random number table method: sham operation group (S group), cerebral I/R group (I/R) and mild hypothermia group (H group). In I/R group and H group, cerebral I/R was induced by middle cerebral artery occlusion using a nylon thread in anesthetized animals, the nylon thread was removed to restore the perfusion after 2 h of occlusion, and the rectal temperature was maintained at 36-37 ℃ during the period. Group H was wiped with 75% alcohol for 3 h starting from the time point immediately after reperfusion, and the rectal temperature was maintained at 32-33℃. Modified neurological severity score (mNSS) was evaluated at 24 h of reperfusion. Animals were then sacrificed for determination of the cerebral infarct size (using TTC staining), expression of M1 marker inducible nitric oxide synthase (iNOS), M2 marker arginase 1(Arg-1), phosphorylated JAK2(p-JAK2)and phosphorylated STAT3(p-STAT3)(by Western blot), expression of iNOS mRNA and Arg-1 mRNA (by quantitative polymerase chain reaction), and contents of interleukin-6 (IL-6) and IL-10 (by enzyme-linked immunosorbent assay). Results:Compared with group S, mNSS and cerebral infarct size were significantly increased, the expression of iNOS, Arg-1 protein and mRNA in cerebral ischemic penumbral zone was up-regulated, and the p-JAK2/JAK2 ratio, p-STAT3/STAT3 ratio, and contents of IL-6 and IL-10 were increased in the other two groups ( P<0.05). Compared with I/R group, mNSS and cerebral infarct size were significantly decreased, the expression of iNOS protein and mRNA in cerebral ischemic penumbral zone was down-regulated, the expression of Arg-1 and mRNA was up-regulated, and the p-JAK2/JAK2 ratio, p-STAT3/STAT3 ratio and IL-6 content were decreased, and the IL-10 content was increased in group H ( P<0.05). Conclusions:Mild hypothermia can promote the polarization shift of microglia from M1 to M2 phenotype during cerebral I/R and inhibit the central inflammatory responses, and the mechanism may be related to inhibition of JAK2/STAT3 signaling pathway in rats.
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Objective:To investigate the effect of Xidi Liangxue recipe on the proliferation and apoptosis of HaCaT cells through the long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) /microRNA (miR) -485-5p/signal transducer and activator of transcription 3 (STAT3) regulatory network. Methods:HaCaT cells were induced by interleukin-17 (IL-17), and the mRNA and protein expression of lncRNA NEAT1, miR-485-5p and STAT3 was detected in IL-17-induced HaCaT cells and normal human epidermal keratinocytes (NHEK) by quantitative PCR (qPCR) and Western blot analysis, respectively. The location of lncRNA NEAT1 and miR-485-5p in IL-17-induced HaCaT cells was observed by fluorescence in situ hybridization (FISH), and the targeted regulatory relationship among lncRNA NEAT1, miR-485-5p and STAT3 was verified by double-luciferase reporter gene assay. Chinese herbs were decocted according to the Xidi Liangxue recipe, SD rats were divided into two groups to be gavaged with the above decoctions (medicated group) or physiological saline (control group) for 5 days, and then serum samples were collected from the above two groups of rats separately. The IL-17-induced HaCaT cells were divided into 4 groups: control group treated with the control sera, lncRNA-NEAT1 overexpression group transfected with lncRNA-NEAT1 overexpression vectors and treated with the control sera, Xidi Liangxue recipe group treated with the medicated sera, and Xidi Liangxue recipe + lncRNA-NEAT1 overexpression group transfected with lncRNA-NEAT1 overexpression vectors and treated with the medicated sera. qPCR, Western blot analysis, flow cytometry, and cell counting kit (CCK8) assay were performed to determine the mRNA and protein expression of lncRNA NEAT1, miR-485-5p and STAT3, and to evaluate cell proliferation and apoptosis. The two independent samples t-test was used for comparisons between two groups, one-way analysis of variance for comparisons among multiple groups, and least significant difference (LSD) t-test for multiple comparisons. Results:The IL-17-induced HaCaT cell group showed significantly increased relative expression levels of lncRNA NEAT1 and STAT3 mRNA (1.84 ± 0.21, 2.20 ± 0.24, respectively) and significantly increased protein expression of STAT3 and p-STAT3 (1.27 ± 0.13, 2.43 ± 0.16, respectively), but significantly decreased expression level of miR-485-5p (0.32 ± 0.04) compared with the NHEK group (lncRNA NEAT1 and STAT3 mRNA: 1.00 ± 0.11, 1.00 ± 0.11, respectively, both P < 0.05; STAT3 and p-STAT3 protein: 1.00 ± 0.11, 1.00 ± 0.10, t = 2.54, 3.02, respectively, both P < 0.05; miR-485-5p: 1.00 ± 0.12, t = 2.94, P = 0.015). FISH demonstrated that miR-485-5p and lncRNA NEAT1 were co-located in the cytoplasm of HaCaT cells. The double-luciferase reporter gene assay showed that the relative activity of luciferase was significantly lower in the miR-485-5p group than in the negative control group (both P < 0.05) after the transfection with wild-type lncRNA NEAT1 or STAT3 recombinant plasmids, while there were no significant differences between the miR-485-5p group and negative control group after the transfection with mutant lncRNA NEAT1 or STAT3 recombinant plasmids (both P > 0.05). Compared with the control group, the lncRNA-NEAT1 overexpression group showed significantly increased expression of lncRNA NEAT1 and STAT3 (including STAT3 mRNA, STAT3 protein, and p-STAT3 protein) in HaCaT cells (all P < 0.05), but significantly decreased miR-485-5p expression ( P < 0.05) ; the Xidi Liangxue recipe group showed significantly decreased expression of lncRNA NEAT1 and STAT3 (all P < 0.05), but significantly increased miR-485-5p expression compared with the control group ( P < 0.05) ; significantly decreased expression of lncRNA NEAT1 and STAT3, but significantly increased miR-485-5p expression was observed in the Xidi Liangxue recipe + lncRNA-NEAT1 overexpression group compared with the lncRNA-NEAT1 overexpression group (all P < 0.05). After 24-, 48-, and 72-hour intervention, CCK8 assay showed that the proliferative activity of HaCaT cells was significantly higher in the lncRNA-NEAT1 overexpression group than in the control group (all P < 0.05), as well as in the Xidi Liangxue recipe + lncRNA-NEAT1 overexpression group than in the Xidi Liangxue recipe group (all P < 0.05), and the cellular proliferative activity was significantly lower in the Xidi Liangxue recipe + lncRNA-NEAT1 overexpression group and Xidi Liangxue recipe group than in the control group (all P < 0.05). The apoptosis rate was significantly lower in the lncRNA-NEAT1 overexpression group (5.84% ± 0.28%) than in the control group (14.75% ± 0.83%, LSD- t = 3.48, P = 0.002), but significantly higher in the Xidi Liangxue recipe group (35.72% ± 3.62%) than in the control group (LSD- t = 5.34, P = 0.001) ; the Xidi Liangxue recipe + lncRNA-NEAT1 overexpression group showed significantly increased apoptosis rate (27.64% ± 2.82%) compared with the lncRNA-NEAT1 overexpression group (LSD- t = 9.06, P < 0.001) . Conclusion:The Xidi Liangxue recipe could inhibit the proliferation of IL-17-induced HaCaT cells and promote their apoptosis, which may be related to the intervention in the lncRNA NEAT1/miR-485-5p/STAT3 regulatory network.
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Background: ZNF460 is a member of the zinc finger protein family transcription factors, and is involved in the regulation of a variety of cellular functions. It has been demonstrated to be closely related to digestive system cancers. Aims: To analyze the expression level of ZNF460 in hepatocellular carcinoma (HCC), and explore the effect and potential mechanisms of ZNF460 on tumor cell proliferation. Methods: Immunohistochemical staining was used to determine the expression level of ZNF460 in tissue microarray of 76 HCC and paired adjacent tissues, and the correlation between ZNF460 expression and TNM staging was analyzed. The effect of ZNF460 on HCC cell proliferation was evaluated by CCK‑ 8 and colony formation assays. Genes positively related to ZNF460 expression in HCC were screened through LinkedOmics database, and the KEGG and GSEA pathway enrichment analyses were performed; the possible downstream molecules of ZNF460 were explored and verified by overexpression or knockdown of ZNF460 in HCC cells combined with luciferase reporter assay and other experiments. Results: ZNF460 was highly expressed in HCC and was positively correlated with TNM staging. ZNF460 promoted the proliferation of HCC cells, and the underlying mechanism might be associated with the transcriptional activation of ZDHHC7, the coding gene of palmitoyl transferase DHHC7 and the subsequent STAT3 palmitoylation and phosphorylation. Conclusions: ZNF460 is highly expressed in HCC and promotes cell proliferation and tumor progression via STAT3 activation. It might be a promising molecular marker and therapeutic target for HCC.
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Objective:To explore the potential mechanism of Tianshui Dichang Decoction in the treatment of ulcerative colitis through network pharmacology and experimental verification.Methods:The active components and targets of Tianshui Dichang Decoction were screened by TCMSP. The related targets of ulcerative colitis were screened by OMIM, GeneCard and TTD databases, and the effective component targets of Tianshui Dichang Decoction were intersected with the potential targets of ulcerative colitis. The PPI network was constructed by STRING database to screen the core targets, and the "Chinese materia medica-disease-active components-target" network was constructed by Cytoscape 3.8.0 software. GO function and KEGG pathway enrichment analysis were carried out using Metascape database. 48 mice were divided into control group, model group, mesalazine group (0.3 g/kg) and Tianshui Dichang Decoction low-, medium-, and high-dosage groups (7.5,15 and 30 g/kg) according to random number table method, with 8 mice in each group. Except the control group, the ulcerative colitis model was established in other groups. After 7 days of intervention with corresponding drugs, the disease activity index (DAI) was scored, the pathological changes of colon were observed by HE staining, and the expressions of IL-6, STAT3mRNA and protein in colon tissue were detected by PCR and Western blot methods.Results:Totally 127 active components in Tianshui Dichang Decoction and 560 targets of ulcerative colitis were obtained. 89 intersecting targets of Tianshui Dichang Decoction and ulcerative colitis were obtained, and the core targets included IL6, TNF, IL1B, AKT1, TP53, VEGFA, JUN, PTGS2, CXCL8, CCL2, STAT3, MMP9 and so on. Oxidative stress response, lipopolysaccharide metabolism, bacterial response, signal transduction and other biological processes were mainly involved, mainly through the cancer pathway, IL17, TNF, MAPK and other signal pathways to play a role in the treatment of ulcerative colitis. The results of experimental verification showed that the DAI score, the expressions of IL-6 and STAT3 protein in colon tissue of Tianshui Dichang Decoction medium- and high-dosage groups.decreased ( P<0.05). The levels of IL-6 and STAT3 mRNA in colon tissue decreased in the Tianshui Dichang Decoction low-, medium- and high-dosage groups.groups ( P<0.05). Conclusion:Tianshui Dichang Decoction has a certain therapeutic effect on UC through component-multitarget-signal pathway, and its mechanism is related to regulating IL-6/STAT3 signal pathway and inhibiting intestinal mucosal inflammation.
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Objective To investigate the effect of 6-paradol on the proliferation, migration, and invasion of human intrahepatic cholangiocarcinoma cells and its mechanism. Methods Human intrahepatic cholangiocarcinoma cell lines HCCC 9810 and HUCCT1 were treated with different concentrations of 6-paradol or an equal volume of DMSO (control group), and then CCK-8 assay, plate colony formation assay, wound healing assay, and Transwell assay were used to measure cell proliferation, migration, and invasion. The bioinformatics software Swiss Target Prediction was used to predict the protein targets of 6-paradol, and Western blot was used to measure the protein expression levels of STAT3, p-STAT3, SRC, p-mTOR, p21, Bcl-2, and p53; Drug Affinity Responsive Target Stability (DARTS) assay was used to investigate the interaction between 6-paradol and STAT3. After cholangiocarcinoma HCCC 9810 and HUCCT1 cells were transfected with STAT3 overexpression plasmid or sh-p21 plasmid, quantitative real-time PCR was used to measure the mRNA expression levels of STAT3 and p21, and Western blot was used to measure the protein expression levels of STAT3 and p21; CCK-8 assay, wound healing assay, and Transwell assay were used to measure cell proliferation, migration, and invasion. The t -test was used for comparison of data between two groups; an analysis of variance was used for comparison between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results Compared with the control group, the 6-paradol treatment groups had significant reductions in cell proliferation, migration, and invasion ( P 0.05). In the 6-paradol treatment groups, the proportion of STAT3 hydrolyzed by protease was reduced by 48.66% and 45.33%, respectively ( t =16.64 and 8.76, both P < 0.05); after transfection with STAT3 overexpression plasmid or p21-silencing plasmid in cholangiocarcinoma cells, there was a significant increase in the mRNA expression level of STAT3 ( t HCCC 9810 =2.82, t HUCCT1 =5.60, both P < 0.05) and a significant reduction in the mRNA expression level of p21 ( t HCCC 9810 =6.84, t HUCCT1 =3.91, both P < 0.05). CCK-8 assay showed that for HCCC 9810 and HUCCT1 cells treated with 6-paradol for 48 and 72 hours, the STAT3 overexpression group had a significantly higher proliferation rate than the single administration group, and the p21 silencing group also had a significantly higher proliferation rate than the single administration group ( P < 0.05). The wound healing assay showed that the HCCC 9810 and HUCCT1 cells with STAT3 overexpression or p21 silencing had a significantly higher wound healing rate than the single administration group (all P < 0.05). Transwell assay showed that the HCCC 9810 and HUCCT1 cells with STAT3 overexpression or p21 silencing had significant increases in migration rate and invasion rate compared with the single administration group (all P < 0.05). Conclusion 6-Paradol inhibits the proliferation, migration, and invasion of cholangiocarcinoma cells by targeting the STAT3-p21 pathway.
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The immune mechanism of chronic hepatitis B (CHB) persistent infection is closely associated with T cells, and the development of T cells requires the coordination of a variety of cytokines. The proteins of the signal transducer and activator of transcription (STAT) family are mainly involved in the signal transduction of cytokines, and STAT5a/b and STAT3 play an important role in the differentiation and development of regulatory T cells (Treg) and T helper 17 cells (Th17). This article analyzes the association of STAT3 and STAT5 with Treg/Th17 balance in CHB and investigates the chronicity of hepatitis B virus infection and the regulatory mechanism of liver inflammation.
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IL-17 is one of more than 30 kinds of interleukin, which plays an important role in many biological processes. Under normal circumstances, IL-17 can cause inflammation. However, under pathological conditions, IL-17 can also promote a variety of tumors. Hepatocellular carcinoma (HCC) is the most common type of liver cancer.It has rapid development, poor prognosis, easy metastasis, and obvious inflammatory reaction in tumor microenvironment. These biological characteristics suggest that the occurrence and development of HCC are related to the expression of IL-17. IL-17 can affect the growth, development and autophagy of hepatocellular carcinoma through a variety of ways. For example, promot the growth of hepatocellular carcinoma through the AKT/IL-6/JAK2/STAT3 signaling pathway and the miR-383/STAT3 axis, increase the expression of MMPs by activating the NF-κB signaling pathway to promote hepatocellular carcinoma metastasis, enhance the expression of PIAS1 through the NF-κB pathway to inhibit the anti-tumor effect of IFN-γ, and by inhibiting the degradation of Bcl2 in Bcl2-Beclin1 to inhibit autophagy of hepatocellular carcinoma cells. This article reviews the research status of IL-17 in the pathogenesis of hepatocellular carcinoma.
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Objective:To investigate the role and mechanism of interleukin (IL)-6/signal transducer and activator of transcription 3 (STAT3) signaling pathway in proliferation, invasion and apoptosis of cervical cancer cells infected by high-risk human papillomavirus (HPV).Methods:HPV-18 positive HeLa cells cultured in vitro were divided into control group (normal cultured), IL-6 group (stimulated with 50 ng/ml IL-6 for 24 h) and IL-6 + AG490 group (stimulated with 50 ng/ml IL-6 and 100 μmol/L STAT3 signaling pathway inhibitor AG490 for 24 h). Methyl thiazolyl tetrazolium (MTT) assay was used to detect the cell survival rate. The ability of cell clone formation was detected by plate clone formation assay. The cell invasion was detected by transwell chamber assay. The cell apoptosis was detected by flow cytometry. Western blot was used to detect the protein expression levels of STAT3, phosphorylation of (p)-STAT3, B-cell lymphoma-2 gene (Bcl-2), CyclinD1 and matrix metalloproteinase-9 (MMP-9). Results:Compared with those in the control group, the cell survival rate, clone formation rate, number of invasive cells and the protein expression levels of p-STAT3, Bcl-2, CyclinD1 and MMP-9 in IL-6 group were significantly higher, while the apoptosis rate was significantly lower ( P<0.05); at the same time, compared with those in IL-6 group, the cell survival rate, clone formation rate, the number of invasive cells and the protein expression levels of p-STAT3, Bcl-2, CyclinD1 and MMP-9 in IL-6 + AG490 group were significantly lower, while the apoptosis rate was significantly higher ( P<0.05). Conclusions:IL-6/STAT3 signaling pathway plays an important role in the malignant progression of high-risk HPV infected cervical cancer cells, and its mechanism may be related to the up-regulation of CyclinD1, MMP-9, Bcl-2 protein expressions.
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Objective:To evaluate the relationship between silence information regulator 1 (SIRT1) and signal transducers and activators of transcription 3 (STAT3) acetylation during high glucose-induced cardiac microvascular endothelial cell injury.Methods:Cardiac microvascular endothelial cells of Sprague-Dawley rats were cultured.The cells at the logarithmic growth phase were selected and divided into 3 groups ( n=24 each) using a random number table method: control group (C group), high glucose group (HG group) and high glucose+ SIRT1 agonist SRT1720 group (HG+ SRT group). The cardiac microvascular endothelial cells were seeded in a 6- or 96-well cell culture plate at a density of 2×10 5 cells/ml.When the cell density reached 50%, the culture medium was then replaced with high-glucose (glucose 33 mmol/L) DMEM culture medium containing with 10% fetal bovine serum and 1% double antibody in HG and HG+ SRT groups.In group HG+ SRT, 20 μmol/L SRT1720 was added simultaneously, and the cells were cultured at 37 ℃ in an incubator with 5% CO 2 for 24 h. The cell viability was determined by CCK-8 assay, the activity of superoxide dismutase (SOD) was detected using a spectrophotometer, the levels of lactic dehydrogenase (LDH), interleukin-6 (IL-6) and tumor necrosis factor-β (TNF-β) in the supernatant were detected by enzyme-linked immunosorbent assay, and the expression of SIRT1, acetylated STAT3 (ac-STAT3) and phosphorylated STAT3 (p-STAT3) was determined by Western blot. Results:Compared with C group, the cell viability and SOD activity were significantly decreased, levels of LDH, IL-6 and TNF-β in the supernatant were increased, expression of SIRT1 was down-regulated, and expression of ac-STAT3 and p-STAT3 was up-regulated in group HG and group HG+ SRT ( P<0.05). Compared with group HG, the cell viability and SOD activity were significantly increased, levels of LDH, IL-6 and TNF-β in the supernatant were decreased, expression of SIRT1 was up-regulated, and expression of ac-STAT3 and p-STAT3 was down-regulated in group HG+ SRT ( P<0.05). Conclusion:SIRT1 can alleviate high glucose-induced cardiac microvascular endothelial cell injury by promoting STAT3 deacetylation.
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ObjectiveTo investigate the role of STAT3 in hepatocyte proliferation after acetaminophen (APAP)-induced hepatocellular injury in mice. MethodsNormal mouse AML12 hepatocytes were cultured in vitro and were stimulated by APAP (1, 2.5, 5, 10, and 20 mmol/L) for 12, 24 or 48 hours, and the hepatocytes treated with an equal volume of phosphate buffered saline were established as control group. After the optimal stimulation concentration and duration of action were screened out, AML12 hepatocytes were treated with AG490 (10, 50, and 100 μmol/L). The CCK-8 assay was used to measure the viability of AML12 hepatocytes; RT-PCR was used to measure the mRNA expression levels of PCNA, CyclinD1, and Ki67 in AML12 hepatocytes, and Western blot was used to measure the protein expression levels of STAT3, p-STAT3, PCNA, and CyclinD1. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsAfter 24 and 48 hours of APAP treatment, compared with the control group, all concentration groups had a significant reduction in the viability of AML12 hepatocytes (all P＜0.05), with a viability of 0.717±0.0271 and 0.752±0.0141, respectively, when the concentration of APAP was 2.5 mmol/L, which was significantly different from that in the control group (all P＜0.05) and met the conditions of subsequent experiment. Compared with the control group, the 24-hour APAP (2.5 mmol/L) group had significant reductions in the mRNA expression of PCNA, CyclinD1, and Ki67 (all P＜0.01); compared with the 24-hour APAP group, the 48-hour APAP (2.5 mmol/L) group had significant increases in the mRNA expression of PCNA, CyclinD1, and Ki67 (all P＜0.01); therefore, a model of hepatocyte regeneration after in vitro AML12 hepatocyte injury was established by stimulation with APAP 2.5 mmol/L for 48 hours. After the addition of AG490, there was no significant difference in viability between the control group and the 10 and 50 μmol/L AG490 groups, and the other groups had a significant reduction in viability (all P＜0.01); compared with the APAP group, the AG490 (50 μmol/L)+APAP group and the AG490 (100 μmol/L)+APAP group had a significant reduction in viability (P＜0.01); therefore, 50 μmol/L AG490 was selected as the concentration for subsequent experiment. Compared with the control group, the APAP group had a significant increase in the protein expression level of p-STAT3 (P＜0.01), while the AG490 group and the APAP+AG490 group had a significant reduction (both P＜0.05); compared with the APAP group, the APAP+AG490 group had significant reductions in the protein expression levels of PCNA and CyclinD1 and the mRNA expression levels of PCNA, CyclinD1, and Ki67 (all P＜0.05). ConclusionSTAT3 participates in hepatocyte proliferation after APAP-induced hepatocyte injury in mice, while AG490, as an STAT3 inhibitor, can inhibit hepatocyte proliferation after APAP-induced hepatocyte injury by inhibiting the phosphorylation of STAT3.
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The present study was aimed to explore the neuroprotective role of imatinib in global ischemia-reperfusion-induced cerebral injury along with possible mechanisms. Global ischemia was induced in mice by bilateral carotid artery occlusion for 20 min, which was followed by reperfusion for 24 h by restoring the blood flow to the brain. The extent of cerebral injury was assessed after 24 h of global ischemia by measuring the locomotor activity (actophotometer test), motor coordination (inclined beam walking test), neurological severity score, learning and memory (object recognition test) and cerebral infarction (triphenyl tetrazolium chloride stain). Ischemia-reperfusion injury produced significant cerebral infarction, impaired the behavioral parameters and decreased the expression of connexin 43 and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in the brain. A single dose administration of imatinib (20 and 40 mg/kg) attenuated ischemia-reperfusion-induced behavioral deficits and the extent of cerebral infarction along with the restoration of connexin 43 and p-STAT3 levels. However, administration of AG490, a selective Janus-activated kinase 2 (JAK2)/STAT3 inhibitor, abolished the neuroprotective actions of imatinib and decreased the expression of connexin 43 and p-STAT3. It is concluded that imatinib has the potential of attenuating global ischemia-reperfusion-induced cerebral injury, which may be possibly attributed to activation of JAK2/STAT3 signaling pathway along with the increase in the expression of connexin 43.
Subject(s)
Animals , Mice , Brain , Carotid Arteries , Cerebral Infarction , Connexin 43 , Imatinib Mesylate , Ischemia , Learning , Memory , Motor Activity , Neuroprotection , Phosphotransferases , Reperfusion , Reperfusion Injury , STAT3 Transcription Factor , Transducers , WalkingABSTRACT
Objective: To investigate the molecular mechanism of polyphyllin (PP) inhibiting proliferation and inducing apoptosis of human gl i oma U251 cel l s by di rectl y targeti ng si gnal transducer and activator of transcription 3 (STAT3). Methods: U251 cells were treated with different concentrations of PP, then the effect of PP on the proliferation of glioma U251 cells was detected by MTT assay, trypan blue dyeing and clone forming assay, respectively. Apoptosis of glioma U251 cells was determined by flow cytometry after Annexin-FITC/PI double staining. The change of mitochondrial membrane potential was detected by flow cytometry after fluorescent probe JC-1 staining. SwissTargetPrediction online prediction software was used to detect the targeting proteins of PP. Western blotting was used to detect the expressions of STAT3 signaling pathway-related proteins. Molecular docking simulated the binding and action modes of PP and STAT3. Results: PPinhibited the proliferation of U251 cells in a time and concentration-dependent manner (all P 0.05). The hydrogen atom of PP was capable of forming hydrogen bonds with the oxygen atom of aspartic acid (Asp)-334 on STAT3, thereby enhancing its ability of targeting STAT3. Conclusion: PP can inhibit the proliferation of glioma U251 cells and induce apoptosis by directly targeting STAT3.
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STAT3, a member of the signal transducer and activator of transcription family, is abnormally activated in chronic inflammation-related tumors including liver cancer. As a key signal molecule in the microenvironment of liver cancer and inflammation, STAT3 not only participates in the inflammation-cancer transformation during the development of liver cancer, but also promotes the proliferation, invasion, and metastasis of hepatoma cells through many ways, and therefore, it may be a potential target for the treatment of liver cancer. This article reviews the recent advances in the association between STAT3 and liver cancer.
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Background: Piperlongumine (PL) is an alkaloid compound extracted from piperlongum. Many studies have shown that PL has anti-tumor effects on a variety of tumor cells in vivo and in vitro. However, the specific mechanism needs to be further explored. Aims: To investigate the regulatory mechanism of PL on the expression of TERT in gastric cancer cells. Methods: Gastric cancer cells were treated with different doses of PL, AG490, respectively. CCK-8 and plate colony formation experiment were used to detect cell viability. Real-time fluorescent quantitative PCR was used to detect the expression of TERT mRNA. Western blotting was used to detect the protein expressions of TERT, STAT3, p-STAT3 and DNMT1. TRAP-ELISA was used to determine the telomerase activity. Luciferase reporter gene was used to detect the TERT promoter activity. Results: Compared with control group, gastric cancer cells viability in PL group was significantly decreased, colony formation ability was significantly reduced, TERT mRNA and protein expressions, as well as telomerase activity were significantly reduced, p-STAT3 and DNMT1 protein expressions were significantly downregulated. AG490 significantly inhibited gastric cancer cells viability, protein expressions of p-STAT3, DNMT1 and TERT. Conclusions: PL may inhibit gastric cancer cells viability through regulation of TERT expression via STAT3-mediated epigenetic regulation and it may become a new target drug for the treatment of gastric cancer in future.