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Aim To study the effect of menthol on hypobaric hypoxia-induced pulmonary arterial hypertension and explore the underlying mechanism in mice. Methods 10 to 12 weeks old wild type (WT) mice and TRPM8 gene knockout (TRPM8
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Krüppel-like transcription factor 2 (KLF2) plays a key regulatory role in endothelial inflammation, thrombosis, angiogenesis and macrophage inflammation and polarization, and up-regulation of KLF2 expression has the potential to prevent and treatment atherosclerosis. In this study, trichostatin C (TSC) was obtained from the secondary metabolites of rice fermentation of Streptomyces sp. CPCC 203909 as a KLF2 up-regulator by using a high throughput screening model based on a KLF2 promoter luciferase reporter assay. TSC significantly inhibited the adhesion of tumor necrosis factor-α (TNFα) induced monocytes (THP-1) to human umbilical vein endothelial cells (HUVECs). Western blot results showed that TSC decreased TNFα induced the protein expression increase of vascular cell adhesion molecule-1 (VCAM-1), and thereby inhibited endothelial inflammation. The results of histone deacetylase (HDAC) overexpression and molecular docking experiments showed that TSC upregulated the expression of KLF2 by inhibiting subtypes of HDAC 4/5/7. In conclusion, this study suggests that TSC up-regulates the expression of KLF2 through inhibiting HDAC 4/5/7 and thus inhibits TNFα induced endothelial inflammation, and it has the potential to prevent and treat atherosclerosis.
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[ Abstract] Objective To explore the role pathway and pattern of the Kruppel-like factor 4 (KLF4) and its mRNA interaction with microRNA (miRNAs) and circular RNA (circRNAs) at 0 hour and the 120 th hour in the rat liver regeneration. Methods The rat 2 / 3 hepatectomy (partial hepatectomy, PH) model was prepared as described by Higgins, the hepatocytes were isolated according to the method of Smedsrod et al, the expression changes of mRNA, miRNA and circRNA together named as competing endogenous RNA (ceRNA) were detected by the large-scale quantitative detection technology, the interaction network of ceRNA was constructed by Cytoscape 3. 2 software, and their correlation in expression and role were analyzed by ceRNA comprehensive analysis. Results It was found that at the 0 hour and the 120th hour PH, the ratio value of KLF4 mRNA showed 1. 00±0. 16 and 3. 14±0. 27, miR-881-3p displays 18. 30±1. 44 and 0. 47±0. 02, circRNA_20298 indicated 0. 32±0. 10 and 4. 24±0. 22, circRNA_14826 showed 0. 42±0. 13 and 0. 61±0. 08. At the same time, the four kinds of cell apoptosis-related genes adrenoceptor beta 2 (ADRB2), dimethylarginine dimethylaminohydrolase 2 (DDAH2), annexin A5 (ANXA5), ect, which were promoted in expression by KLF4, were down-regulated at 0 hour after PH, but the cell apoptosis-related genes synuclein gamma (SNCG), glutathione-disulfide reductase (GSR), FYVE, RhoGEF and PH domain containing 4 (FGD4), ect, which were inhibited in expression by KLF4, were up-regulated at 0 hour after PH. On the other hand, the cell apoptosis-related genes ANXA5 and thymosin beta 10 (TMSB10), which are promoted in expression by KLF4, were up-regulated at the 120th hour after PH, but the cell apoptosis-related genes chloride intracellular channel 4 (CLIC4) and ataxin 3 (ATXN3), ect, which were inhibited in expression by KLF4, were down-regulated at the 120th hour after PH. Conclusion The correlation in expression and role of the miRNAs, which are inhibited by circRNAs, KLF4, its mRNA is inhibited by miRNAs, and the cell apoptosis-related genes, which are regulated by KLF4, are helpful for the hepatocyte to be in active state 0 hour after PH and to be in apoptotic state 120-hour after PH.
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Overexpression of Krüppel like factor 2 (Klf2) or Klf7 inhibits adipocyte formation. However, it remains unclear whether Klf2 regulates klf7 expression in adipose tissue. In this study, oil red O staining and Western blotting were employed to study the effect of Klf2 overexpression on the differentiation of chicken preadipocytes. The results showed that Klf2 overexpression inhibited the differentiation of chicken preadipocytes induced by oleate and the expression of pparγ, while promoted klf7 expression in chicken preadipocytes. Spearman correlation analysis was used to study the correlation between the expression data of klf2 and klf7 in the adipose tissue of both human and chicken. The results showed that there was a significantly positive correlation between the expression of klf2 and klf7 in adipose tissues (r > 0.1). Luciferase reporter assay showed that overexpression of Klf2 significantly promoted the activity of chicken klf7 promoter (-241/-91, -521/-91, -1 845/-91, -2 286/-91, -1 215/-91; P < 0.05). In addition, the activity of klf7 promoter (-241/-91) reporter in chicken preadipocytes was significantly positively correlated with the amount of klf2 overexpression plasmid transfected (Tau=0.917 66, P=1.074×10-7). Moreover, Klf2 overexpression significantly promoted the mRNA expression of klf7 in chicken preadipocytes (P < 0.05). In conclusion, upregulation of klf7 expression might be one of the pathways that Klf2 inhibits chicken adipocyte differentiation, and the sequence from -241 bp to -91 bp upstream chicken klf7 translation start site might mediate the regulation of Klf2 on klf7 transcription.
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Animals , Humans , Chickens/genetics , Kruppel-Like Transcription Factors/metabolism , Transcription Factors/metabolism , Adipocytes/metabolism , Adipose Tissue/metabolismABSTRACT
Objective To explore the effect of microRNA-22-3p (miR-22-3p) regulating the expression of Kruppel-like factor 6 (KLF6) on the cardiomyocyte-like differentiation of bone marrow mesenchymal stem cell (BMSC). Methods Rat BMSC was isolated and cultured,and the third-generation BMSC was divided into a control group,a 5-azacytidine(5-AZA)group,a mimics-NC group,a miR-22-3p mimics group,a miR-22-3p mimics+pcDNA group,and a miR-22-3p mimics+pcDNA-KLF6 group.Real-time fluorescent quantitative PCR (qRT-PCR) was carried out to determine the expression of miR-22-3p and KLF6 in cells.Immunofluorescence staining was employed to detect the expression of Desmin,cardiac troponin T (cTnT),and connexin 43 (Cx43).Western blotting was employed to determine the protein levels of cTnT,Cx43,Desmin,and KLF6,and flow cytometry to detect the apoptosis of BMSC.The targeting relationship between miR-22-3p and KLF6 was analyzed by dual luciferase reporter gene assay. Results Compared with the control group,5-AZA up-regulated the expression of miR-22-3p (q=7.971,P<0.001),Desmin (q=7.876,P<0.001),cTnT (q=10.272,P<0.001),and Cx43 (q=6.256,P<0.001),increased the apoptosis rate of BMSC (q=12.708,P<0.001),and down-regulated the mRNA (q=20.850,P<0.001) and protein (q=11.080,P<0.001) levels of KLF6.Compared with the 5-AZA group and the mimics-NC group,miR-22-3p mimics up-regulated the expression of miR-22-3p (q=3.591,P<0.001;q=11.650,P<0.001),Desmin (q=5.975,P<0.001;q=13.579,P<0.001),cTnT (q=7.133,P<0.001;q=17.548,P<0.001),and Cx43 (q=4.571,P=0.037;q=11.068,P<0.001),and down-regulated the mRNA (q=7.384,P<0.001;q=28.234,P<0.001) and protein (q=4.594,P=0.036;q=15.945,P<0.001) levels of KLF6.The apoptosis rate of miR-22-3p mimics group was lower than that of 5-AZA group (q=8.216,P<0.001).Compared with the miR-22-3p mimics+pcDNA group,miR-22-3p mimics+pcDNA-KLF6 up-regulated the mRNA(q=23.891,P<0.001) and protein(q=13.378,P<0.001)levels of KLF6,down-regulated the expression of Desmin (q=9.505,P<0.001),cTnT (q=10.985,P<0.001),and Cx43 (q=8.301,P<0.001),and increased the apoptosis rate (q=4.713,P=0.029).The dual luciferase reporter gene experiment demonstrated that KLF6 was a potential target gene of miR-22-3p. Conclusion MiR-22-3p promotes cardiomyocyte-like differentiation of BMSC by inhibiting the expression of KLF6.
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Animals , Rats , Myocytes, Cardiac , Kruppel-Like Factor 6 , Connexin 43 , Desmin , Cell Differentiation , Azacitidine/pharmacology , Mesenchymal Stem Cells , RNA, Messenger , MicroRNAsABSTRACT
ObjectiveTo study the underlying mechanism of Liuwei Dihuangwan in inhibiting triple-negative breast cancer through mitogen-activated protein kinase kinase kinase 1 （MAPKKK1） and Krüppel-like factor 4 （KLF4）. MethodFour hundreds SPF female Kunming mice aged 11.5 months were palpated once every 3 days. The model mice of spontaneous tumors were randomly divided into a model group （normal saline）， a paclitaxel group （0.025 g·kg-1·d-1， ip， 21 days）， and high-， medium- and low-dose Liuwei Dihuangwan groups （7.2， 3.6， 1.8 g·kg-1·d-1， ig）. Tumor tissues were separated until the moribund stage. The tumor volume and weight were measured， and the tumor inhibition rate and the survival time of the tumor mice were calculated （after 6 months， tumor-free mice were assigned into the normal group）. SPF SD rats were selected to prepare serum samples containing Liuwei Dihuangwan of different concentrations for cell culture， and MAPKKK1 in MDA-MB-231 cells was silenced. The protein expression of MAPKKK1 and KLF4 was detected by immunofluorescence and Western blot. ResultThe in vivo experimental results showed that compared with the conditions of the normal group， the protein expression of MAPKKK1 and KLF4 in tumor tissues of the model group dropped （P<0.01）. Compared with the model group， all medication groups showed reduced tumor volume and weight （P<0.05， P<0.01）， increased tumor inhibition rate， prolonged survival time of tumor mice （P<0.05）， and increased protein expression of MAPKKK1 （P<0.01）. Additionally， the paclitaxel group and the high-dose Liuwei Dihuangwan group exhibited increased protein expression of KLF4 （P<0.01）. The in vitro experiments showed that compared with the conditions of the normal group， the fluorescence intensities of MAPKKK1 and KLF4 in MDA-MB-231 cells in all medication groups were potentiated， and the protein expression of MAPKKK1 in the paclitaxel group and the high- and medium-dose Liuwei Dihuangwan groups， and the protein expression of KLF4 in the paclitaxel group and high-dose Liuwei Dihuangwan group increased （P<0.01）. After silencing of MAPKKK1， compared with the conditions of the negative plasmid group （unsilenced MAPKKK1）， the fluorescence intensities of MAPKKK1 and KLF4 and the protein expression decreased in the RNAi-27 positive plasmid group （silenced MAPKKK1） （P<0.05， P<0.01）. Compared with the RNAi-27 positive plasmid group， all medication groups had enhanced fluorescence intensities of MAPKKK1 and KLF4 and protein expression to different degrees （P<0.05， P<0.01）. ConclusionLiuwei Dihuangwan can inhibit the growth of triple-negative breast cancer， and the underlying molecular mechanism is related to the up-regulation of MAPKKK1 and activation of KLF4 expression.
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Objective To investigate the protective effect of micro RNA(miR)-98-5p targeting Kruppel-like factor 9(KLF9) against myocardial isehemia-reperfusion(MI/R) injury in rats. Methods Totally 50 rats were randomly divided into sham operation group, model group, miR-98-5p agomir group, agomir-NC group, and miR-98-5p agomir+pcDNA-3. 1-KLF9 group, 10 in each group. MI/R model was established by coronary artery ligation. The pathological changes of myocardial tissue were detected by HE staining. The myocardial apoptosis were detected by TUNEL. The levels of creatine kinase (CK), creatine kinase isoenzymes (CK-MB) and lactate dehydrogenase (LDH) in serum were detected by ELISA. The expression levels of miR-98-5p and KLF9 mRNA were detected by Real-time PCR. The expression of KLF9, Bax and JAK2/STAT3 pathway relative protein were detected by Western blotting. Dual luciferase assay verified the relationship between miR-98-5p and KLF9. Results Compared with the sham operation group, the arrangement of myocardial cells in the model group was disordered, and the myocardial cells appeared necrosis. The apoptosis rate of myocardial cells, serum CK, CK-MB and LDH contents increased, the expression level of miR-98-5p decreased, and the expression levels of KLF9 mRNA and protein, p-JAK2 and p-STAT3 protein increased (P < 0. 05) . After the overexpression of miR-98-5p, the myocardial cells arranged more orderly and the myocardial cell necrosis decreased. The apoptosis rate of myocardial tissue, the contents of CK, CK-MB and LDH in serum and the expression of p-JAK2 and p-STAT3 protein were decreased (P< 0. 05) . The result of dual luciferase assay showed that KLF9 was the target gene of miR-98-5p. The overexpression of KLF9 reversed the effects of miR-98-5p agomir on myocardial injury. Conclusion MiR-98-5p targeting KLF9 can improve the myocardial injury of MI/R rats. The mechanism may be related to the regulation of JAK2/STAT3 signal pathway by miR-98-5p which inhibit myocardial cell apoptosis.
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Krüppel-like factor 7 (KLF7) is a negative regulator of adipogenesis, whereas hypoxia-inducible factor 1 (HIF1) promotes anoxic-induced adipose tissue development in mammals. Our previous ChIP-seq analysis showed that one of the KLF7 binding peaks was present upstream of hypoxia-inducible factor 1 alpha (HIF1α), indicating that KLF7 may regulate HIF1α transcription. For this purpose, ChIP-PCR was used to verify ChIP-seq results, which showed that KLF7 directly bound to the HIF1α upstream region. Dual luciferase reporter and qRT-PCR results showed that KLF7 overexpression significantly decreased the luciferase reporter activity of HIF1α (- 4 432/- 4 182) (P < 0. 01) and inhibited HIF1α expression. After the deletion of KLF7 binding motif “TGCGCAGCAA” (- 4 300/-4 290) predicted by bioinformatics, the luciferase reporter activity of HIF1α (-4 432/-4 182) was significantly enhanced compared with wild-type plasmid (P<0. 01). Furthermore, Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF) at the age of 1-7 weeks from the 19
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BACKGROUND: LINC01234, a long noncoding RNA (lncRNA), is overexpressed in several cancers, including colorectal cancer (CRC). We investigated the role of LINC01234 in CRC development and confirmed its correlation with Krüppel-like factor 6 (KLF6), a tumor suppressor gene that is dysregulated in CRC. METHODS: We tested mRNA levels using quantitative reverse transcription PCR (qRT-PCR). Tissue samples from patients with CRC, inflammatory bowel disease (IBD), hyperplastic polyp, and adenoma were included. Correlations between clinicopathological parameters, overall survival (OS) rate, and LINC01234 were analyzed using Kruskal-Wallis H test. Additionally, cell proliferation, apoptosis, and tumor formation in nude mice were tested to investigate the mechanism of LINC01234. Western blotting was used to determine protein levels. RESULTS: LINC01234 expression was significantly upregulated in CRC tissues and CRC cell lines than in non-tumor tissues and normal epithelial cells, respectively. LINC01234 was associated with high tumor stage, larger tumor size, and metastasis. Patients with higher LINC01234 expression showed reduced OS. Cell proliferation was inhibited by LINC01234 knockdown, whereas apoptosis was enhanced. Mice injected with SW480 cells with LINC01234 knockdown displayed decreased tumor volume, weight, and Ki-67 levels compared with those injected with control cells. KLF6 was negatively regulated by LINC01234. Overexpression of KLF6 showed effects similar to those observed following LINC01234 knockdown on cell proliferation and apoptosis. CONCLUSIONS: LINC01234 could be a prognostic biomarker in CRC patients. Upregulation of LINC01234 in CRC promotes tumor development through negative regulation of KLF6.
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Animals , Humans , Mice , Adenoma , Apoptosis , Blotting, Western , Cell Line , Cell Proliferation , Colorectal Neoplasms , Epithelial Cells , Genes, Tumor Suppressor , Inflammatory Bowel Diseases , Mice, Nude , Neoplasm Metastasis , Polymerase Chain Reaction , Polyps , Prognosis , Reverse Transcription , RNA, Long Noncoding , RNA, Messenger , Tumor Burden , Up-RegulationABSTRACT
Objective: To explore the possibility of promoting tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis in prostate cancer PC-3 cell by inhibiting Krüppel-like factor 5 (KLF5). Methods: MTT assay, flow cytometry, Western blot assay and qRT-PCR assay were deployed to detect the cell viability, apoptosis and apoptotic markers in KLF5-inhibited and TRAIL-induced PC-3 cells. Results: After KLF5 was inhibited in TRAIL-induced PC-3 cells, cell viability reduced, apoptosis enhanced, the expressions of DR4 and DR5 increased while the expression of cellular fas-associated death domain-like interleukin-1β converting enzyme inhibitory protein (c-FLIP) decreased. Conclusion: Inhibiting KLF5 suppresses cell viability by promoting TRAIL-induced apoptosis in prostate cancer cell PC-3. It may be a potential means to treat hormone-insensitive prostate cancer.
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[Abstract] Objective To explore the relationship between Kruppel-like factor 4 (KLF-4) and E-cadherin in human peritoneal mesothelial cells (HPMCs), and the expression and function of KLF-4 in the animal model of peritoneal fibrosis induced by high glucose peritoneal dialysate. Methods Co-transfection in HPMCs with the plasmid of KLF-4 and the bind site or mutant in the promoter region of E-cadherin, and then the luciferase activity was measured of the each bind site and its matched mutants to estimate whether KLF-4 can combine with the bind site in the promoter region of E-cadherin; Chromatin immunocoprecipitation (CHIP) was exploited to verify if KLF-4 can combine with the bind site in the promoter region of E-cadherin; Real-time PCR and Western blotting were performed to detect the expression of E-cadherin at the bind site and matched mutants of b, d, f and g. Thirty SD rats were randomly divided into saline group, peritoneal dialysate group and experimental group (10 each). Rats in saline group were given intraperitoneal injection with 0.9% NaCl, in peritoneal dialysate group were given with 4.25% high glucose peritoneal dialysate, and in experimental group were given via tail vein with 4.25% high glucose peritoneal dialysate and the mixture of KLF-4 plasmid suspension containing ultrasound microbubble. To observe the peritoneal tissue thickness of the 3 groups of rats by Hematoxylin and Eosin staining. Masson trichrome staining was performed to detect the deposition of collagen fibers in peritoneal tissue of the 3 groups of rats. Immunohistochemistry was used to detect the expression level of KLF-4, E-cadherin, α-SMA and fibronectin (FN) in peritoneal tissue of the 3 groups of rats. Results Promoter luciferase reporter gene and CHIP results showed that KLF-4 can combine with the bind site in the promoter region of E-cadherin in HPMCs. Real-time PCR and Western blotting showed that KLF-4 can positively regulate the expression of E-cadherin. HE staining showed that the peritoneal tissue was obviously thickened in rats of peritoneal dialysate group [(105.91±12.0) μm] than in rats of saline group [(20.89±5.39) μm] and of experimental group [(23.05±6.07) μm] with statistical significance (P0.05). Masson staining showed that the deposition of collagen fiber significantly increased in peritoneal dialysate group (0.89±0.09) than in saline group (0.19±0.03) and experimental group (0.15±0.06) with statistical significance (P0.05). Immunohistochemistry results showed that the expressions of KLF-4 and E-cadherin were obviously lower in peritoneal dialysate group (0.27±0.09, 0.31±0.03) than in saline group (0.79±0.19, 0.83±0.13) and experimental group (0.85±0.11, 0.76±0.11) with statistically significant difference (P0.05). In contrast, the expressions of α-SMA and FN were evidently higher in peritoneal dialysate group (0.83±0.09, 0.63±0.09) than in saline group (0.22±0.08, 0.30±0.07) and experimental group (0.19±0.05, 0.11±0.03) with statistically significant difference (P0.05). Conclusion KLF-4 may positive regulate the expression of E-cadherin by combining with the bind site in the promoter region of E-cadherin, and inhibit the peritoneal fibrosis induced via high glucose peritoneal dialysate.
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Objective To screen the critical genes related to the development of esophageal squamous cell carcinoma ( ESCC ) by weighted gene co-expression network analysis ( WGCNA ) and to verify by experiments.Methods Gene expression data of ESCC were downloaded from gene expression omnibus (GEO) database based on gene chip platform ( GPL) 570, GPL571, GPL96/97 or GPL14613 platform, respectively. Meanwhile, the obtained differentially expressed genes together with gene expression data of 81 ESCC patients from the cancer genome atlas ( TCGA ) and clinical data were analyzed by WGCNA to set up co-expression networks including mRNA and microRNA ( miRNA ) . The expression of miRNA in ESCC tissues and paracancerous tissues was examined by quantitative real-time polymerase chain reaction ( RT-PCR ) .And the expression of target protein Kruppel like factor 4 ( KLF4 ) and desmocollin 2 ( DSC2 ) were detected by immunohistochemistry .After ESCC cell line ECA-109 cells were transfected with miRNA-92b-3p mimic, cell cycle was tested by flow cytometry ,the cell invasion and migration ability was measured by Transwell chamber assay and scratch-wound assay.The expression of KLF4 and DSC2 was observed by confocal laser scanning microscopy and Western blotting .The target genes were verified by luciferase assay .T-test, rank sum test, chi-square test and Pearson correlation analysis were performed for statistical analysis .Results A total of 4023 differential expression gene ( DEG) and 328 differential expression miRNA ( DEM) were screened and 11 gene modules were set up by WGCNA .Among them, the brown modules were negatively associated with tumor grade and T stage (r=-0.340 and -0.268, P=0.002 and 0.016).Meanwhile, has-miR-92b and the potential target genes KLF4 and DSC2 were all in the brown module .Furthermore, the results of RT-PCR showed the expression of miRNA-92b-3p in ESCC tissues was higher than that in paracancerous tissues (3.052(1.652, 5.371) vs.0.985(0.558, 2.032)), and the difference was statistically significant (Z=-4.021,P<0.01). The results of immunohistochemistry demonstrated that the positive rates of KLF 4 and DSC2 in ESCC tissues were 43.3％(13/30) and 20.0％(6/30), respectively, which were lower than those of paracancerous tissues (70.0％(21/30) and 63.3％(19/30)), and the differences were statistically significant (χ2 =4.344 and 1.589, both P<0.05).After ECA-109 cells were transfected with miRNA-92b-3p mimics, the percentage of cells at G0/G1 phase decreased ((63.71 ±2.83)％vs.(54.62 ±4.00)％) and the percentage of cells at the S phase and G2/M phase increased ((31.81 ±2.88)％vs.(41.20％±2.87)％, and (3.87 ±1.75)％vs. (8.10 ±1.71)％, respectively), and the differences were statistically significant (t =3.215, 4.000 and 2.998;P=0.032, 0.016 and 0.040).The invasion and migration ability of the cells were significantly improved (79.67 ±27.54 vs.280.33 ±46.18, (69.72 ±3.91)％ vs.(84.90 ±5.25)％), and the differences were statistically significant (t=6.465 and 4.019, P=0.003 and 0.016).The results of Western blotting indicated that, compared with control mimic group , the expression of KLF4 and DSC2 was both dramatically downregulated after transfected with miRNA-92b-3p mimics transfected (1.00 ±0.23 vs.0.42 ±0.03, 1.00 ±0.20 vs.0.55 ± 0.21), and differences were statistically significant (t=4.470 and 5.493, P=0.042 and 0.032).The results of luciferase assay demonstrated that miRNA-92b-3p could directly bind KLF4 and DSC2. Conclusion WGCNA is an efficient systemic biological approach by which miRNA-92b-3p is identified as a new cancer-promoting gene .
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Objective To explore the effect of Kruppel like factor 4 (KLF4) on epithelial-to-mesenchymal transition and invasion ability of pancreatic cancer.Methods The expression of KLF4 in 70 pancreatic cancer tissues and 10 normal pancreatic tissues was detected by immunohistochemistry,and the correlations between KLF4 expression and pathological characteristics were analyzed.Small hairpin RNA targeting KLF4 (sh-KLF4) and negative control shRNA were constructed.After the transfection of shRNA,qRT-PCR and western blot were used to detect the mRNA and protein expression of KLF4,E-cadherin and vimentin,and cell scratch-wound assay and transwell assay were utilized to determine the ability of invasion and metastasis.Results KLF4 expression (47.1％) was lower in pancreatic cancer tissues compared with normal pancreatic tissues (80.0％),and negatively correlated with cell differentiation,tumor stage and distant metastasis.Down-regulated KLF4 expression in PANC1 cell caused decreased mRNA and protein expression of E-cadherin (F =25.71,P =0.0011) and increased mRNA and protein expression of vimentin (F =24.95,P=0.0012).Knockdown of KLF4 in PANC1 cell promoted the transition from epithelial morphology to mesenchymal morphology,and enhanced the healing ability (F =47.82,P ＜ 0.001),migration (F =53.68,P=0.0001) and invasion (F=27.65,P=0.0009).Conclusions Knockdown of KLF4 can promote EMT and enhance the invasion ability of pancreatic cancer.
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Objective: To investigate the effect of Krüppel-like factor 4 (KLF4) on the expression of miR-106a and their interaction on the invasive activity of human gastric cancer cells. Methods: The JASPAR database was used to screen the transcriptional factor combined with miR-106a promoter. KLF4 over-expression vector KLF4-pcDNA and miR-106a reporter gene pmiR-106a-WT/MUT-luc were both constructed, and the dual luciferase reporter assay was used to detect the effect of KLF4 on the activity of miR-106a promoter. Forty specimens of human gastric cancer and their adjacent formalin-fixed paraffin-embedded (FFPE) specimens were collected, and Real-time PCR and immunohistochemistry were both used to detect the expression of KLF4. Human gastric cancer cell line BGC-823 was divided into four groups: miR-106a mimic, mimic NC, KLF4+pcDNA, and pcDNA basic. Transwell assay was employed to detect the invasive ability of the gastric cancer cells after four different treatments. Results: Dual luciferase reporter assay indicated that KLF4-pcDNA could antagonize the activity of miR-106a promoter, which suggested that the activity of pmiR-106a-WT-luc luciferase was inhibited (pmiR-106a-WT+pcDNA-basic compared with pmiR-106a-WT+pcDNA-KLF4, P0.05). FFPE samples showed that the relative expression of KLF4 in gastric cancer was 0.69±0.59, which significantly differed from that in adjacent paracancerous tissues (P<0.001). The expression of KLF4 was correlated with differentiation degree, lymph node metastasis, and infiltration depth of human gastric cancer (P<0.05). The positive expression of KLF4 was mainly located in the nucleus and cytoplasm of gastric mucosal epithelial cells. Transwell essay showed that the number of invasive cells in the four different treatment groups was not entirely the same (P<0.001): 89.00±14.85 for miR-106a mimic, 50.90±17.94 for mimic NC, 37.70±12.60 for KLF4+pcDNA, and 66.20±3.19 for pcDNA basic. Conclusion: Transcription factor KLF4 at least has direct binding with miR-106a promoter in vitro, which may be involved in the invasion and metastasis process of gastric cancer cell by negatively regulating the expression of miR-106a at the upstream transcription level.
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Objective To investigate whether microRNA-34a (miR-34a) participates in lipopolysaccharide (LPS) mediated sepsis related renal function impairment via Kruppel-like factor 4 (KLF4). Methods Thirty healthy male Sprague-Dawley (SD) rats, weighing 180-200 g, were randomly divided into two groups: control group and model group, with 15 rats in each group. The SD rats from model group were injected with LPS 7.5 mg/kg to induce sepsis related renal function impairment model, the SD rats from control group were injected with normal saline. The serum creatinine concentration (SCr) and blood urine nitrogen (BUN) content was detected by multifunction biochemical analyzer; the morphological changes of renal tissue were observed by hematoxylin and eosin stain (HE) staining; the expression of miR-34a and KLF4 gene in plasma and renal tissue were detected by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR); the protein expression of KLF4 in renal tissue was detected by Western Blot; the target gene of miR-34a was verified by double luciferase reporter gene analysis. Results Compared with control group, inflammatory cell infiltration in renal tissue was increased in model group, the SCr and BUN were significantly increased [SCr (μmol/L): 142.5±10.6 vs. 46.4±5.6, BUN (mmol/L): 31.6±6.2 vs. 8.5±1.2, both P < 0.01], the gene expression of miR-34 in plasma and renal tissue were significantly increased (2 -ΔΔCt: 2.26±0.11 vs. 1.14±0.05 in plasma, 4.23±0.12 vs. 1.12±0.04 in renal tissue, both P < 0.01), the gene and protein expressions of KLF4 were significantly decreased [KLF4 gene (2 -ΔΔCt): 0.52±0.03 vs. 1.21±0.06, KLF4 protein (A value): 0.72±0.03 vs. 1.05±0.04, both P < 0.01], which indicated that kidney injury occurred in rats. Pearson correlation analysis showed that plasma miR-34a was positively correlated with SCr and BUN (r value were 0.678, 0.721, respectively, both P < 0.05). Double luciferase reporter assay confirmed that KLF4 was the target gene of miR-34a. Conclusion The miR-34a participates in LPS mediated sepsis related renal function impairment via KLF4.
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Objective To construct and identify a lentiviral vector carrying mouse Krüppel-like factor 4 (KLF4) gene, and establish RAW264.7 cell line of peritoneal macrophages that over-expressed KLF4. Methods KLF4 gene was cloned using the measure of polymerase chain reaction (PCR). Then the recombinant transfer vector pLVX-KLF4 (pLVX-KLF4-mCMV-ZsGreen-PGK-Puro) was constructed. The pLVX-KLF4 was confirmed through PCR, restriction enzyme digestion and sequencing. The correct recombinant transfer vector together with its two helper virus vectors (psPAX2 and pMD2.G) were cotransfected into the 293T cells by Lipofectamine? 3000. The supernatant of 293T was harvested to infect RAW264.7 cells. Flow cytometry (FCM) was used to test the viral titer of the expression level of green fluorescent protein. The expression of KLF4 mRNA in RAW264.7 cells was measured by real-time PCR. Results The restriction enzyme digestion, PCR and sequencing confirmed that the transfer lentiviral vector pLVX-KLF4 was constructed successfully. KLF4 mRNA was over-expressed in Lenti-KLF4 transfected RAW264.7 cells than that of wild type RAW264.7 cells (P<0.05). In transfected RAW264.7 cells, KLF4 mRNA was over-expressed (P<0.05). The recombinant lentivirus of KLF4(Lenti-KLF4)titer was 2.05×108 TU/mL measured by FCM.The flow cytometry results showed that the S phase fraction was prolonged and G0/G1 was arrested in the over-expressed KLF4 of RAW264.7 cells. The EdU showed that the up-regulated expression of KLF4 gene stimulated the proliferation of RAW264.7 cells. Conclusion The recombinant lentiviral vector, which can effectively express KLF4 mRNA, has been successfully constructed. The up-regulated KLF4 gene may increase the proliferation of RAW264.7 cells.
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AIM:To investigate the role of Krüppel-like factor 17(KLF17)in nude mouse xenograft model, and to explore the target genes regulated by KLF 17, the target gene functions and the signaling pathways involved.ME-THODS:The KLF17 was stably up-regulated in human lung adenocarcinoma A 549 cells and down-regulated in human lung adenocarcinoma H322 cells by lentiviral infection.BLAB/c nu/nu nude mice(n=11)were divided into KLF17 up-regual-tion group(n=5)and KLF17 down-regulation group(n=6).The right and left bodies of the nude mice were subcutane-ously injected with KLF17-up-/down-regulating cells and the counterpart empty vectors were used as control cells,respec-tively.The effects of KLF17 on the growth of the cell-derived xenografts in nude mice were analyzed.The mRNA and pro-tein expression levels of KLF17 in xenograft tumor tissues were analyzed by real-time PCR and immunohistochemical stai-ning,respectively.Transcriptome sequencing was used to explore the differentially expressed genes in the xenograft tumors derived from KLF17-up-regulating A549 cells,and the functions of the potential target genes were analyzed using the lung adenocarcinoma data from The Cancer Genome Atlas(TCGA)database.Gene Ontology and KEGG PATHWAY enrichment analyses were performed to analyze the functions of the differentially expressed genes and the involved signal pathways.RE-SULTS:The growth rate of KLF17-up-regulating A549 cell-derived xenograft tumors in the nude mice was significantly lower than that in empty control group(P<0.05),while the growth rate and the weight of KLF 17-down-regulating H322 cell-derived xenograft tumors in nude mice were significantly higher than those in empty control group(P<0.01 and P<0.05,respectively).In the A549 cell-derived xenograft tumor model,the KLF17 mRNA and protein were significantly in-creased in KLF17 up-regualtion group.The transcriptome sequencing showed the potential target genes regulated by KLF 17 were ras homolog family member V(RHOV)and coronin 1C(CORO1C).Ten-year cumulative survival time of the patients with lung adenocarcinoma from TCGA database was significantly different between high and low expression of RHOV and CORO1C at mRNA level.Increased expression levels of RHOV and CORO1C were correlated with short survival time in the patients with lung adnocarcinoma.The results of Gene Ontology and KEGG PATHWAY enrichment analyses indicated that the target genes(differentially expressed genes)regulated by KLF17 were related to the stimulation response,growth and adhesion of tumor cells,and participated in chemotaxis-,adhesion-and extracellular matrix receptor-related signaling path-ways.CONCLUSION:KLF17 inhibits the xenograft tumor growth in nude mice,and inhibits the oncogenes such as RHOV and CORO1C.The target genes regulated by KLF17 participate in the regulation of tumor adhesion-and growth-related sig-naling pathways.
ABSTRACT
AIM:To observe the effect of Yiqi Huayu Huatan decoction(YHHD)on unilaterral ureteral ob-struction(UUO)-induced renal interstitial fibrosis in rats, and to investigate the possible mechanism.METHODS: Fe-male SD rats(n=48)were randomly divided into sham group, model group, telmisartan group, and low-, middle-and high-dose YHHD groups,with 8 rats in each group.The UUO model rats was established by ligating left ureter.The rats in sham group and model group were treated with equal volume of normal saline, others were treated with the corresponding drugs daily.After 12 weeks,the rats were sacrificed.The serum samples were collected for determining the concentrations of cystatin C(Cys-C)and uric acid(UA).The morphological changes of the renal tissue were observed by PAS staining. The collagen fiber was observed by Masson staining.The mRNA expression of Krüppel-like factor 15(KLF15),high-mo-bility group box protein 1(HMGB1),nuclear factor-κB(NF-κB),IκB,monocyte chemotactic protein-1(MCP-1),inter-leukin-1β(IL-1β), tumor necrosis factor-α(TNF-α),fibronectin(FN),collagen type I(Col I)and Col-Ⅳwas detec-ted by real-time PCR.The protein expression of KLF15, HMGB1 and NF-κB was detected by Western blot.The protein expression of MCP-1 was determined by the method of immunohistochemistry.RESULTS:Compared with sham group,the deposition rate of collagen fibers and the concentration of Cys-C in model group were significantly increased(P<0.05), the mRNA and protein expression of KLF15 was significantly down-regulated(P<0.05), while the mRNA expression of HMGB1,NF-κB,IκB,MCP-1,IL-1β,TNF-α,FN,Col I and Col Ⅳand the protein expression of HMGB1,NF-κB and MCP-1 were significantly up-regulated(P<0.05).Compared with model group,the deposition rates of collagen fibers in middle-and high-dose YHHD groups and telmisartan group were significantly decreased(P<0.05),with down-regulated protein expression of HMGB1 and NF-κB and mRNA expression of IL-1βand TNF-α(P<0.05).The protein expression of KLF15 was significantly up-regulated in high-dose YHHD group and telmisartan group(P<0.05),while the protein ex-pression of MCP-1 and the mRNA expression of FN were significantly down-regulated(P<0.05).The mRNA expression of KLF15 was significantly up-regulated in high-dose YHHD group(P<0.05), while the mRNA expression of MCP-1, Col I and Col IV was significantly down-regulated(P<0.05).The mRNA expression of NF-κB and IκB was significantly down-regulated and the concentration of Cys-C was significantly decreased in each dose of YHHD groups and telmisartan group(P<0.05).No significant difference of UA level among the groups was observed.CONCLUSION:YHHD allevi-ates renal interstitial fibrosis in a dose-dependent manner, and YHHD at high dose shows the most obvious effect.The mechanism may be associated with the up-regulation of KLF15 and the down-regulation of HMGB1, NF-κB and its down-stream inflammation-related factors in the renal tissue.
ABSTRACT
Objective To investigate the regulating effects of Krüppel-like factor 6 (KLF6) on the apoptosis of human lens epithelial cells (HLECs) by activating transcription factor 4 (ATF4) pathway and explore the bio-molecular mechanism of KLF6/ATF4-induced HLECs apoptosis.Methods HLECs (HLE-B3) were cultured using high glucose DMEM medium.The eukaryotic expression plasmid pEGFP-C2-ATF4 was transfected into the cells by liposome 2000 in the ATF4-transfected group,and pEGFP-C2 was transfected in the empty plasmid group.Then the cells were exposed to 20 mJ/cm2 ultraviolet ray B (UVB) for 200 seconds,The morphological changes of the cells were observed by hematoxylin & eosin staining and Hoechst33258 fluorescein staining.Cultured cells were transfected using pEGFP-C2-KLF6 and pEGFP-C2 plasmid and pSilencer-KLF6 (siKLF6) and pSilencer plasmid,respectively,and the expression of ATF4 protein in the cells was detected by Western blot assay.Culture cells were divided into four groups.pEGFP-C2 and pSilencer plasmids were co-transfected into the cells in the empty plasmid group;pEGFP-C2-KLF6 and pSilencer empty plasmid were co-transfected into the cells of the KLF6 + pSilencer group;pEGFP-C2 empty plasmid and pSilencer-ATF4 were co-transfected in the cells of the siATF4 + pEGFP-C2 group;pEGFP-C2-KLF6 and pSilencer-ATF4 plasmids were co-transfected in the cells of the KLF6 + siATF4 group,and then the cells were exposed to UVB.The apoptosis of the cells were detected by ELISA assay.Results Cultured cells grew well in the normal control group with the uniform morphology and regular arrangement.The karyopyknosis,karyorrhexis and enlargement of intercellular space were found in the cells exposed to UVB.In the ATF4 transfected group,the number of cells was decreased.The relative expression level of the ATF4 protein in the cells was 0.99±0.06 and 0.13±0.02 in the UVB+ATF4 transfected group and UVB+pEGFP-C2 plasmid group,respectively,with a significant difference between them (t =23.13,P＜0.01).The relative expression levels of KLF6 and ATF4 proteins in the KLF6 transfected group were higher than those in the empty plasmid group,and the relative expression levels of KLF6 and ATF4 proteins in the siKLF6 group were significantly lower than those in the empty plasmid group (all at P＜0.01).ELISA assay showed that the apoptotic rate in the ATF4 transfected group was 1.37± 0.11,which was significantly higher than 0.31 ±0.11 in the normal control group (t =8.034,P =0.001);the apoptotic rate of the cells was increased in the KLF6+pSilencer group and decreased in the siATF4+pEGFP-C2 group in comparison with the empty plasmid group (P＜0.01,P=0.02).In addition,the apoptotic rate in the KLF6+ siATF4 group was remarkably lower than that in the KLF6 + pSilencer group (P＜ 0.01).Conclusions KLF6 promotes the apoptosis of HLECs induced by UVB radiation.Silence of ATF4 gene reduces the apoptotic rate of the cells.ATF4 is probably a target factor in the regulating oathwav of KLF6 to apoptosis.
ABSTRACT
Regulation of vascular smooth muscle cell (VSMC) phenotype plays an essential role in many cardiovascular diseases. In the present study, we provide evidence that krüppel-like factor 8 (KLF8) is essential for tumor necrosis factor α (TNFα)-induced phenotypic conversion of VSMC obtained from thoracic aorta from 4-week-old SD rats. Stimulation of the contractile phenotype of VSMCs with TNFα significantly reduced the VSMC marker gene expression and KLF8. The gene expression of KLF8 was blocked by TNFα stimulation in an ERK-dependent manner. The promoter region of KLF8 contained putative Sp1, KLF4, and NFκB binding sites. Myocardin significantly enhanced the promoter activity of KLF4 and KLF8. The ectopic expression of KLF4 strongly enhanced the promoter activity of KLF8. Moreover, silencing of Akt1 significantly attenuated the promoter activity of KLF8; conversely, the overexpression of Akt1 significantly enhanced the promoter activity of KLF8. The promoter activity of SMA, SM22α, and KLF8 was significantly elevated in the contractile phenotype of VSMCs. The ectopic expression of KLF8 markedly enhanced the expression of SMA and SM22α concomitant with morphological changes. The overexpression of KLF8 stimulated the promoter activity of SMA. Stimulation of VSMCs with TNFα enhanced the expression of KLF5, and the promoter activity of KLF5 was markedly suppressed by KLF8 ectopic expression. Finally, the overexpression of KLF5 suppressed the promoter activity of SMA and SM22α, thereby reduced the contractility in response to the stimulation of angiotensin II. These results suggest that cross-regulation of KLF family of transcription factors plays an essential role in the VSMC phenotype.