Objective: To explore the expression characteristics and role of Krüppel-like factor 4 (KLF4) in macrophage inflammatory response and its effects on inflammatory response and organ injury in septic mice, so as to lay a theoretical foundation for targeted treatment of burns and trauma sepsis. Methods: The method of experimental research was used. Mouse RAW264.7 macrophages and primary peritoneal macrophages (PMs) isolated from 10 male C57BL/6J mice aged 6-8 weeks were used for the experiments. RAW264.7 macrophages and PMs were treated with endotoxin/lipopolysaccharide (LPS) for 0 (without treatment), 1, 2, 4, 6, 8, 12, and 24 h, respectively, to establish macrophage inflammatory response model. The mRNA expression of interleukin 1β (IL-1β), IL-6, CC chemokine ligand 2 (CCL2) and tumor necrosis factor-α (TNF-α) were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR), and the LPS treatment time was determined for some of the subsequent experiments. RAW264.7 macrophages were treated with LPS for 0 and 8 h, the localization and protein expression of KLF4 were detected by immunofluorescence method, transcriptome sequencing of the cells was performed using the high-throughput sequencing technology platform, and the differently expressed genes (DEGs) between the two time points treated cells were screened by DESeq2 software. RAW264.7 macrophages and PMs were treated with LPS for 0, 1, 2, 4, 6, 8, 12, and 24 h, respectively, and the mRNA and protein expressions of KLF4 were detected by real-time fluorescence quantitative RT-PCR and Western blotting, respectively. RAW264.7 macrophages were divided into negative control (NC) group and KLF4-overexpression group according to the random number table, which were treated with LPS for 0 and 8 h respectively after transfection of corresponding plasmid. The mRNA expressions of KLF4, IL-1β, IL-6, CCL2, and TNF-α were detected by real-time fluorescence quantitative RT-PCR, while the protein expression of KLF4 was detected by Western blotting. The number of samples in aforementioned experiments was all 3. Forty male C57BL/6J mice aged 6-8 weeks were divided into KLF4-overexpression group and NC group (with 20 mice in each group) according to the random number table, and the sepsis model of cecal ligation perforation was established after the corresponding transfection injection was injected respectively. Twelve mice were selected from each of the two groups according to the random number table, and the survival status within 72 hours after modeling was observed. Eight hours after modeling, the remaining 8 mice in each of the two groups were selected, the eyeball blood samples were collected to detect the levels of IL-1β and IL-6 in serum by enzyme-linked immunosorbent assay, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum by dry chemical method. Subsequently, the heart, lung, and liver tissue was collected, and the injury was observed after hematoxylin-eosin staining. Data were statistically analyzed with independent sample t test, Cochran & Cox approximate t test, one-way analysis of variance, Dunnett test, Brown-Forsythe and Welch one-way analysis of variance, Dunnett T3 test, log-rank (Mantel-Cox) test. Results: Compared with that of LPS treatment for 0 h, the mRNA expressions of IL-1β in RAW264.7 macrophages treated with LPS for 6 h and 8 h, the mRNA expressions of IL-6 in RAW264.7 macrophages treated with LPS for 4-12 h, the mRNA expressions of CCL2 in RAW264.7 macrophages treated with LPS for 8 h and 12 h, and the mRNA expressions of TNF-α in RAW264.7 macrophages treated with LPS for 4-8 h were significantly up-regulated (P<0.05 or P<0.01), while the mRNA expressions of IL-1β and CCL2 in PMs treated with LPS for 4-8 h, the mRNA expressions of IL-6 in PMs treated with LPS for 2-24 h, and the mRNA expressions of TNF-α in PMs treated with LPS for 2-12 h were significantly up-regulated (P<0.05 or P<0.01). Eight hours was selected as the LPS treatment time for some of the subsequent experiments. KLF4 mainly located in the nucleus of RAW264.7 macrophages. Compared with those of LPS treatment for 0 h, the protein expression of KLF4 in RAW264.7 macrophages treated with LPS for 8 h was obviously decreased, and there were 1 470 statistically differentially expressed DEGs in RAW264.7 macrophages treated with LPS for 8 h, including KLF4 with significantly down-regulated transcriptional expression (false discovery rate<0.05, log₂ (fold change)=-2.47). Compared with those of LPS treatment for 0 h, the mRNA expressions of KLF4 in RAW264.7 macrophages treated with LPS for 6-24 h, the protein expressions of KLF4 in RAW264.7 macrophages and PMs treated with LPS for 1-24 h, and the mRNA expressions of KLF4 in PM treated with LPS for 4-24 h were significantly decreased (P<0.05 or P<0.01). Compared with those in NC group, the mRNA (with t' values of 17.03 and 8.61, respectively, P<0.05 or P<0.01) and protein expressions of KLF4 in RAW264.7 macrophages treated with LPS for 0 h and 8 h in KLF4-overexpression group were significantly increased, the mRNA expressions of IL-6 and CCL2 increased significantly in RAW264.7 macrophages treated with LPS for 0 h (with t values of 6.29 and 3.40, respectively, P<0.05 or P<0.01), while the mRNA expressions of IL-1β, IL-6, CCL2, and TNF-α decreased significantly in RAW264.7 macrophages treated with LPS for 8 h (with t values of 10.52, 9.60, 4.58, and 8.58, respectively, P<0.01). The survival proportion of mice within 72 h after modeling in KLF4-overexpression group was significantly higher than that in NC group (χ²=4.01, P<0.05). Eight hours after modeling, the serum levels of IL-1β, IL-6 and ALT, AST of mice in KLF4-overexpression group were (161±63), (476±161) pg/mL and (144±24), (264±93) U/L, respectively, which were significantly lower than (257±58), (654±129) pg/mL and (196±27), (407±84) U/L (with t values of 3.16, 2.44 and 4.04, 3.24, respectively, P<0.05 or P<0.01) in NC group. Eight hours after modeling, compared with those in NC group, the disorder of tissue structure of heart, lung, and liver, inflammatory exudation, and pathological changes of organ parenchyma cells in KLF4-overexpression group were obviously alleviated. Conclusions: The expression of KLF4 is significantly down-regulated in LPS-induced macrophage inflammatory response, which significantly inhibits the macrophage inflammatory response. KLF4 significantly enhances the survival rate of septic mice and alleviates inflammatory response and sepsis-related organ injury.
Male ; Mice ; Animals ; Mice, Inbred C57BL ; Lipopolysaccharides ; Tumor Necrosis Factor-alpha ; Kruppel-Like Factor 4 ; Interleukin-6 ; Wound Infection ; Sepsis

OBJECTIVETo study the effects of rosiglitazone on Kruppule-like factor 6 (KLF6) and its target gene TGFâ1 during the development of nonalcoholic fatty liver fibrosis.

METHODThirty-six Wistar rats were divided into three groups: a control group, a high fat model group and an intervention group. Their blood serum TG, FFA, AST, ALT, HA, LN and CIV were measured. Hepatic expressions of KLF6, TGFbeta1 and alpha-SMA were detected by RT-PCR and immunohistochemistry. The pathological features and the degree of liver fibrosis before and after the rosiglitazone intervention were also studied.

RESULTSThe contents of TG, FFA, AST, ALT, HA, LN and CIV, the expression of KLF6, TGFbeta1 and alpha-SMA mRNA, and the degree (score) of liver fibrosis at the 24th week in the model group were significantly higher than those in the control group (P<0.01) but they were lower in the rosiglitazone intervention group (P<0.05). The protein expression of a-SMA was also lower in the intervention group compared with that of the model group.

CONCLUSIONRosiglitazone, to a certain extent, can inhibit KLF6-TGFbeta1 signal transduction by inducing expression of PPAR-gamma, and then inhibit the activation of hepatic stellate cells and minimize hepatic fibrosis.

Animals ; Fatty Liver ; metabolism ; pathology ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; metabolism ; Liver ; metabolism ; Male ; PPAR gamma ; metabolism ; Proto-Oncogene Proteins ; metabolism ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Thiazolidinediones ; pharmacology ; Transforming Growth Factor beta1 ; metabolism

To investigate the expression of KLF6mRNA in primary hepatocellular carcinoma (HCC), nomal liver tissues and the tissues adjacent to the cancers, reverse-transcription polymerase chain reaction (RT-PCR) was employed to investigate the expression of the KLF6 gene in HCC, the corresponding adjacent non-cancerous tissues and normal liver tissue. Our results showed that an amplified fragment of 427 bp DNA was detected in 18 of 19 (94.7%) adjacent non-cancerous tissues and normal liver tissue, and in 12 (85.7%) of 14 HCC. There were no significant differences in the levels of KLF6 mRNA between normal liver and liver tumors (P>0.05). It is concluded that KLF6 mRNA is generally expressed in HCC.
Carcinoma, Hepatocellular ; metabolism ; Humans ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; biosynthesis ; genetics ; Liver ; metabolism ; Liver Neoplasms ; metabolism ; Proto-Oncogene Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo explore the mutated KLF6 gene in hepatocellular carcinoma (HCC) and to characterize its behavior in human hepatocellular carcinoma cell line HepG2.

METHODSWe analyzed the DNA isolated from 23 hepatocellular carcinoma tissues and their adjacent nontumor tissues by polymerase chain reaction (PCR). Direct sequencing was used to establish the incidence of mutation in exon2 of the KLF6 gene. Loss of growth suppressive function of the HCC-derived KLF6 mutants was characterized by in vitro analyzing alteration of cell cycle and MTT assay. Expression of p21WAF1, a possible downstream gene of KLF6, was detected in human hepatocellular carcinoma cell line HepG2 transiently transfected with KLF6 genes.

RESULTSMutations of KLF6 were found in 2 of the 23 (8.7%) hepatocellular carcinomas. The two mutations were located in the transactivation domain and one of them resulted in single amino acid substitution of TGG (W) by GGG (G) at codon 162. Unlike the wild-type KLF6, cancer-derived KLF6 mutants neither suppressed growth nor induced p21WAF1 following transfection into culture cells.

CONCLUSIONSMutations of the KLF6 gene may play a role in the pathogenesis of HCC, but are not the dominating mechanism resulting in inactivation of KLF6 functions. KLF6 suppresses hepatocellular carcinoma cell proliferation partly through upregulating expression of the p21WAF1 gene.

Base Sequence ; Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; genetics ; physiology ; Liver Neoplasms ; genetics ; pathology ; Molecular Sequence Data ; Point Mutation ; Proto-Oncogene Proteins ; genetics ; physiology ; Sequence Analysis, DNA

OBJECTIVETo investigate the roles of Kruppel-like factor 6 (KLF6) and its splice variant KLF6V on suppressing growth and inducing differentiation of human hepatocellular carcinoma hepG2 cells.

METHODKLF6V cDNA was amplificated by RT-PCR from human hepatocellular carcinoma (HCC) tissue and then sequenced. The recombinant vectors expressing KLF6 variant (KLF6V) were constructed using molecular clone technology based on established plasmid pcDNA3.1A(-)/wtKLF6. KLF6V or KLF6-transfected HepG2 cells were established after being screened with G418. Growth activity of HepG2/KLF6 or HepG2/KLF6V cells was detected by in vitro MTT assay. Expression of p21WAF1 or cyclin D1 protein was detected by Western blot, and expressions of AFP or ALB protein were measured by radioimmunoassay.

RESULTSA novel alternatively spliced transcript of the human KLF6 gene was found and its sequencing revealed that the variant form of KLF6 lacked 126nt and its encoded protein products had a deletion of 42 aa near the COOH-terminal amino acid in comparison with full-length KLF6. Although KLF6 alternative splicing was present in both normal and cancerous tissues, expression of the KLF6 splice variants seemed to be up-regulated in HCCs tissues. The isoform of KLF6 proteins antagonized the ability of wild-type KLF6 to up-regulate p21 expression or down-regulate cyclin D1 expression and suppress HepG2 cell proliferation. KLF6 gene increased albumin production and decreased alpha fetoprotein production of the cells.

CONCLUSIONThe isoform of KLF6 protein, present in HCC tissue, antagonizes the ability of wild-type KLF6 to suppress cell proliferation and induce cellular differentiation.

Cell Differentiation ; Cell Proliferation ; DNA, Complementary ; Gene Expression Regulation, Neoplastic ; Hep G2 Cells ; Humans ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; genetics ; Protein Isoforms ; genetics ; Proto-Oncogene Proteins ; genetics ; Transfection

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.
Animals ; Rats ; Myocytes, Cardiac ; Kruppel-Like Factor 6 ; Connexin 43 ; Desmin ; Cell Differentiation ; Azacitidine/pharmacology* ; Mesenchymal Stem Cells ; RNA, Messenger ; MicroRNAs

Animals ; Cells, Cultured ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Hepatocytes ; drug effects ; metabolism ; Humans ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; NF-kappa B ; metabolism ; Proto-Oncogene Proteins ; RNA, Messenger ; metabolism ; Trans-Activators ; genetics ; metabolism ; Transcription Factor AP-1 ; metabolism ; Transcription Factors ; pharmacology

BACKGROUND AND AIMThe Krüppel-like transcription factor KLF6 is a novel tumor-suppressor gene. It was inactivated in human prostate cancer and other tumors tissue, as the result of frequent mutation and loss of heterozygosity (LOH). However, there is no data reporting the levels of KLF6 both mRNA and protein in hepatocellular carcinomas (HCCs). We therefore detected mutations and expression of KLF6 in HCC tissues and further observed the effect of it on cell growth in HCC cell lines.

METHODSWe analyzed the exon-2 of KLF6 gene by direct DNA sequencing, and detected the expression of KLF6 by RT-PCR and Western blot in 23 HCC tissues and corresponding nontumorous tissues. Loss of growth suppressive effect of the HCC-derived KLF6 mutant was characterized by in vitro growth curves plotted, flow cytometry and Western blotting.

RESULTSKLF6 mutations were found in 2 of 23 HCC tissues and one of mutations was missense. Expression of KLF6 mRNA or protein was down-regulated in 8 (34.7%) or 9 (39.1%) of 23 HCC tissues. Wild-type KLF6 (wtKLF6) inhibited cellular proliferation and prolonged G1-S transition by inducing the expression of p21WAF1 following stable transfection into cultured HepG2 cells, but tumor-derived KLF6 mutant (mKLF6) had no effects.

CONCLUSIONOur findings suggest that KLF6 may be involved in pathogenesis of HCC.

Blotting, Western ; Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; genetics ; Liver Neoplasms ; genetics ; pathology ; Mutation ; Proto-Oncogene Proteins ; genetics ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Transfection

OBJECTIVETo observe the effect of KLF6 on prostate cancer cell line PC-3 by transgenic method.

METHODSWe obtained KLF6 cDNA by RT-PCR method from the liver cell, transfected plasmid pEGFP-C, recombinated with KLF6 into PC-3 cells, and used them as a transfection group and a control group. MTT, flow cytometer and immunocytochemical methods were used to observe the effect of anti-oncogene wild type KLF6 on prostate cancer cell line PC-3 by transgenic method for 48 hours.

RESULTSAfter transfected into PC-3 cells, KLF6 enhanced growth suppression, (30.0 +/- 5.4)% in the transfection group and 0% in the control, P < 0.01, apoptosis, (24.3 +/- 2.3)% in the transfection group and (5.2 +/- 0.7)% in the control, P < 0.01, the down-regulation of the expression of cyclin D1, (25.3 +/- 3.7)% in the transfection group and (38.5 +/- 4.6)% in the control, P < 0.05 and Bcl-2, (18.7 +/- 3.2)% in the transfection group, and (41.8 +/- 5.9)% in the control, P < 0.01 in PC-3 cells. It also decreased the ratio of the cell phase G2/M, increased the ratio of G0/G1 from (58.6 +/- 7.3)% in the control to (80.0 +/- 9.8)% in the transfection group, P < 0.05.

CONCLUSIONPC-3 cells transfected with wild type KLF6 can enhance its growth suppression and apoptosis. It shows great potential for the gene therapy of androgen-independent carcinoma of the prostate.

Apoptosis ; physiology ; Cell Cycle ; physiology ; Cell Line, Tumor ; Cyclin D1 ; biosynthesis ; Down-Regulation ; Flow Cytometry ; Humans ; Immunohistochemistry ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; genetics ; physiology ; Male ; Prostatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins ; genetics ; physiology ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo investigate the expressions and clinical implications of kruppel-like factor 6(KLF-6) and APC in human colorectal carcinoma.

METHODSThe expressions of KLF-6 and APC in tumor and normal tissues from 32 patients with colorectal carcinoma were investigated by RT-PCR and immunohistochemical technique.

RESULTSThe expression rates of KLF-6 and APC mRNA were 37.5% and 34.3% in tumor tissue, 96.9% and 93.8% respectively in normal tissues (both P< 0.05). The expression rates of KLF-6 and APC protein were 28.1% and 25.0% in colorectal carcinomas, 81.3% and 84.43% respectively in normal tissues (both P< 0.05). There was a significant correlation between the expressions of KLF-6 and APC in colorectal carcinomas (P < 0.05). The expressions of KLF-6 and APC were significantly correlated with tumor differentiation, depth of infiltration, lymph node metastasis and clinical stage (P< 0.05).

CONCLUSIONDown-regulations of KLF-6 and APC might play an important role in the carcinogenesis, development, metastasis of human colorectal carcinoma.

Adenomatous Polyposis Coli Protein ; genetics ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Female ; Gene Expression ; Genes, APC ; Humans ; Intestinal Mucosa ; metabolism ; pathology ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; genetics ; Male ; Middle Aged ; Neoplasm Staging ; Proto-Oncogene Proteins ; genetics ; RNA, Messenger ; genetics