This study was aimed to construct a recombinant adenovirus vector for antisense klf4 gene through AdEasy system. Human klf4 cDNA was reversely inserted into the multiple cloning sites (MCS)of the pShuttle-CMV by using the backbone plasmid AdEasy-l, the antisense klf4 gene was constructed through homologous recombination in E.coli BJ5183, then the adenoviruses were packaged and amplified in the HEK 293 ce1ls. The adenovirus vector for antisense klf4 gene confirmed by PCR, restriction analysis and DNA sequencing. After being transfected with the adenoviruses at 200 MOI for 48 hours, total RNA and protein were extracted from human umbilical vein endothelial cells (HUVEC). Klf4 mRNA and KLF4 protein expression levels were evaluated by Real-time PCR and Western-blot. The results showed that the recombinant adenovirus vector for antisense klf4 gene was successfully constructed, recombinant adenovirus could suppress the expression of klf4 mRNA and KLF4 protein in HUVECs. It is concluded that the adenovirus vector for antisense klf4 gene has been constructed successfully, which provides the material basis for further studying the biologic function and potential application of klf4.
Adenoviridae ; genetics ; Antisense Elements (Genetics) ; Genetic Vectors ; Humans ; Kruppel-Like Transcription Factors ; genetics ; Plasmids ; Transfection

OBJECTIVETo study the frequency of rare blood group Lu(a-b-) phenotype in a population from Shanghai region, and to explore the molecular basis of Lu(a-b-) by detecting the Lu and Lu relative mediator gene EKLF/KLF1.

METHODSDonors from Shanghai region were screened for Lutheran blood group by monoclonal anti-Lub using serological methods. Individuals with Lu(b-) were determined Lua, P1 and i antigens. Fifteen exons of the LU gene and 3 exons of the EKLF/KLF1 gene for the identified Lu(a-b-) samples were amplified and sequenced.

RESULTSTen Lu(a-b-) donors were obtained from 44 331 donors from Shanghai region. No homozygous or heterozygous mutations were found in the LU gene, whilst 7 mutations in EKLF/KLF1 gene were identified in the 10 samples.

CONCLUSIONThe frequency of rare Lu(a-b-) blood group in Shanghai was approximately 0.02%, and all the individuals had an In(Lu) phenotype. The molecular basis of such samples may be related to mutations in the EKLF/KLF1 gene.

China ; ethnology ; Humans ; Kruppel-Like Transcription Factors ; genetics ; Lutheran Blood-Group System ; genetics ; Mutation ; Phenotype

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 (T_au=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.
Animals ; Humans ; Chickens/genetics* ; Kruppel-Like Transcription Factors/metabolism* ; Transcription Factors/metabolism* ; Adipocytes/metabolism* ; Adipose Tissue/metabolism*

OBJECTIVE: To observe the effect of Krüppel-like factor 4 (KLF4) overexpression on heat stress-induced apoptosis of Raw264.7 macrophages. METHODS: The fragment containing full length mouse KLF4 cDNA coding sequence was inserted into the pcDNA3.1 vector and Raw264.7 macrophages were transfected with pcDNA3.1-KLF4 plasmids using lipofectamine.The expression of KLF4 was examined by Western blot in the Raw264.7 macrophages stably transfected with pcDNA3.1- KLF4 plasmids. Raw264.7 cells transfected with pcDNA3.1 and pcDNA3.1-KLF4 were exposed to heat stress (42 degrees C) for 1h and recovered at 37 degrees C for 12h. Flow cytometry, Hoechest 33258 staining assay, and DNA ladder assays were performed to assess the apoptosis. RESULTS: The KLF4 overexpressed Raw264.7 macrophages were established. After the heat stress, flow cytometry showed that apoptotic cells increased significantly in KLF4 overexpressed cells compared with the vector control; Hoechest 33258 staining was characterized with classical changes including apoptotic body, and nuclear condensation. Agarose gel electrophoresis showed that "DNA ladder" could be observed clearly. CONCLUSION KLF4 overexpression can increase heat stress-induced apoptosis of Raw264.7 macrophages.
Animals ; Apoptosis ; genetics ; Cell Line ; Genetic Vectors ; Heat-Shock Response ; Kruppel-Like Transcription Factors ; genetics ; Macrophages ; cytology ; Mice ; Plasmids ; Transfection

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 ; Kruppel-Like Transcription Factors/*biosynthesis ; Kruppel-Like Transcription Factors/genetics ; Liver/metabolism ; Liver Neoplasms/*metabolism ; Proto-Oncogene Proteins/*biosynthesis ; Proto-Oncogene Proteins/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/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

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 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

Animals ; Astrocytes ; cytology ; Cell Differentiation ; Cellular Reprogramming ; Induced Pluripotent Stem Cells ; cytology ; Kruppel-Like Transcription Factors ; genetics ; metabolism ; Mice ; Neurons ; cytology ; Octamer Transcription Factor-3 ; genetics ; metabolism

OBJECTIVE: To explore the clinical and genetic characteristics of a child with Pallister-Hall syndrome (PHS). METHODS: DNA was extracted from peripheral blood sample from the child and subjected to whole exome sequencing. Suspected variants were verified by Sanger sequencing of his family members. RESULTS: Genetic testing revealed that the child has harbored a heterozygous c.3320_3330delGGTACGAGCAG (p.G1107Afs×18) variant of the GLI3 gene. Neither parent was found to carry the same variant. CONCLUSION The c.3320_3330delGGTACGAGCAG (p.G1107Afs×18) frameshift variant of the GLI3 gene probably underlay the pathogenesis of PHS in this child. Genetic testing should be considered for patients featuring hypothalamic hamartoma and central polydactyly.
Humans ; Child ; Pallister-Hall Syndrome/genetics* ; Kruppel-Like Transcription Factors/genetics* ; Zinc Finger Protein Gli3/genetics* ; Polydactyly/genetics* ; Hamartoma/pathology* ; Nerve Tissue Proteins/genetics*