Krüppel-like factor 15 deficiency exacerbates osteoarthritis through reduced expression of peroxisome proliferator-activated receptor gamma signaling in mice.

OBJECTIVE: Krüppel-like zinc finger transcription factors (KLFs) play diverse roles in mammalian cell differentiation and development. In this study, we investigated the function of KLF15 in the progression of osteoarthritis (OA). METHODS: 0Destabilization of the medial meniscus (DMM) surgery was performed in 10-week-old male wild-type control (WT) mice and cartilage-specific KLF15 knockout (KO) mice. Histological analysis, immunohistochemistry, and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling staining were performed. Morphological changes were measured using microcomputed tomography. Six mice from each group were analyzed (total number of mice analyzed: 60). In vitro, immunofluorescence, quantitative reverse transcription-polymerase chain reaction, and western blot analyses were performed. RESULTS: KLF15 KO DMM mice exhibited significant cartilage degradation compared to WT mice. According to the Osteoarthritis Research Society International cartilage OA-histopathology scoring system, the mean sum score in KLF15 KO mice was significantly higher than that in WT mice at 8 weeks after surgery. Immunohistochemistry results revealed KLF15 KO mice exhibited reduced peroxisome proliferator-activated receptor gamma (PPARγ) expression, increased pIKKα/ß, a disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTS) 5, and Matrix metalloproteinases (MMP13) expression, and reduced Forkhead box O (FOXO1) and Light chain 3B (LC3B) expression. Inhibition of PPARγ phosphorylation accelerated the effects of interleukin (IL) 1ß-treatment in both KLF15 KO and WT chondrocytes, and activation of PPARγ expression canceled the IL1ß-induced catabolic effects. CONCLUSION: Our results indicated that the OA phenotype of KLF15 KO DMM mice was influenced by reduced PPARγ expression, including enhanced pIKKα/ß, ADAMTS5, and MMP13 expression, reduced autophagy, and increased apoptosis. KLF15 regulation may constitute a possible therapeutic strategy for the treating OA.

LncRNA SLC1A5-AS/MZF1/ASCT2 Axis Contributes to Malignant Progression of Hepatocellular Carcinoma.

BACKGROUND: Hypoxia is a pivotal factor influencing cellular gene expression and contributing to the malignant progression of tumors. Metabolic anomalies under hypoxic conditions are predominantly mediated by mitochondria. Nonetheless, the exploration of hypoxia-induced long noncoding RNAs (lncRNAs) associated with mitochondria remains largely uncharted. METHODS: We established hypoxia cell models using primary human hepatocytes (PHH) and hepatocellular carcinoma (HCC) cell lines. We isolated mitochondria for high-throughput sequencing to investigate the roles of candidate lncRNAs in HCC progression. We employed in vitro and in vivo assays to evaluate the functions of solute carrier family 1 member 5 antisense lncRNA (SLC1A5-AS). RNA-seq was utilized to scrutinize the comprehensive genome profile regulated by SLC1A5-AS in HCC. Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis were utilized to validate the expression of alanine-serine-cysteine transporter 2 (ASCT2, encoded by the SLC1A5 gene), and a glutamine uptake assay was employed to estimate the glutamine uptake capacity of Huh-7 cells after SLC1A5-AS overexpression. To delve into the mechanisms governing the regulation of SLC1A5 expression by SLC1A5-AS, we employed a biotin-labeled SLC1A5-AS probe in conjunction with a western blot assay to confirm the interactions between SLC1A5-AS and candidate transcription factors. Luciferase reporter assays and chromatin immunoprecipitation (ChIP) were utilized to authenticate the effects of the predicted transcription factors on SLC1A5 promoter activity. RESULTS: Following the screening, we identified CTB-147N14.6, derived from the antisense strand of the SLC1A5 gene, which we have named SLC1A5-AS. SLC1A5-AS exhibited significantly elevated expression levels in HCC tissue and was associated with poor prognosis in HCC patients. In vitro and in vivo assays revealed that the overexpression of SLC1A5-AS significantly heightened cell invasion and metastasis. RNA-seq data unveiled SLC1A5-AS involvement in glutamine metabolism, left-handed amino (L-amino) acid transmembrane transporter activity, and the nuclear factor kappa-B (NF-κB) signaling pathway. Overexpression of SLC1A5-AS markedly increased ASCT2 mRNA/protein levels, thereby enhancing glutamine uptake and promoting the growth and metastasis of HCC cells. Mechanistically, higher RNA levels of SLC1A5-AS directly bound with myeloid zinc finger 1 (MZF1), acting as a transcriptional repressor, thus diminishing its binding to the SLC1A5 promoter region. CONCLUSIONS: Our findings unveil a novel role for the lncRNA SLC1A5-AS in glutamine metabolism, suggesting that targeting SLC1A5-AS/MZF1, in conjunction with ASCT2 inhibitor treatment, could be a potential therapeutic strategy for this disease.

The active phthalate metabolite, DHEP, induces proliferation and metastasis of prostate cancer cells via upregulation of ß-catenin and downregulation of KLF7.

Phthalates such as DHEP are among the widely used compounds in industry. It has been shown that DHEP can convey various biological consequences in mammalian cells, among them, the carcinogenic effects of DHEP are emphasized. The present study aimed to assess the impact of DHEP exposure on the proliferation and invasiveness of DU145 prostate cancer cells through in vitro and in vivo models. The DU145 cells were treated with increasing concentrations of DHEP and the tumorigenic parameters were analyzed. KLF7 as a probable mediator of the effect of DHEP was either overexpressed or knocked down in DU145 to evaluate the probable impact of KLF7 on the biological effects of DHEP. The effect of DHEP was also studied in a DU145 xenograft tumor model. The moderate doses of DHEP increased the proliferation and migration of DU145 cells. In the case of gene expression patterns, DHEP reduced the levels of p53 and KLF7 while elevated the expression of ß-catenin. The knock-down of KLF7 conveyed comparable effects to that of DHEP to some degree and increased the proliferation of DU145 cells, while the transduction of KLF7 increased the expressions of p53 and p21 along with controlling the tumor size. The present study demonstrated the potential of DHEP in increasing the tumorigenic properties of DU145 cells along with a focus on the underlying mechanisms. Sustained exposure to DHEP can cause a dysregulation in balance between oncogenes and tumor suppressor genes which is the hallmark of malignant transformation. Thus, special considerations seem necessary for the safe exploitation of phthalates.

Simvastatin inhibits proliferation and promotes apoptosis of oral squamous cell carcinoma through KLF2 signal.

OBJECTIVES: This study aimed to explore the role and specific mechanism of the cholesterol-lowering drug simvastatin in inhibiting oral squamous cell carcinoma (OSCC). METHODS: The proliferation, apoptosis, and migration levels of OSCC cells were detected by CCK8, quantitative real-time polymerase chain reaction, Western blot, colony formation, TdT-mediated dUTP Nick-End Labeling assay, and wound healing assay. The inhibitory effect of simvastatin in vivo was detected by a mouse xenograft tumor model. Immunohistochemistry and immunofluorescence staining were used to assess the KLF2 and ß-catenin expressions in cells and tissues. RESULTS: KLF2 expression in OSCC cells and tissues was downregulated. The addition of KLF2 inducer, GGTI298, inhibited the proliferation and migration of OSCC cells. Simvastatin played a role in inhibiting the proliferation and promoting the apoptosis of OSCC cells. Moreover, it inhibited ß-catenin expression and promoted KLF2 expression in OSCC cells. KLF2 siRNA reversed the effect of simvastatin on the proliferation and apoptosis of OSCC cells. CONCLUSIONS: KLF2, as a tumor suppressor gene, may be an important marker for diagnosing and treating OSCC. Simvastatin inhibits the progression of OSCC by regulating the KLF2 signal.

TRAF7 inhibits glycolysis to potentiate growth inhibition and apoptosis of myeloid leukemia cells via regulating the KLF2-PFKFB3 axis.

Tumor necrosis factor receptor-related factor 7 (TRAF7) can regulate cell differentiation and apoptosis, but its specific functional mechanism in the pathological process of acute myeloid leukemia (AML) closely related to differentiation and apoptosis disorders is largely unclear. In this study, TRAF7 was found to be lowly expressed in AML patients and a variety of myeloid leukemia cells. TRAF7 was overexpressed in AML Molm-13 and chronic myeloid leukemia (CML) K562 cells by transfection with pcDNA3.1-TRAF7. CCK-8 assay and flow cytometry analysis showed that TRAF7 overexpression induced growth inhibition and apoptosis in K562 and Molm-13 cells. Measurements of glucose and lactate suggested that TRAF7 overexpression impaired glycolysis of K562 and Molm-13 cells. Cell cycle analysis indicated that most of K562 and Molm-13 cells were captured in G0/G1 phase by TRAF7 overexpression. PCR and western blot assay revealed that TRAF7 increased Kruppel-like factor 2 (KLF2) expression but decreased 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression in AML cells. KLF2 knockdown can counteract TRAF7-triggered PFKFB3 inhibition, and abolish TRAF7-mediated glycolysis inhibition and cell cycle arrest. KLF2 knockdown or PFKFB3 overexpression both can partially neutralize TRAF7-induced growth inhibition and apoptosis of K562 and Molm-13 cells. Moreover, Lv-TRAF7 decreased human CD45+ cells in mouse peripheral blood in the xenograft mice established by NOD/SCID mice. Taken together, TRAF7 exerts anti-leukemia effects by impairing glycolysis and cell cycle progression of myeloid leukemia cells via modulating the KLF2-PFKFB3 axis.

Kruppel-like Factors 3 Regulates Migration and Invasion of Gastric Cancer Cells Through NF-κB Pathway.

Context: The poorly understood regulatory mechanisms impede gastric cancer therapy. Kruppel-like factors (KLFs) are associated with the development of various tumors, The studies on the role of the KLF transcription factor 13 (KLF13) in gastric cancer progression haven't been studied. Objective: The current study aimed to investigate the role of KLF13 in the migration and invasion of gastric cancer and the regulatory mechanism of KLF13 in gastric cancer progression. Design: The research team performed a laboratory study. Setting: The study took place at the Zengcheng District People's Hospital of Guangzhou in Zengcheng, China. Participants: In addition to using normal gastric cells, GES1, and seven gastric cancer cell lines, the research team compared the fresh, gastric cancer tissues (T) and paired, adjacent, noncancerous gastric tissues (ANT) from eight patients undergoing surgical resection at the hospital. The research team also downloaded the data for 33 gastric cancer tissues and adjacent, normal gastric tissues from the Cancer Genome Atlas' TCGA database. Intervention: The research team used: (1) short hairpin RNAs (shRNAs) to knock down KLF13, (2) wound healing and transwell invasion analyses to determine the effects of KLF13 on the migration and invasion of gastric cancer, and (3) a Luciferase reporter assay to determine the effects of KLF13 on nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity. Results: KLF13 was upregulated in gastric cancer cells and tissues, and the patients with a high KLF13 expression had poor outcome. Downregulation of KLF13 significantly inhibited the migration and invasion of gastric cancer cells. Mechanistically, downregulation of KLF13 significantly inhibited NF-κB activity, and its targets such as: (1) snail family transcriptional repressor 1 (SNAI1 or Snail), (2) snail family transcriptional repressor 2 (SNAI2 or Slug), (3) zinc finger e-box binding homeobox 1 (ZEB1), (4) Smad interacting protein 1 (Sip1), (5) twist family basic helix-loop-helix (BHLH) transcription factor (Twist), (6) matrix metallopeptidase 2 (MMP2), and (7) MMP9. Tumor necrosis factor alpha (TNF-α) can activate NF-κB. Treating with TNF-α can reverse the effects of KLF13 downregulation on migration and invasion, confirming that KLF13 promotes the migration and invasion of gastric cancer cells through activating the NF-κB pathway. Conclusions: KLF13 promoted the migration and invasion of gastric cancer cells through activating the NF-κB pathway, providing a new target for gastric cancer therapy.

KLF5-mediated aquaporin 3 activated autophagy to facilitate cisplatin resistance of gastric cancer.

BACKGROUND: Resistance to chemotherapeutic drugs limits the control of gastric cancer (GC) development. The study intended to probe into the mechanism of aquaporin 3 (AQP3) on the chemoresistance of GC. METHODS: Cisplatin (CDDP)-resistant cells were constructed. Parental AGS and HGC-27 cells and their respective CDDP-resistant cells were transfected with AQP3 overexpression plasmid, AQP3 short hairpin RNA (sh-AQP3) and sh-Kruppel-like factor 5 (shKLF5). The expressions of AQP3 and factors related to autophagy (LC3 I, LC3 II, Atg5, Beclin-1, p62)/epithelial-mesenchymal transition (EMT; E-cadherin and snail) were assessed by Western blot and qRT-PCR. Cell counting kit-8 assay was adopted to test cell viability and half maximal inhibitory concentration (IC 50) was determined. Transwell assay was used for the examination of cell migration and invasion. The regulatory relationship of AQP3 and KLF5 was tested by chromatin immunoprecipitation (ChIP) and dual luciferase reporter assays. RESULTS: AQP3 was highly-expressed in GC cells and its level was even higher in CDDP-resistant GC cells. AQP3 silencing inhibited viability, autophagy and EMT in CDDP-resistant GC cells, while AQP3 overexpression had the opposite effect. KLF5 positively modulated AQP3 in GC cells resistant to CDDP. KLF5 knockdown reversed AQP3-induced autophagy, viability, migration, invasion and EMT in CDDP-resistant GC cells. CONCLUSION: KLF5-modulated AQP3 activated autophagy to facilitate the resistance of GC to CDDP.

KLF9 positively regulates TRIM33 to inhibit abnormal synovial fibroblast proliferation, migration as well as inflammation in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) can cause irreversible joint injury and serious disability. This study aimed to investigate how TRIM33 regulated by KLF9 affects the aggressive behaviors of synovial fibroblasts induced by tumor necrosis factor-α (TNF-α). MATERIALS AND METHODS: TNF-α-induced MH7A cells were used to simulate the in vitro model of RA. TRIM33 and KLF9 expression in TNF-α-challenged MH7A cells and transfection efficiency were analyzed via real-time reverse transcription polymerase chain reaction together with western blot. The viability, proliferation, invasion, and migration of TNF-α-induced MH7A cells after transfection was respectively detected by CCK-8, EdU staining, transwell, and wound-healing assays. The expression of invasion and migration-related proteins and inflammation-related proteins was determined by western blot and the levels of inflammatory factors were detected by enzyme-linked immunosorbent assay. The combination between TRIM33 and KLF9 was substantiated through dual-luciferase reporter assay and chromatin immunoprecipitation. RESULTS: TRIM33 and KLF9 expression in TNF-α-challenged MH7A cells was downregulated. TRIM33 elevation inhibited TNF-α-elicited proliferation, metastasis as well as inflammation of MH7A cells. Moreover, KLF9 was combined with TRIM33 and KLF9 promoted transcription of TRIM33. The inhibitory effect of TRIM33 overexpression on proliferation, invasion and migration and inflammation of MH7A cells induced by TNF-α was alleviated by the downregulation of KLF9. CONCLUSION: KLF9 positively regulates TRIM33 to suppress the abnormal MH7A cell proliferation, migration, and reduce inflammation upon exposure to TNF-α, which was reversed by inhibiting KLF9.

LncRNA Airn maintains LSEC differentiation to alleviate liver fibrosis via the KLF2-eNOS-sGC pathway.

BACKGROUND: Long noncoding RNAs (lncRNAs) have emerged as important regulators in a variety of human diseases. The dysregulation of liver sinusoidal endothelial cell (LSEC) phenotype is a critical early event in the fibrotic process. However, the biological function of lncRNAs in LSEC still remains unclear. METHODS: The expression level of lncRNA Airn was evaluated in both human fibrotic livers and serums, as well as mouse fibrotic livers. Gain- and loss-of-function experiments were performed to detect the effect of Airn on LSEC differentiation and hepatic stellate cell (HSC) activation in liver fibrosis. Furthermore, RIP, RNA pull-down-immunoblotting, and ChIP experiments were performed to explore the underlying mechanisms of Airn. RESULTS: We have identified Airn was significantly upregulated in liver tissues and LSEC of carbon tetrachloride (CCl4)-induced liver fibrosis mouse model. Moreover, the expression of AIRN in fibrotic human liver tissues and serums was remarkably increased compared with healthy controls. In vivo studies showed that Airn deficiency aggravated CCl4- and bile duct ligation (BDL)-induced liver fibrosis, while Airn over-expression by AAV8 alleviated CCl4-induced liver fibrosis. Furthermore, we revealed that Airn maintained LSEC differentiation in vivo and in vitro. Additionally, Airn inhibited HSC activation indirectly by regulating LSEC differentiation and promoted hepatocyte (HC) proliferation by increasing paracrine secretion of Wnt2a and HGF from LSEC. Mechanistically, Airn interacted with EZH2 to maintain LSEC differentiation through KLF2-eNOS-sGC pathway, thereby maintaining HSC quiescence and promoting HC proliferation. CONCLUSIONS: Our work identified that Airn is beneficial to liver fibrosis by maintaining LSEC differentiation and might be a serum biomarker for liver fibrogenesis.

CircCDK14 ameliorates interleukin-1ß-induced chondrocyte damage by the miR-1183/KLF5 pathway in osteoarthritis.

BACKGROUND: The pathogenesis of osteoarthritis (OA), an endemic and debilitating disease, remains unclear. The study aimed to reveal the role of circular RNA cyclin dependent kinase 14 (circCDK14) in OA development and the underlying mechanism. METHODS: Human chondrocytes were stimulated by 10 ng/mL interleukin-1ß (IL-1ß) to mimic OA cell model. The RNA expression of circCDK14, microRNA-1183 (miR-1183) and kruppel like factor 5 (KLF5) was checked through quantitative real-time polymerase chain reaction. Western blot was employed to detect protein expression. Cell viability, proliferation and apoptosis were investigated by cell counting kit-8, 5-Ethynyl-29-deoxyuridine and flow cytometry analysis, respectively. Starbase online database was performed to identify the interaction between miR-1183 and circCDK14 or KLF5. Exosomes were isolated by differential centrifugation and identified by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. RESULTS: CircCDK14 and KLF5 expression were significantly decreased, while miR-1183 was increased in OA cartilage tissues and IL-1ß-treated chondrocytes in comparison with controls. CircCDK14 overexpression attenuated the inhibitory effect of IL-1ß treatment on cell proliferation and the promoting effects on cell apoptosis and extracellular matrix degradation. Additionally, miR-1183 was targeted by circCDK14, and miR-1183 mimics reversed circCDK14-mediated actions in IL-1ß-treated chondrocytes. The knockdown of KLF5, a target mRNA of miR-1183, also rescued the effects of miR-1183 inhibitors in IL-1ß-induced chondrocytes. Moreover, circCDK14 could induce KLF5 expression by interacting with miR-1183. Further, exosomal circCDK14 had a high diagnostic value in OA. CONCLUSION: CircCDK14 reintroduction assuaged IL-1ß-caused chondrocyte damage by the miR-1183/KLF5 pathway, providing a diagnostic biomarker for OA.

The effects of extracellular vesicles derived from Krüppel-Like Factor 2 overexpressing endothelial cells on the regulation of cardiac inflammation in the dilated cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is one of the common causes of heart failure. Myocardial injury triggers an inflammatory response and recruits immune cells into the heart. High expression of Krüppel-like factor 2 (KLF2) in endothelial cells (ECs) potentially exerts an anti-inflammatory effect. However, the role of extracellular vesicles (EVs) from KLF2-overexpressing ECs (KLF2-EVs) in DCM remains unclear. METHODS AND RESULTS: EVs were separated from the supernatant of KLF2-overexpressing ECs by gradient centrifugation. Mice were repeatedly administered low-dose doxorubicin (DOX) and then received KLF2-EVs through an intravenous injection. Treatment with KLF2-EVs prevented doxorubicin-induced left ventricular dysfunction and reduced the recruitment of Ly6high Mo/Mø in the myocardium. We used flow cytometry to detect Ly6high monocytes in bone marrow and spleen tissues and to elucidate the mechanisms underlying this beneficial effect. KLF2-EVs increased the retention of Ly6Chigh monocytes in the bone marrow but not in the spleen tissue. KLF2-EVs also significantly downregulated C-C chemokine receptor 2 (CCR2) protein expression in cells from the bone marrow. CONCLUSIONS: EVs derived from KLF2-overexpressing ECs reduced cardiac inflammation and ameliorated left ventricular dysfunction in DCM mice by targeting the CCR2 protein to inhibit Ly6Chigh monocyte mobilization from the bone marrow.

Calycosin ameliorates atherosclerosis by enhancing autophagy via regulating the interaction between KLF2 and MLKL in apolipoprotein E gene-deleted mice.

BACKGROUND AND PURPOSE: Atherosclerosis is one of the underlying causes of cardiovascular disease. Formation of foam cells and necrotic core in the plaque is a hallmark of atherosclerosis, which results from lipid deposition, apoptosis, and inflammation in macrophages. Macrophage autophagy is a critical anti-atherogenic process and defective autophagy aggravates atherosclerosis by enhancing foam cell formation, apoptosis, and inflammation. Hence, enhancing autophagy can be a strategy for atherosclerosis treatment. Calycosin, a flavonoid from Radix Astragali, displays anti-oxidant and anti-inflammatory activities and therefore is potential to reduce the risk of cardiovascular disease. However, the anti-atherogenic effect of calycosin and the involved mechanism remains unclear. In this study, we assessed the potential benefits of calycosin on autophagy and atherosclerosis, and revealed the underlying mechanism. EXPERIMENTAL APPROACH: In this study, apoE-/- mice were fed high-fat diet for 16 weeks in the presence of calycosin and/or autophagy inhibitor chloroquine, which was followed by determination of atherosclerosis development, autophagy activity, and involved mechanisms. KEY RESULTS: Calycosin protected against atherosclerosis and enhanced plaque stability via promoting autophagy. Calycosin inhibited foam cell formation, inflammation, and apoptosis by enhancing autophagy. MLKL was demonstrated as a new autophagy regulator, which can be negatively regulated by KLF2. Mechanistically, inhibitory effects of calycosin on atherogenesis were via improved autophagy through KLF2-MLKL signalling pathway modulation. CONCLUSIONS AND IMPLICATIONS: This study demonstrated the atheroprotective effect of calycosin was through upregulating KLF2-MLKL-mediated autophagy, which not only proposed novel mechanistic insights into t atherogenesis but also identified calycosin as a potential drug candidate for atherosclerosis treatment.

Krüppel-like factor KLF9 inhibits chicken intramuscular preadipocyte differentiation.

1. Poultry meat quality is affected by many factors, among which intramuscular fat (IMF) is predominant. IMF content affects tenderness, juiciness and flavour of meat. Krüppel-like transcriptional factors (KLFs) are important regulators of adipocyte differentiation. However, little is known about the KLF9 gene associated with poultry IMF deposition, especially intramuscular adipocyte differentiation.2. Previous work has shown that chicken KLF9 was differentially expressed during adipogenesis of intramuscular preadipocytes differentiation. In this study, the function of KLF9 in chicken intramuscular preadipocytes differentiation was investigated.3. In the chicken preadipocyte differentiation model, KLF9 expression showed a major increase with adipogenic induction. Overexpression of KLF9 down-regulated the expression of the adipogenic marker gene AP2, and impaired triglyceride accumulation. Knockdown of KLF9 in chicken intramuscular preadipocytes increased the expression of PPARG, CEBPA and AP2. In addition, it was proposed that KLF9 may regulate adipogenesis via lncRNAs NONGGAT002209.2, NONGGAT003346.2, NONGGAT000436.2 and NONGGAT006302.2 in chicken.4. The data supported a novel role of KLF9 in regulating chicken intramuscular preadipocyte differentiation. Such findings may contribute to a more thorough understanding of chicken IMF deposition and the improvement of poultry meat quality.

Functional Odontoblastic-Like Cells Derived from Human iPSCs.

The induced pluripotent stem cells (iPSCs) have an intrinsic capability for indefinite self-renewal and large-scale expansion and can differentiate into all types of cells. Here, we tested the potential of iPSCs from dental pulp stem cells (DPSCs) to differentiate into functional odontoblasts. DPSCs were reprogrammed into iPSCs via electroporation of reprogramming factors OCT-4, SOX2, KLF4, LIN28, and L-MYC. The iPSCs presented overexpression of the reprogramming genes and high protein expressions of alkaline phosphatase, OCT4, and TRA-1-60 in vitro and generated tissues from 3 germ layers in vivo. Dentin discs with poly-L-lactic acid scaffolds containing iPSCs were implanted subcutaneously into immunodeficient mice. After 28 d from implantation, the iPSCs generated a pulp-like tissue with the presence of tubular dentin in vivo. The differentiation potential after long-term expansion was assessed in vitro. iPSCs and DPSCs of passages 4 and 14 were treated with either odontogenic medium or extract of bioactive cement for 28 d. Regardless of the passage tested, iPSCs expressed putative markers of odontoblastic differentiation and kept the same mineralization potential, while DPSC P14 failed to do the same. Analysis of these data collectively demonstrates that human iPSCs can be a source to derive human odontoblasts for dental pulp research and test bioactivity of materials.

Effect of TIEG1 on apoptosis and expression of Bcl-2/Bax and Pten in leukemic cell lines.

We examined the effect of transforming growth factor-b inducible early gene-1 (TIEG1) on the apoptosis of leukemic cell lines and expression of B-cell lymphoma 2 (Bcl-2) and phosphatase and tensin homolog (Pten). Four leukemic cell lines (HL-60, U937, Raji, and K562) were treated with 0, 1, 5, 10, and 20 ng/mL TIEG1, respectively. The cell growth inhibitory ratio was assessed using the MTT assay. An inhibitory curve was drawn, and half-maximal inhibitory concentration was calculated. Additionally, 1640 culture medium containing 10 ng/mL TIEG1 was used to culture leukemic cell lines for 0, 6, 12, 24, and 48 h. The apoptosis of each cell line at different time points was detected by flow cytometry. Total RNA was extracted before reverse transcription-polymerase chain reaction. The products of this reaction were analyzed by electrophoresis, and the expression of Bcl-2/Bcl-2-associated X protein (Bax) and Pten were detected. After treatment with TIEG1, proliferation of the 4 leukemic cell lines was inhibited both time- and dose-dependently. During apoptosis induction, the expression of Bcl-2 was decreased and the expressions of Bax and Pten were increased in the 4 leukemic cell lines induced by TIEG1 (P < 0.05). TIEG1 can inhibit the proliferation of leukemic cells and induce their apoptosis in a time- and dose-dependent manner. A close relationship exists between Bcl-2/Bax and Pten expression and cell apoptosis induced by TIEG1.

Optimization of culture conditions for maintaining porcine induced pluripotent stem cells.

Ground state porcine induced pluripotent stem cells (piPSCs), which retain the potential to generate chimeric animal and germline transmission, are difficult to produce. This study investigated morphological and biological progression at the early stage of porcine somatic cell reprogramming, and explored suitable conditions to increase the induction efficiency of piPSCs. A cocktail of defined transcription factors was used to generate piPSCs. The amphotropic retrovirus, which carried human OCT4 (O), SOX2 (S), KLF4 (K), C-MYC (M), TERT (T), and GFP, were used to infect porcine embryonic fibroblasts (PEFs). The number of clones derived from OSKM (4F) and OSKMT (4F+T) was significantly higher than that from SKM (3F) and SKMT (3F+T), suggesting that OCT4 played a critical role in regulating porcine cell reprogramming. The number of alkaline phosphatase-positive clones from a medium with leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) (M1 medium) was significantly higher than that with insulin and 2i PD0325901/CHIR99021 (M2 medium), indicating that insulin and 2i could not effectively maintain piPSC propagation. In the M1 medium, piPSC lines could not maintain the typical self-renewal morphology on gelatin-coated and Matrigel-coated plates. Without the mouse embryonic fibroblast (MEF) feeder, piPSCs started to simultaneously differentiate. Based on the potential for self-renewal and activation of pluripotent markers, we found that the culture condition of 4F+T plus LIF and bFGF plus MEF feeder promoted PEF reprogramming more efficiently than the other conditions tested here. Two piPSC lines (IB-1 and IB-2) were derived and maintained for up to 20 passages in vitro.

[Influence of TIEG1 on apoptosis of HL-60 cells and expression of Bcl-2/Bax].

This study was aimed to investigate the influence of TIEG1 on apoptosis of HL-60 cells and the expression of Bcl-2/Bax. Different concentration of TIEG1 were used to treat HL-60 cells, the cell growth inhibition rate was detected by MTT method. After treating HL-60 cells with 12.03 ng/ml TIEG1, cell apoptosis was detected with flow cytometry. Bcl-2 and Bax was detected with RT-PCR. The results showed that TIEG1 had inhibitory effect on HL-60 cell proliferation, and in time-and dose-dependent manners. The more obvious inhibitory effect was observed in HL-60 cells treated with TIEG1 of 12.03 ng/ml. During the course of cell apoptosis, Bax expression increased, but Bcl-2 expression decreased (P < 0.05). It is concluded that TIEG1 inhibits HL-60 cell proliferation and induces apoptosis in time and dose-dependent manners. During the course of HL-60 cells apoptosis induced by TIEG1, Bcl-2/Bax are associated with HL-60 cell apoptosis induced by TIEG1.

MicroRNA-302 increases reprogramming efficiency via repression of NR2F2.

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression through translational inhibition and RNA decay and have been implicated in the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. In this study, we analyzed global miRNA and mRNA microarrays to predict novel miRNA-mRNA interactions in human embryonic stem cells and induced pluripotent stem cells (iPSCs). In particular, we demonstrate a regulatory feedback loop between the miR-302 cluster and two transcription factors, NR2F2 and OCT4. Our data show high expression of miR-302 and OCT4 in pluripotent cells, while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of miR-302, which we experimentally confirm by reporter luciferase assays and real-time polymerase chain reaction. We also demonstrate that NR2F2 directly inhibits the activity of the OCT4 promoter and thus diminishes the positive feedback loop between OCT4 and miR-302. Importantly, higher reprogramming efficiencies were obtained when we reprogrammed human adipose-derived stem cells into iPSCs using four factors (KLF4, C-MYC, OCT4, and SOX2) plus miR-302 (this reprogramming cocktail is hereafter referred to as "KMOS3") when compared to using four factors ("KMOS"). Furthermore, shRNA knockdown of NR2F2 mimics the over-expression of miR-302 by also enhancing reprogramming efficiency. Interestingly, we were unable to generate iPSCs from miR-302a/b/c/d alone, which is in contrast to previous publications that have reported that miR-302 by itself can reprogram human skin cancer cells and human hair follicle cells. Taken together, these findings demonstrate that miR-302 inhibits NR2F2 and promotes pluripotency through indirect positive regulation of OCT4. This feedback loop represents an important new mechanism for understanding and inducing pluripotency in somatic cells.

Derivation and cardiomyocyte differentiation of induced pluripotent stem cells from heart failure patients.

AIMS: Myocardial cell replacement therapies are hampered by a paucity of sources for human cardiomyocytes and by the expected immune rejection of allogeneic cell grafts. The ability to derive patient-specific human-induced pluripotent stem cells (hiPSCs) may provide a solution to these challenges. We aimed to derive hiPSCs from heart failure (HF) patients, to induce their cardiomyocyte differentiation, to characterize the generated hiPSC-derived cardiomyocytes (hiPSC-CMs), and to evaluate their ability to integrate with pre-existing cardiac tissue. METHODS AND RESULTS: Dermal fibroblasts from two HF patients were reprogrammed by retroviral delivery of Oct4, Sox2, and Klf4 or by using an excisable polycistronic lentiviral vector. The resulting HF-hiPSCs displayed adequate reprogramming properties and could be induced to differentiate into cardiomyocytes with the same efficiency as control hiPSCs (derived from human foreskin fibroblasts). Gene expression and immunostaining studies confirmed the cardiomyocyte phenotype of the differentiating HF-hiPSC-CMs. Multi-electrode array recordings revealed the development of a functional cardiac syncytium and adequate chronotropic responses to adrenergic and cholinergic stimulation. Next, functional integration and synchronized electrical activities were demonstrated between hiPSC-CMs and neonatal rat cardiomyocytes in co-culture studies. Finally, in vivo transplantation studies in the rat heart revealed the ability of the HF-hiPSC-CMs to engraft, survive, and structurally integrate with host cardiomyocytes. CONCLUSIONS: Human-induced pluripotent stem cells can be established from patients with advanced heart failure and coaxed to differentiate into cardiomyocytes, which can integrate with host cardiac tissue. This novel source for patient-specific heart cells may bring a unique value to the emerging field of cardiac regenerative medicine.

Differentiation of reprogrammed mouse cardiac fibroblasts into functional cardiomyocytes.

Fibroblasts can be reprogrammed by ectopic expression of reprogramming factors to yield induced pluripotent stem (iPS) cells that are capable of transdifferentiating into diverse types of somatic cell lines. In this study, we examined if functional cardiomyocytes (CMs) can be produced from mouse cardiac fibroblasts (CFs), using iPS cell factor-based reprogramming. CFs were isolated from Oct4-GFP-C57 mice and infected with a retrovirus expressing the Yamanaka reprogramming factors, Oct4, Sox2, Klf4, and c-Myc to reprogram the CFs into a CF-iPS cell line. Primary mouse embryonic fibroblast cells (MEFs) were used as a control. We found that the dedifferentiated CF-iPS cells showed similar biological characteristics (morphology, pluripotent factor expression, and methylation level) as embryonic stem cells (ESs) and MEF-iPS cells. We used the classical embryoid bodies (EBs)-based method and a transwell CM co-culture system to simulate the myocardial paracrine microenvironment for performing CF-iPS cell cardiogenic differentiation. Under this simulated myocardial microenvironment, CF-iPS cells formed spontaneously beating EBs. The transdifferentiated self-beating cells expressed cardiac-specific transcription and structural factors and also displayed typical myocardial morphology and electrophysiological characteristics. CFs can be dedifferentiated into iPS cells and further transdifferentiated into CMs. CFs hold great promise for CM regeneration as an autologous cell source for functional CM in situ without the need for exogenous cell transplantation in ischemic heart disease.