Krüppel-like factor 12 regulates aging ovarian granulosa cell apoptosis by repressing SPHK1 transcription and sphingosine-1-phosphate (S1P) production.

Oxidative stress triggered by aging, radiation, or inflammation impairs ovarian function by inducing granulosa cell (GC) apoptosis. However, the mechanism inducing GC apoptosis has not been characterized. Here, we found that ovarian GCs from aging patients showed increased oxidative stress, enhanced reactive oxygen species activity, and significantly decreased expression of the known antiapoptotic factor sphingosine-1-phosphate/sphingosine kinase 1 (SPHK1) in GCs. Interestingly, the expression of Krüppel-like factor 12 (KLF12) was significantly increased in the ovarian GCs of aging patients. Furthermore, we determined that KLF12 was significantly upregulated in hydrogen peroxide-treated GCs and a 3-nitropropionic acid-induced in vivo model of ovarian oxidative stress. This phenotype was further confirmed to result from inhibition of SPHK1 by KLF12. Interestingly, when endogenous KLF12 was knocked down, it rescued oxidative stress-induced apoptosis. Meanwhile, supplementation with SPHK1 partially reversed oxidative stress-induced apoptosis. However, this function was lost in SPHK1 with deletion of the binding region to the KLF12 promoter. SPHK1 reversed apoptosis caused by hydrogen peroxide-KLF12 overexpression, a result further confirmed in an in vitro ovarian culture model and an in vivo 3-nitropropionic acid-induced ovarian oxidative stress model. Overall, our study reveals that KLF12 is involved in regulating apoptosis induced by oxidative stress in aging ovarian GCs and that sphingosine-1-phosphate/SPHK1 can rescue GC apoptosis by interacting with KLF12 in negative feedback.

CRC Therapy Identifies Indian Hedgehog Signaling in Mouse Endometrial Epithelial Cells and Inhibition of Ihh-KLF9 as a Novel Strategy for Treating IUA.

Intrauterine adhesion (IUA) causes menstrual disturbance and infertility. There is no effective treatment available for moderate to severe IUA cases. Stem cell-based therapy has been investigated for treating IUA but is limited in clinical applications due to issues including the precise induction of differentiation, tumorigenesis, and unclear molecular mechanisms. In our recent study, we isolated and expanded the long-term cultures of conditional reprogrammed (CR) mouse endometrial epithelial cells. Treating IUA mice with these CR cells (CRCs) restored the morphology and structure of the endometrium and significantly improved the pregnancy rate. In this study, our data with high-throughput sequencing, CRISPR knockout Ihh-/-CRCs, and transplantation identified for the first time that the Indian hedgehog (Ihh) gene plays a critical role in the regulation of endometrial epithelial cell proliferation. We also found that aberrant activated Ihh-krüppel-like factor 9 (KLF9) signaling contributes to the inhibition of normal progesterone receptor (PR) function in IUA mice. Thus, we hypothesized that inhibition of the Ihh-KLF9 pathway may be a novel strategy to treat IUA. Our data demonstrated that treatment with the hedgehog signaling inhibitor Vismodegib restored the morphology, structure, and microenvironment of the endometrium, and greatly improved the pregnancy rate in IUA mice. This study suggests a promising application of hedgehog inhibitors as a targeted drug in the IUA clinic.

Schisandrae chinensis Fructus Extract Ameliorates Muscle Atrophy in Streptozotocin-Induced Diabetic Mice by Downregulation of the CREB-KLF15 and Autophagy-Lysosomal Pathways.

Type 1 diabetes mellitus is an autoimmune disease caused by the destruction of pancreatic beta cells. Many patients with type 1 diabetes experience skeletal muscle wasting. Although the link between type 1 diabetes and muscle wasting is not clearly known, insulin insufficiency and hyperglycemia may contribute to decreased muscle mass. In this study, we investigated the therapeutic effect of the ethanolic extract of Schisandrae chinensis Fructus (SFe) on muscle wasting in streptozotocin (STZ)-induced diabetic mice. STZ-diabetic C57BL/6 mice (blood glucose level ≥300 mg/dL) were orally administered SFe (250 or 500 mg/kg/day) for 6 weeks. We observed that SFe administration did not change blood glucose levels but increased gastrocnemius muscle weight, cross-sectional area, and grip strength in STZ-induced diabetic mice. Administration of SFe (500 mg/kg) decreased the expression of atrophic factors, such as MuRF1 and atrogin-1, but did not alter the expression of muscle synthetic factors. Further studies showed that SFe administration decreased the expression of KLF15 and p-CREB, which are upstream molecules of atrophic factors. Examination of the expression of molecules involved in autophagy-lysosomal pathways (e.g., p62/SQSTM1, Atg7, Beclin-1, ULK-1, LC3-I, and LC3-II) revealed that SFe administration significantly decreased the expression of p62/SQSTM1, LC3-I, and LC3-II; however, no changes were observed in the expression of Atg7, Beclin-1, or ULK-1. Our results suggest that SFe ameliorated muscle wasting in STZ-induced diabetic mice by decreasing protein degradation via downregulation of the CREB-KLF15-mediated UPS system and the p62/SQSTM1-mediated autophagy-lysosomal pathway.

CircATRNL1 protects against osteoarthritis by targeting miR-153-3p and KLF5.

BACKGROUND: Osteoarthritis (OA) is characterized by chondrocyte injury. Circular RNAs (circRNAs) are involved in the pathogenesis of various diseases, including OA. The purpose of this study was to determine the potential role of circATRNL1 in OA pathology in vitro. METHODS: Human chondrocytes were isolated and treated with interleukin-1 beta (IL-1ß) to mimic OA in vitro. High-throughput RNA sequencing was performed to identify differentially expressed circRNAs, miRNAs and mRNAs between IL and 1ß-treated chondrocytes and normal chondrocytes. The expression of circATRNL1, miR-153-3p and KLF5 was measured using quantitative real-time polymerase chain reaction (qRT-PCR). For functional analyses, cell apoptosis was assessed using a flow cytometry assay. Extracellular matrix (ECM) degradation was monitored by measuring the levels of ECM-associated proteins by Western blot. The potential target miRNAs of circATRNL1 were screened by bioinformatics analysis and verified by dual-luciferase reporter assay. RESULTS: The expression of circATRNL1 was decreased in IL-1ß-treated chondrocytes. CircATRNL1 overexpression ameliorated cell apoptosis and ECM degradation, which were promoted by IL-1ß treatment. Mechanistic analysis revealed that circATRNL1 directly targeted miR-153-3p and that miR-153-3p could reverse the inhibitory effects of circATRNL1 overexpression on inflammatory responses, cell apoptosis and ECM degradation. KLF5 is a target of miR-153-3p. CONCLUSION: Taken together, the results in this study suggested that circATRNL1 might ameliorate the development and progression of OA through regulating miR-153-3p/KLF5 axis. Our study increased the understanding of circRNAs as therapeutic targets in the treatment of OA.

EZH2-mediated inhibition of KLF14 expression promotes HSCs activation and liver fibrosis by downregulating PPARγ.

OBJECTIVES: Induction of deactivation and apoptosis of hepatic stellate cells (HSCs) are principal therapeutic strategies for liver fibrosis. Krüppel-like factor 14 (KLF14) regulates various biological processes, however, roles, mechanisms and implications of KLF14 in liver fibrosis are unknown. MATERIALS AND METHODS: KLF14 expression was detected in human, rat and mouse fibrotic models, and its effects on HSCs were assessed. Chromatin immunoprecipitation assays were utilized to investigate the binding of KLF14 to peroxisome proliferator-activated receptor γ (PPARγ) promoter, and the binding of enhancer of zeste homolog 2 (EZH2) to KLF14 promoter. In vivo, KLF14-overexpressing adenovirus was injected via tail vein to thioacetamide (TAA)-treated rats to investigate the role of KLF14 in liver fibrosis progression. EZH2 inhibitor EPZ-6438 was utilized to treat TAA-induced rat liver fibrosis. RESULTS: KLF14 expression was remarkably decreased in human, rat and mouse fibrotic liver tissues. Overexpression of KLF14 increased LD accumulation, inhibited HSCs activation, proliferation, migration and induced G2/M arrest and apoptosis. Mechanistically, KLF14 transactivated PPARγ promoter activity. Inhibition of PPARγ blocked the suppressive role of KLF14 overexpression in HSCs. Downregulation of KLF14 in activated HSCs was mediated by EZH2-regulated histone H3 lysine 27 trimethylation. Adenovirus-mediated KLF14 overexpression ameliorated TAA-induced rat liver fibrosis in PPARγ-dependent manner. Furthermore, EPZ-6438 dramatically alleviated TAA-induced rat liver fibrosis. Importantly, KLF14 expression was decreased in human with liver fibrosis, which was significantly correlated with EZH2 upregulation and PPARγ downregulation. CONCLUSIONS: KLF14 exerts a critical anti-fibrotic role in liver fibrosis, and targeting the EZH2/KLF14/PPARγ axis might be a novel therapeutic strategy for liver fibrosis.

An inhibitor of Krüppel-like factor 5 suppresses peritoneal fibrosis in mice.

BACK GROUND: Krüppel-like transcription factor 5 (KLF5) is a transcription factor regulating cell proliferation, angiogenesis and differentiation. It has been recently reported that Am80, a synthetic retinoic acid receptor α-specific agonist, inhibits the expression of KLF5. In the present study, we have examined the expression of KLF5 in fibrotic peritoneum induced by chlorhexidine gluconate (CG) in mouse and evaluated that Am80, as an inhibitor of KLF5, can reduce peritoneal fibrosis. METHODS: Peritoneal fibrosis was induced by intraperitoneal injection of CG into peritoneal cavity of ICR mice. Am80 was administered orally for every day from the start of CG injection. Control mice received only a vehicle (0.5% carboxymethylcellulose solution). After 3 weeks of treatment, peritoneal equilibration test (PET) was performed and peritoneal tissues were examined by immunohistochemistry. RESULTS: The expression of KLF5 was less found in the peritoneal tissue of control mice, while KLF5 was expressed in the thickened submesothelial area of CG-injected mice receiving the vehicle. Am80 treatment reduced KLF5 expression and remarkably attenuated peritoneal thickening, accompanied with the reduction of type III collagen expression. The numbers of transforming growth factor ß-positive cells, α-smooth muscle actin-positive cells and infiltrating macrophages were significantly decreased in Am80-treated group. PET revealed the increased peritoneal permeability in CG mice, whereas Am80 administration significantly improved the peritoneal high permeability state. CONCLUSIONS: These results indicate the involvement of KLF5 in the progression of experimental peritoneal fibrosis and suggest that Am80 may be potentially useful for the prevention of peritoneal fibrosis through inhibition of KLF5 expression.

MiR-375 silencing attenuates pro-inflammatory macrophage response and foam cell formation by targeting KLF4.

Macrophage mediated inflammation and foam cell formation play crucial roles in the development of atherosclerosis. MiR-375 is a small noncoding RNA that significantly implicated in multiple tumor regulation and has been emerged as a novel biomarker for type 2 diabetes. However, the exact role of miR-375 on macrophage activation remains unknown. In the present study, we observed that miR-375 expression showed an up-regulated expression in atherosclerotic aortas, as well as in bone marrow derived macrophages (BMDMs) and mouse peritoneal macrophages (MPMs) isolated from ApoE deficiency mice and was gradually increased followed the Ox-LDL treated time. Functionally, miR-375 inhibition significantly decreased foam cell formation accompanied by up-regulated genes expression involved in cholesterol efflux but reduced genes expression implicated in cholesterol influx. Moreover, miR-375 silencing increased resolving M2 macrophage but reduced pro-inflammatory M1 macrophage markers expression. Such above effects can be reversed by miR-375 overexpression. Mechanistically, we noticed that miR-375 knockdown promoted KLF4 expression which was required for the ameliorated effect of miR-375 silencing on macrophage activation. Importantly, the consistent results in mRNA expression of M1 and M2 markers were observed in vivo, and miR-375-/-ApoE-/- mice significant decreased atherosclerotic lesions in the whole aorta and aortic sinus. Taken together, these evidences suggested that miR-375 knockdown attenuated macrophage activation partially through activation of KLF4-dependent mechanism.

Autophagy-induced p62 accumulation is required for curcumol to regulate KLF5-mediated angiogenesis in liver sinusoidal endothelial cells.

Liver pathological angiogenesis is considered to be one of the key events in the development of liver fibrosis. Autophagy is a defense and stress regulation mechanism. However, whether autophagy regulates pathological angiogenesis in liver fibrosis is still questionable. Here, we aimed to study how curcumol regulated liver sinusoidal endothelial cells (LSECs) angiogenesis through autophagy. We found that curcumol (10, 20 and 40 µM) could inhibit the expression of angiogenesis markers in the LSECs. Importantly, we showed that curcumol might influence LSEC pathological angiogenesis by regulating autophagy level. Furthermore, we indicated that the transcription factor Krüppel-like factor 5 (KLF5) was considered as a key target for curcumol to regulate LSEC angiogenesis. Interestingly, we also suggested that autophagy was as a potential mechanism for curcumol to restrain KLF5 expression. Increased autophagy level could impair the suppression effect of curcumol on KLF5. Fascinatingly, our results indicated that curcumol inhibited autophagy and led to p62 accumulation, which might be a regulation mechanism of KLF5 degradation. Finally, in mice liver fibrosis model, we unanimously showed that curcumol (30 mg/kg) inhibited pathological angiogenesis by reducing LSEC autophagy level and suppressing KLF5 expression. Collectively, these results provided a deeper insight into the molecular mechanism of curcumol to inhibit LSEC pathological angiogenesis during liver fibrosis.

Atorvastatin Combined with Low-Dose Dexamethasone Treatment Protects Endothelial Function Impaired by Chronic Subdural Hematoma via the Transcription Factor KLF-2.

OBJECTIVE: Our previous study showed that the combination therapy with atorvastatin and low-dose dexamethasone protected endothelial cell function in chronic subdural hematoma (CSDH) injury. In this study, we aimed to investigate the mechanism underlying the effects of this combination therapy on CSDH-induced cell dysfunction. METHODS: Monocytes and endothelial cells were cocultured with CSDH patient hematoma samples to mimic the pathological microenvironment of CSDH. Monocytes (THP-1 cells) and endothelial cells (hCMEC/D3 cells) were cocultured in a transwell system for 24 h before stimulation with hematoma samples diluted in endothelial cell medium (ECM) at a 1:1 ratio. Tight junction markers were detected by Western blotting, PCR and immunofluorescence. hCMEC/D3 cells were collected for Western blot and PCR analyses to detect changes in the expression levels of vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule (ICAM-1), and Kruppel-like factor 2 (KLF-2). The IL-6, IL-10 and VEGF levels in the supernatant were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: KLF-2 expression in endothelial cells was decreased after stimulation with CSDH patient hematoma samples, but combination therapy with atorvastatin and low-dose dexamethasone reversed this trend. KLF-2 protected injured cells by increasing the expression of VE-cadherin and ZO-1; attenuating the expression of VCAM-1, ICAM-1, IL-6 and VEGF; and enhancing the expression of IL-10, all of which play pivotal roles in endothelial inflammation. Moreover, the effect of combination therapy with atorvastatin and low-dose dexamethasone was obviously reduced in endothelial cells with KLF-2 knockdown compared with normal cells. CONCLUSION: Coculture with hematoma samples decreased KLF-2 expression in human cerebral endothelial cells. Combination therapy with atorvastatin and low-dose dexamethasone counteracted hematoma-induced KLF-2 suppression in human cerebral endothelial cells to attenuate robust endothelial inflammation and permeability. KLF-2 plays an important role in drug therapy for CSDH and may become the key factor in treatment and prognosis.

KLF16 overexpression deleteriously affects the proliferation and migration of retinoblastoma by transcriptionally repressing BCL2L15.

Krüppel-like factors (KLFs) are transcription factors that control the expression of downstream genes. The role of KLFs has been reported in cancers. KLF16 promotes the proliferation of gastric cancer cells by upregulating p21, while suppresses the tumorigenesis of glioma through targeting TFAM. The function of KLF16 is controversial in cancer development. In this study, we aimed to investigate the role of KLF16 in retinoblastoma (RB). KLF16 was highly expressed in RB tissues and cells. Overexpression of KLF16 promoted the proliferation, growth and migration of RB cells. By contrast, KLF16 interference showed opposite effects. Cell cycle arrest and apoptosis were induced or repressed by KLF16 knockdown or overexpression, respectively. Mechanistically, BCL2 like 15 (BCL2L15), an apoptosis gene, was negatively regulated by KLF16. Luciferase reporter and ChIP assay showed that KLF16 transcriptionally repressed the expression of BCL2L15 by binding to its promoter. BCL2L15 was lowly expressed in RB tissues. Additionally, overexpression of BCL2L15 inhibited the proliferation and increased the apoptosis in RB cells. Our study identifies that KLF16 contributes to RB cell proliferation and migration by negatively regulating BCL2L15.

Salvia miltiorrhiza bunge exerts anti-oxidative effects through inhibiting KLF10 expression in vascular smooth muscle cells exposed to high glucose.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicinal herb Salvia miltiorrhiza Bunge(Danshen) and its components have been widely used to treat cardiovascular diseases for hundreds of years in China, including hypertension, diabetes, atherosclerosis, and chronic heart failure. Salvia miltiorrhiza injection (SMI), an aqueous extracts of Salvia miltiorrhiza Bunge, is one of most widely used traditional Chinese medicine injections. SMI is widely used in the treatment of diabetic vascular complications, However, the mechanisms remain to be defined. AIM OF THE STUDY: To investigate protective mechanism of Salvia miltiorrhiza Bunge against ROS generation in VSMCs of diabetic mice and patients. MATERIALS AND METHODS: Salvia miltiorrhiza injection (hereinafter referred to as SMI, 1.5 g mL-1), which was approved by the State Food and Drug Administration (approval number: Z32020161), was obtained from Shenlong Pharmaceutical Co., Ltd. (batch number: 11040314). SMI or vehicle were intraperitoneally administrated to the HFD-fed db/db mice, artery was harvested after 24weeks later. qRT-PCR and Western blot analysis were used to detect the expression of KLF6, KLF5, KLF4, KLF10, KLF12, and HO-1. DCFH-DA staining detected intracellular ROS production. Loss- and gain-of-function experiments of KLF10 were used to investigate the effect of KLF10 on the expression of HO-1. Dual-luciferase reporter assay evaluated the effect of KLF10 on the activity of the HO-1 promoter. RESULTS: KLF10 expression and ROS generation are significantly increased in the arteries of HFD-fed db/db mice, VSMCs of diabetic patients, as well as in high glucose-treated VSMCs. KLF10 overexpression suppresses, while its knockdown facilitates the expression of heme oxygenase (HO-1) mRNA and protein. Further, Salvia miltiorrhiza injection (SMI) abrogates KLF10 upregulation and reduces ROS generation induced by high glucose in VSMCs. Mechanistically, KLF10 negatively regulates the HO-1 gene transcription via directly binding to its promoter. Accordingly, SMI treatment of VSMCs reduces ROS generation through inhibiting KLF10 expression and thus relieving KLF10 repression of the expression of HO-1 gene, subsequently contributing to upregulation of HO-1. CONCLUSION: SMI exerts anti-oxidative effects on VSMCs exposed to high glucose through inhibiting KLF10 expression and thus upregulating HO-1.

Teneligliptin Promotes Bile Acid Synthesis and Attenuates Lipid Accumulation in Obese Mice by Targeting the KLF15-Fgf15 Pathway.

Bile acids (BAs) play essential physiological roles not only by facilitating the absorption and transport of nutrients but also by acting as a complex molecular signaling system. Reduced levels of BAs have been observed in obesity and other metabolic disorders. In the present study, we explored the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor teneligliptin on BA synthesis, both in vitro and in vivo. In our in vivo experiments, we found that teneligliptin increased the liver, ileal, and serum BA concentrations in mice undergoing teneligliptin treatment for 10 weeks. We further found that in mice fed a high-fat diet, teneligliptin prevented an increase in markers of obesity (body weight, total cholesterol, total triglyceride, adipocyte size) while increasing the total serum and ileal levels of BA. Mechanistically, teneligliptin increased BA synthesis through the alternative synthesis pathway, as the levels of both 7α-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1) along with downstream oxysterol 7α-hydroxylase (CYP7B1) but not sterol 12α-hydroxylase (CYP8B1) were increased. Importantly, teneligliptin suppressed the expression of the BA synthesis inhibitory factor Fgf15, which was mediated through phosphatidylinositol 3-kinase (PI3K)/AKT/Krüppel-like factor 15 (KLF15) signaling. Inhibition of KLF15 abolished this effect. Together, our results provide evidence of the potential benefit of teneligliptin in the treatment of metabolic disorders via increased BA production.

A study of the effects of SGLT-2 inhibitors on diabetic cardiomyopathy through miR-30d/KLF9/VEGFA pathway.

OBJECTIVE: The aim of this study was to observe the protective effects of SGLT-2 inhibitors on diabetic cardiomyopathy. MATERIALS AND METHODS: After constructing a diabetic rat model, the effects of SGLT-2 (Sodium-Glucose Cotransporter-2) inhibitors and miR-30d on cardiac function in rats were investigated by cardiac echocardiography, hematoxylin-eosin (HE) staining, and immunohistochemical methods. At the same time, changes in autophagy levels in rats were detected by Western blot (WB) experiments. RESULTS: SGLT-2 inhibitors improved the cardiac function of diabetic rats, and alleviated the pathological damage of myocardial tissue. Besides, knocking down miR-30d prevented the decrease of cardiac function in diabetic rats. Moreover, miR-30d could regulate the expression of the KLF9/VEGFA pathway and inhibit autophagy in rats. CONCLUSIONS: SGLT-2 inhibitors can regulate the autophagy level in diabetic rats through the miR-30d/KLF9/VEGFA pathway, thereby improving cardiac function.

Knockdown of KLF9 promotes the differentiation of both intramuscular and subcutaneous preadipocytes in goat.

KLF9 is reported to promote adipocyte differentiation in 3T3-L1 cells and pigs. However, the roles of KLF9 in adipocytes differentiation of goat remain unknown. In this study, the expression profiles of KLF9 were different between subcutaneous and intramuscular preadipocytes of goat during differentiation process. After silencing KLF9 gene, the lipid droplets were increased in both two types of adipocytes. In subcutaneous preadipocyte with silencing KLF9, the expressions of C/EBPß, PPARγ, LPL, KLF1-2, KLF5, and KLF17 genes were up-regulated, while KLF12, KLF4, and KLF13 genes were down-regulated in expression level. In intramuscular preadipocyte, aP2, C/EBPα, KLF2-3, KLF5, and KLF7 gene were up-regulated, and Pref-1 gene was down-regulated. In addition, the binding sites of KLF9 existed in the promoters of aP2, C/EBPα, C/EBPß, LPL and Pref-1. Taken together, KLF9 play a negative role in the differentiation of both intramuscular and subcutaneous preadipocytes in goats, but the functional mechanism may be different.

GPS2 promotes erythroid differentiation by control of the stability of EKLF protein.

Erythropoiesis is a complex multistage process that involves differentiation of early erythroid progenitors to enucleated mature red blood cells, in which lineage-specific transcription factors play essential roles. Erythroid Krüppel-like factor (EKLF/KLF1) is a pleiotropic erythroid transcription factor that is required for the proper maturation of the erythroid cells, whose expression and activation are tightly controlled in a temporal and differentiation stage-specific manner. Here, we uncover a novel role of G-protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor corepressor complex, in erythrocyte differentiation. Our study demonstrates that knockdown of GPS2 significantly suppresses erythroid differentiation of human CD34+ cells cultured in vitro and xenotransplanted in nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor γ-chain null mice. Moreover, global deletion of GPS2 in mice causes impaired erythropoiesis in the fetal liver and leads to severe anemia. Flow cytometric analysis and Wright-Giemsa staining show a defective differentiation at late stages of erythropoiesis in Gps2-/- embryos. Mechanistically, GPS2 interacts with EKLF and prevents proteasome-mediated degradation of EKLF, thereby increasing EKLF stability and transcriptional activity. Moreover, we identify the amino acids 191-230 region in EKLF protein, responsible for GPS2 binding, that is highly conserved in mammals and essential for EKLF protein stability. Collectively, our study uncovers a previously unknown role of GPS2 as a posttranslational regulator that enhances the stability of EKLF protein and thereby promotes erythroid differentiation.

High Expression of Krüppel-like Factor 7 Indicates Unfavorable Clinical Outcomes in Patients with Lung Adenocarcinoma.

BACKGROUND: Krüppel-like factor 7 (KLF7), which belongs to the KLF family of zinc finger transcription factors, plays a critical role in regulating gene expression. It was reported that KLF7 overexpression was closely related to the progression of gastric cancer. However, the role of KLF7 in lung adenocarcinoma (LAC) has not been elucidated. The aim of our study is to investigate the expression pattern of KLF7 and explore whether the KLF7 expression is correlated with unfavorable clinical outcome of patients with LAC. MATERIALS AND METHODS: The protein and mRNA levels of KLF7 were examined in LAC tissues by using immunohistochemistry staining and quantitative reverse transcription polymerase chain reaction, respectively. The prognostic role of KLF7 in patients with LAC was assessed using univariate and multivariate analyses. Clinical outcomes were evaluated by Kaplan-Meier analysis and logrank test. The effects of KLF7 on lung cancer cells were investigated through cellular experiments. RESULTS: KLF7 expression was elevated in LAC tissues compared with adjacent normal tissues. High protein level of KLF7 was correlated with larger tumor size, positive lymph node metastasis, and advanced TNM stage. Moreover, patients with LAC with higher expression level of KLF7 had poorer overall survival, and KLF7 was identified as an unfavorable independent prognosis factor. Knockdown of KLF7 can suppress the proliferation and invasion abilities of cancer cells. CONCLUSIONS: Our studies revealed that high KLF7 expression level was significantly associated with the poorer clinical outcomes of patients with LAC, indicating the potential role of KLF7 as a novel prognostic biomarker and therapeutic target.

Helicobacter pylori infection leads to KLF4 inactivation in gastric cancer through a TET1-mediated DNA methylation mechanism.

Krüppel-like factor 4 (KLF4) has a tumor suppressor role in the progression of gastric cancer (GC), and inhibition or loss of KLF4 expression was identified in GC. The aim of this study was to explore the new molecular mechanism of KLF4 inactivation in gastric cancer. Herein, we report that Helicobacter pylori infection or Cag pathogenicity island protein A (CagA) gene transduction resulted in KLF4 expression downregulation and promoted gastric epithelial cell and gastric cancel cell proliferation, migration, and colony formation. Mechanistically, we found that CagA gene transduction led to DNA methylation of the KLF4 promoter, an effect that was relevant to the significant downregulation of TET1 expression. Causally, knockdown of TET1 expression decreased KLF4 expression, whereas overexpression of TET1 had the opposite effect. Clinically, we found that KLF4 expression and the 5-hmC levels were lower in GC cells with H pylori infection than in GC cells without H pylori infection. Thus, our study not only sheds new light on how H pylori infection promotes the progression of GC but also elucidates a novel mechanism of KLF4 inactivation in GC pathogenesis. During pathogenesis, an alteration in the H pylori/CagA-TET1-KLF4 signaling pathway plays a critical role, suggesting that this pathway may be a prospective target for gastric carcinoma intervention and therapy.

Long non-coding RNA AFAP1-AS1 promotes proliferation and migration of gastric cancer by downregulating KLF2.

OBJECTIVE: To clarify the function of actin filament associated protein 1-antisense RNA1 (AFAP1-AS1) to promote the proliferation and migration of gastric cancer (GC) cells by downregulating Krüppel-like factor 2 (KLF2). MATERIALS AND METHODS: Expression level of AFAP1-AS1 in GC tissues and matched paracancerous tissues was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Besides, its level in GC either with lymphatic metastasis or not, and those in different tumor stages were determined as well. Regulatory roles of AFAP1-AS1 in cellular behaviors of GC cells were evaluated by functional experiments. The ability of AFAP1-AS1 to recruit EZH2 was evaluated through chromatin immunoprecipitation (ChIP) assay. The expression level of KLF2 in GC cells influenced by AFAP1-AS1 and EZH2 was detected by Western blot. Finally, a series of rescue experiments were conducted to clarify the role of AFAP-AS1/KLF2 in GC cell performances. RESULTS: AFAP1-AS1 was upregulated in GC tissues, and its expression in lymph node metastasis and progressive gastric cancer tissues were much higher. Knockdown of AFAP1-AS1 reduced the viability, proliferative and migratory abilities, but induced apoptosis of GC cells. AFAP1-AS1 was verified to bind to EZH2. After knockdown of AFAP1-AS1, the ability of AFAP1-AS1 to recruit EZH2 was remarkably attenuated. Knockdown of AFAP1-AS1 or EZH2 upregulated KLF2 expression in GC cells. Notably, knockdown of KLF2 partially reversed the effect of AFAP1-AS1 on GC cell performances. CONCLUSIONS: LncRNA AFAP1-AS1 accelerates the proliferative and migratory abilities of GC cells by downregulating the expression of KLF2, thus promoting the progression of GC.

Ophiopogonin D promotes bone regeneration by stimulating CD31hi EMCNhi vessel formation.

OBJECTIVES: CD31hi EMCNhi vessels (CD31, also known as PECAM1 [platelet and endothelial cell adhesion molecule 1]; EMCN, endomucin), which are strongly positive for CD31 and endomucin, couple angiogenesis and osteogenesis. However, the role of CD31hi EMCNhi vessels in bone regeneration remains unknown. In the present study, we investigated the role of CD31hi EMCNhi vessels in the process of bone regeneration. MATERIALS AND METHODS: We used endothelial-specific Krüppel like factor 3 (Klf3) knockout mice and ophiopogonin D treatment to interfere with CD31hi EMCNhi vessel formation. We constructed a bone regeneration model by surgical ablation of the trabecular bone. Immunofluorescence and micro-computed tomography (CT) were used to detect CD31hi EMCNhi vessels and bone formation. RESULTS: CD31hi EMCNhi vessels participate in the process of bone regeneration, such that endothelial-specific Klf3 knockout mice showed increased CD31hi EMCNhi vessels and osteoprogenitors in the bone regeneration area, and further accelerated bone formation. We also demonstrated that the natural compound, ophiopogonin D, acts as a KLF3 inhibitor to promote vessels formation both in vitro and in vivo. Administration of ophiopogonin D increased the abundance of CD31hi Emcnhi vessels and accelerated bone healing. CONCLUSIONS: Our findings confirmed the important role of CD31hi Emcnhi vessels in bone regeneration and provided a new target to treat bone fracture or promote bone regeneration.

MiR-137-3p exacerbates the ischemia-reperfusion injured cardiomyocyte apoptosis by targeting KLF15.

Ischemia-reperfusion (I/R) injury is a kind of the tissue damage caused by an abrupt re-supplying tissue with blood after a period of ischemia or hypoxia. It contributes to a wide range of pathological processes including kidney injury, circulatory arrest, and especially cardiovascular disease. However, the underlying pathological mechanism is not fully elucidated. Previously, extensive studies demonstrated that miRNAs participate in the pathogenesis of I/R injury, such as I/R-induced cardiomyocyte apoptosis. Here, we found that miR-137-3p, a mature form of miR-137, was up-regulated in I/R-injured cardiomyocytes of myocardial infarction patients. Deficiency of miR-137-3p partly alleviated the cardiomyocyte apoptosis and oxidative stress induced by hypoxia-reoxygenation (H/R) treatment in H9c2 cells. Also, we provided evidences that miR-137-3p directly targeted the 3' UTR of KLF15 mRNA to down-regulate its expression, and loss function of KLF15 significantly abolished the deleterious effects of ectopic miR-137-3p on cardiomyocytes both in vitro and in vivo. Collectively, these observations highlight a molecular perturbation in the pathogenesis of I/R injury in cardiomyocytes.