Krüppel-like Factor 4 Protein Research Articles

Machine learning-based classification of deubiquitinase USP26 and its cell proliferation inhibition through stabilizing KLF6 in cervical cancer

ObjectiveWe aim to accurately distinguish ubiquitin-specific proteases (USPs) from other members within the deubiquitinating enzyme families based on protein sequences. Additionally, we seek to elucidate the specific regulatory mechanisms through which USP26 modulates Krüppel-like factor 6 (KLF6) and assess the subsequent effects of this regulation on both the proliferation and migration of cervical cancer cells. MethodsAll the deubiquitinase (DUB) sequences were classified into USPs and non-USPs. Feature vectors, including 188D, n-gram, and 400D dimensions, were extracted from these sequences and subjected to binary classification via the Weka software. Next, thirty human USPs were also analyzed to identify conserved motifs and ascertained evolutionary relationships. Experimentally, more than 90 unique DUB-encoding plasmids were transfected into HeLa cell lines to assess alterations in KLF6 protein levels and to isolate a specific DUB involved in KLF6 regulation. Subsequent experiments utilized both wild-type (WT) USP26 overexpression and shRNA-mediated USP26 knockdown to examine changes in KLF6 protein levels. The half-life experiment was performed to assess the influence of USP26 on KLF6 protein stability. Immunoprecipitation was applied to confirm the USP26-KLF6 interaction, and ubiquitination assays to explore the role of USP26 in KLF6 deubiquitination. Additional cellular assays were conducted to evaluate the effects of USP26 on HeLa cell proliferation and migration. Results1. Among the extracted feature vectors of 188D, 400D, and n-gram, all 12 classifiers demonstrated excellent performance. The RandomForest classifier demonstrated superior performance in this assessment. Phylogenetic analysis of 30 human USPs revealed the presence of nine unique motifs, comprising zinc finger and ubiquitin-specific protease domains. 2. Through a systematic screening of the deubiquitinase library, USP26 was identified as the sole DUB associated with KLF6. 3. USP26 positively regulated the protein level of KLF6, as evidenced by the decrease in KLF6 protein expression upon shUSP26 knockdown in both 293T and Hela cell lines. Additionally, half-life experiments demonstrated that USP26 prolonged the stability of KLF6. 4. Immunoprecipitation experiments revealed a strong interaction between USP26 and KLF6. Notably, the functional interaction domain was mapped to amino acids 285–913 of USP26, as opposed to the 1–295 region. 5. WT USP26 was found to attenuate the ubiquitination levels of KLF6. However, the mutant USP26 abrogated its deubiquitination activity. 6. Functional biological assays demonstrated that overexpression of USP26 inhibited both proliferation and migration of HeLa cells. Conversely, knockdown of USP26 was shown to promote these oncogenic properties. Conclusions1. At the protein sequence level, members of the USP family can be effectively differentiated from non-USP proteins. Furthermore, specific functional motifs have been identified within the sequences of human USPs. 2. The deubiquitinating enzyme USP26 has been shown to target KLF6 for deubiquitination, thereby modulating its stability. Importantly, USP26 plays a pivotal role in the modulation of proliferation and migration in cervical cancer cells.

Identification of genetic associations and functional SNPs of bovine KLF6 gene on milk production traits in Chinese holstein

BackgroundOur previous research identified the Kruppel like factor 6 (KLF6) gene as a prospective candidate for milk production traits in dairy cattle. The expression of KLF6 in the livers of Holstein cows during the peak of lactation was significantly higher than that during the dry and early lactation periods. Notably, it plays an essential role in activating peroxisome proliferator-activated receptor α (PPARα) signaling pathways. The primary aim of this study was to further substantiate whether the KLF6 gene has significant genetic effects on milk traits in dairy cattle.ResultsThrough direct sequencing of PCR products with pooled DNA, we totally identified 12 single nucleotide polymorphisms (SNPs) within the KLF6 gene. The set of SNPs encompasses 7 located in 5′ flanking region, 2 located in exon 2 and 3 located in 3′ untranslated region (UTR). Of these, the g.44601035G > A is a missense mutation that resulting in the replacement of arginine (CGG) with glutamine (CAG), consequently leading to alterations in the secondary structure of the KLF6 protein, as predicted by SOPMA. The remaining 7 regulatory SNPs significantly impacted the transcriptional activity of KLF6 following mutation (P < 0.005), manifesting as changes in transcription factor binding sites. Additionally, 4 SNPs located in both the UTR and exons were predicted to influence the secondary structure of KLF6 mRNA using the RNAfold web server. Furthermore, we performed the genotype-phenotype association analysis using SAS 9.2 which found all the 12 SNPs were significantly correlated to milk yield, fat yield, fat percentage, protein yield and protein percentage within both the first and second lactations (P < 0.0001 ~ 0.0441). Also, with Haploview 4.2 software, we found the 12 SNPs linked closely and formed a haplotype block, which was strongly associated with five milk traits (P < 0.0001 ~ 0.0203).ConclusionsIn summary, our study represented the KLF6 gene has significant impacts on milk yield and composition traits in dairy cattle. Among the identified SNPs, 7 were implicated in modulating milk traits by impacting transcriptional activity, 4 by altering mRNA secondary structure, and 1 by affecting the protein secondary structure of KLF6. These findings provided valuable molecular insights for genomic selection program of dairy cattle.

Krüppel-like factor 4 promotes autophagy in macrophages under high glucose concentration by inhibiting the AKT/mTOR signaling pathway

Background: Diabetic atherosclerosis (AS) is the main cause of disability and death in diabetes. In the progression of AS, autophagic activity plays an important role. Krüppel-like factor 4 (KLF4) is a member of the zinc finger protein transcription factor family and is believed to play a protective role in the pathogenesis of atherosclerosis. This study aimed to explore the role of KLF4 in diabetic atherosclerosis and the autophagic mechanism. Methods: A diabetic mouse model was established and the expression level of KLF4 protein in the aorta of the mice was detected after a high-fat diet. The effects of KLF4 on cholesterol content, apoptosis, autophagy-related proteins, and the AKT/mTOR signaling pathway of THP-1 macrophages were also evaluated. Results: The expression level of KLF4 protein in the aorta of diabetic mice was decreased. Meanwhile, overexpression of KLF4 in THP-1 macrophages significantly decreased cholesterol accumulation, increased beclin-1 expression, decreased P62 expression, enhanced LC3 fluorescence intensity decreased cell apoptosis and p-mTOR and p-AKT expression were decreased under the condition of high glucose. After the reduction of KLF4 expression, the result is reversed. Conclusion: KLF4 induces autophagy by inhibiting the AKT/mTOR pathway and alleviates cholesterol deposition in THP-1 macrophages under high glucose concentration.

Cardiac Fibroblast Activation Induced by Oxygen-Glucose Deprivation Depends on the HIF-1α/miR-212-5p/KLF4 Pathway.

It is widely accepted that miRNAs play an important role in the pathogenesis of myocardial fibrosis. This study aimed to identify a new pathway of miR-212-5p in the activation of human cardiac fibroblasts (HCFs) induced by oxygen-glucose deprivation (OGD). First, we found that KLF4 protein was markedly decreased in OGD-induced HCFs. Then, bioinformatics analysis and verification experiments were used to identify the existence of an interaction of KLF4 with miR-212-5p. Functional experiments indicated that OGD significantly upregulated the expression of hypoxia inducible factor-1 alpha (HIF-1α) in HCFs, which positively regulated miR-212-5p transcription by binding to its promoter. MiR-212-5p inhibited the expression of Krüppel-like factor 4 (KLF4) protein by binding to the 3' untranslated coding regions (UTRs) of KLF4 mRNA. Inhibition of miR-212-5p effectively inhibited the activation of OGD-induced HCFs by upregulating KLF4 expression and inhibited cardiac fibrosis in vivo and in vitro.

Protocatechuic acid boosts continual efferocytosis in macrophages by derepressing KLF4 to transcriptionally activate MerTK.

Macrophages clear apoptotic cells through a process called continual efferocytosis. We found that protocatechuic acid (PCA), a polyphenolic compound abundant in fruits and vegetables, increased the continual efferocytic capacity of macrophages and inhibited the progression of advanced atherosclerosis. PCA reduced the intracellular amounts of microRNA-10b (miR-10b) by promoting its secretion in extracellular vesicles, which led to an increase in the abundance of the miR-10b target Krüppel-like factor 4 (KLF4). In turn, KLF4 transcriptionally induced the gene encoding Mer proto-oncogene tyrosine kinase (MerTK), an efferocytic receptor for the recognition of apoptotic cells, resulting in increased continual efferocytic capacity. However, in naive macrophages, the PCA-induced secretion of miR-10b did not affect KLF4 and MerTK protein abundance or efferocytic capacity. In mice, oral administration of PCA increased continual efferocytosis in macrophages residing in the peritoneal cavities, thymi, and advanced atherosclerotic plaques through the miR-10b-KLF4-MerTK pathway. In addition, pharmacological inhibition of miR-10b with antagomiR-10b also increased the efferocytic capacity of efferocytic but not naive macrophages in vitro and in vivo. Together, these data describe a pathway that promotes continual efferocytosis in macrophages through miR-10b secretion and a KLF4-dependent increase in MerTK abundance, which can be activated by dietary PCA and which has implications for understanding the regulation of continual efferocytosis in macrophages.

Lnc956 regulates mouse embryonic stem cell differentiation in response to DNA damage in a p53-independent pathway.

Maintaining genomic stability is crucial for embryonic stem cells (ESCs). ESCs with unrepaired DNA damage are eliminated through differentiation and apoptosis. To date, only tumor suppressor p53 is known to be implicated in this quality control process. Here, we identified a p53-independent quality control factor lncRNA NONMMUT028956 (Lnc956 for short) in mouse ESCs. Lnc956 is prevalently expressed in ESCs and regulates the differentiation of ESCs after DNA damage. Mechanistically, Ataxia telangiectasia mutated (ATM) activation drives m6A methylation of Lnc956, which promotes its interaction with Krüppel-like factor 4 (KLF4). Lnc956-KLF4 association sequestrates the KLF4 protein and prevents KLF4's transcriptional regulation on pluripotency. This posttranslational mechanism favors the rapid shutdown of the regulatory circuitry of pluripotency. Thus, ATM signaling in ESCs can activate two pathways mediated by p53 and Lnc956, respectively, which act together to ensure robust differentiation and apoptosis in response to unrepaired DNA damage.

CircHIPK3 promotes bone microvascular endothelial cell proliferation, migration and angiogenesis by targeting miR-7 and KLF4/VEGF signaling in steroid-induced osteonecrosis of the femoral head.

Circular RNA homeodomain interacting protein kinase 3 (circHIPK3) has been implicated in facilitating angiogenesis in various conditions. However, its role in steroid-induced osteonecrosis of the femoral head (ONFH) remains unclear. To investigate whether circHIPK3 promotes bone microvascular activity and angiogenesis by targeting miR-7 and Krüppel-like factor 4 (KLF4)/vascular endothelial growth factor (VEGF) signaling in ONFH. Fifty patients with steroid-induced ONFH undergoing hip-preserving surgery or total hip arthroplasty were included in this study. The expression of circHIPK3, miR-7 and KLF4 was evaluated using reverse transcription polymerase chain reaction (RT-PCR) in necrotic and healthy samples of the femoral head. Bone microvascular endothelial cells (BMECs) were extracted and cultured with 0.1 mg/mL hydrocortisone to create a hormonally deficient cell model. These BMECs were then transfected with either circHIPK3 overexpressing or silencing plasmids with or without miR-7 mimics. The MTT assays were used to detect cell proliferation. Scratch assays were used to assess the migration ability of the BMECs. The tube formation was carried out using an in vitro Matrigel angiogenesis assay. Annexin V-FITC/PI and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used to assess the degree of apoptosis. Western blot assays were carried out to discern KLF4 and VEGF expression. The interactions of circHIPK3, miR-7 and KLF4 were confirmed using luciferase, RNA-binding protein immunoprecipitation (RIP), RNA pull-down, and fluorescence in situ hybridization (FISH) assays. The circHIPK3 and KLF4 expression was decreased, whereas miR-7 expression was increased in necrotic tissues compared to non-necrotic samples. Both circHIPK3 and KLF4 expression correlated negatively with miR-7. The overexpression of circHIPK3 promoted the proliferative, migratory and angiogenic capabilities of the BMECs, while adding an miR-7 mimic reversed these effects. At the same time, the overexpression of circHIPK3 reduced the apoptosis rate of the BMECs and increased KLF4 and VEGF protein expression, but adding an miR-7 mimic reversed these effects. The FISH, RNA pull-down, RIP, and luciferase assays revealed an interaction between circHIPK3, miR-7 and KLF4. The circHIPK3 promotes BMEC proliferation, migration and angiogenesis by targeting miR-7 and KLF4/VEGF signaling.

DANCR Induces Cisplatin Resistance of Triple-Negative Breast Cancer by KLF5/p27 Signaling

An increasing body of evidence suggests that long noncoding RNAs play critical roles in human cancer. Breast cancer is a heterogeneous disease and the potential involvement of long noncoding RNAs in breast cancer remains poorly understood. Herein, the study identified a long noncoding RNA, DANCR, which promotes cisplatin chemoresistance in triple-negative breast cancer (TNBC) cells. Mechanistically, binding of DANCR to Krüppel-like factor 5 (KLF5) induced acetylation of KLF5 at lysine 369 (K369), and DANCR knockdown resulted in down-regulation of KLF5 protein levels. Furthermore, DANCR/KLF5 signaling pathway induced hypersensitivity to cisplatin in chemoresistant patients by inhibiting p27 transcription. In summary, this study reinforced the potential presence of a growth regulatory network in TNBC cells, and documented a DANCR/KLF5/p27 signaling pathway mediating cisplatin chemoresistance in TNBC.

DANCR acetylated‐KLF5 induces cisplatin resistance in triple‐negative breast cancer by inhibiting the transcription of p27Kip1

Cisplatin is used as a common chemotherapy drug in triple‐negative breast cancer. However, although growing evidence demonstrates that its chemoresistance becomes a important obstacle for curing of triple‐negative breast cancer, its molecular mechanism remains unclear. Here we detected that Krüppel‐like factor 5 (KLF5) depletion reduce cisplatin chemoresistance both in triple‐negative breast cancer cells in vitro and in vivo. Further experiments showed that long non‐coding RNA DANCR binds with KLF5 and then induces the acetylation of KLF5 at lysine 369 (K369), which upregulates KLF5 protein level, contributing to cisplatin chemoresistant. Additionally, DANCR/KLF5 signalling pathway renders chemoresistant hypersensitive to cisplatin that inhibiting p27 transcription. Our study uncover a novel DANCR/KLF5/p27 signalling pathway to mediate cisplatin chemoresistant in triple‐negative breast cancer and provide a new molecular target for treatment of triple‐negative breast cancer.

DUB3/KLF4 combats tumor growth and chemoresistance in hepatocellular carcinoma

This study aimed to investigate the role of deubiquitinating enzyme 3 (DUB3) in the regulation of Krüppel-like factor 4 (KLF4) expression in hepatocellular carcinoma (HCC). Gain- and loss-of-function assay, luciferase reporter assay, co-immunoprecipitation, and intracellular and extracellular deubiquitination assays were conducted in vitro. A tumor xenograft mouse model was established. The expression of DUB3 and KLF4 was examined in HCC patient specimens. The results showed that DUB3 upregulated KLF4 expression by deubiquitinating and stabilizing KLF4 protein in HCC cells through binding with KLF4. DUB3 inhibited HCC cell proliferation in vitro and tumor growth in vivo while enhancing the chemosensitivity of HCC cells in a KLF4-dependent manner. Furthermore, KLF4 promoted DUB3 transcription by binding to the DUB3 promoter. In HCC patients, DUB3 expression positively correlated with KLF4 expression in HCC tissues. Low DUB3 expression predicted worse overall survival and recurrence in HCC patients. In conclusion, this study revealed a positive DUB3/KLF4 feedback loop that inhibits tumor growth and chemoresistance in HCC. These results suggest that DUB3/KLF4 activation might be a potential therapeutic approach for HCC treatment.

Abstract 9817: Krüppel-Like Factor 4 Integrates Immunometabolism of Human Arterial Endothelial Cells

Introduction: Inflammation and metabolic alterations are two common hallmarks of cardiovascular diseases. Yet, the molecular mechanisms integrating inflammation and cell metabolism remain elusive. Objective: We investigated whether Krüppel-like factor 4 (KLF4) serves as a link between cellular glucose metabolism and inflammation in human arterial endothelial cells. Methods and Results: Primary human pulmonary artery and aortic endothelial cells (PAEC and AoEC, respectively) were stimulated with tumor necrosis factor α (TNFα) or lipopolysaccharide (LPS). Results from fluorescent microscopy and Seahorse extracellular flux analysis revealed that TNFα and LPS enhanced glucose uptake by 2.2-fold, glycolysis by 2.3-fold, and mitochondrial respiration by 1.6-fold in PAEC and AoEC. The increase in glycolysis correlated with a 2-4-fold upregulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and PFKFB4, two key regulatory enzymes of glycolysis. Silencing PFKFB3 but not PFKFB4 entirely prevented TNFα-induced enhancement of glycolysis with a concomitant metabolic shift to mitochondrial respiration. Mechanistically, TNFα and LPS stimulated KLF4 mRNA and protein expression (2-4-fold), which functions as a transcriptional repressor of PFKFB3 . KLF4 knockdown potentiated TNFα-induced stimulation of PFKFB3 expression by 1.6-fold and glycolysis by 2.2-fold, effects which were completely prevented by co-silencing of PFKFB3 . Functionally, KLF4 silencing exacerbated TNFα-induced vascular cell adhesion molecule 1 (VCAM1) expression (by 2-3-fold), E-selectin secretion (by 2.8-fold), and monocyte adhesion (by 1.6-fold) in PAEC, all of which were attenuated by over 50% when PFKFB3 was inhibited by siRNA or with 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO). By contrast, KLF4 overexpression-mediated inhibition of inflammation was significantly counteracted in PAEC with co-overexpression of PFKFB3 . Conclusion: These results indicate that KLF4 inhibits inflammation by normalizing PFKFB3-mediated enhancement of glycolysis and that the KLF4-PFKFB3 signaling axis integrates immunometabolism in human arterial endothelial cells.

Palmitic acid up regulates Gal-3 and induces insulin resistance in macrophages by mediating the balance between KLF4 and NF-κB.

Insulin resistance is the main sign of type 2 diabetes mellitus and is often accompanied by the infiltration of inflammatory factors. These inflammatory factors are mainly produced and secreted by macrophages. The purpose of the current study was to explore the relationship between macrophages and insulin resistance, and to determine its underlying mechanism. The insulin resistance of macrophages was induced by palmitic acid (PA) in vitro. The glucose uptake rate of macrophages, the expression levels of inflammatory cytokines and the expression levels of insulin resistance-related proteins were detected. The protein expression levels of Krüppel-like factor 4 (KLF4), toll-like receptor 4 (TLR4), NF-κB and Galectin-3 (Gal-3) were detected via western blotting and recovery experiments were performed by combining the Gal-3 and TLR4 inhibitors GB1107 and TAK242. The results revealed that PA-induced macrophages demonstrated insulin resistance. Additionally, KLF4 protein was inhibited and the sugar uptake rate was significantly lower than that of the control group. Western blotting and immunofluorescence assays revealed that the expression of Gal-3 in PA-induced macrophages was significantly upregulated. The addition of the Gal-3 inhibitor GB1107 significantly increased glucose utilization and reduced insulin resistance in PA-treated cells. Inhibitor of TLR4 inhibited the protein expression level of the TLR4/NF-κB pathway. In conclusion, PA promoted the TLR4/phosphorylated-NF-κB signaling pathway by inhibiting KLF4, promoted the upregulation of Gal-3 expression and improved the insulin resistance of macrophages.

H. pylori infection induces CXCL8 expression and promotes gastric cancer progress through downregulating KLF4

Tumour-derived CXCL8 facilitates the movement of myeloid-derived suppressor cells, which are able to restrain antitumour immune responses to the tumour microenvironment. Kruppel-like factor 4 (KLF4) is a potential tumour suppressor in gastric cancer (GC). However, knowledge regarding correlations between KLF4 and CXCL8 in GC is limited. We use cellular and molecular biological methods to assess whether these two factors interact in GC. Expression CXCL8 and KLF4 was altered in human GC tissues compared to normal gastric tissues in opposite ways. Additionally, cytotoxin-associated gene A protein (CagA) gene transduction or Helicobacter pylori(H. pylori) infection upregulated CXCL8 expression. Knockdown of KLF4 expression increased CXCL8 protein and RNA expression, whereas its overexpression had the opposite effect. CXCL8-mediated enhancement of GC cell migration and proliferation was reversed by upregulation of KLF4 expression. Further mechanistic research revealed that KLF4 binds the CXCL8 promoter, suppressing CXCL8 transcription. Moreover, CXCL8 stimulation reduced KLF4 protein expression and promoted GC cell proliferation and migration, eventually promoting neoplasm growth in vivo. Together, our findings demonstrate that CagA promotes CXCL8 and inhibits KLF4. CXCL8 is a decisive downstream target gene of KLF4, and KLF4 negatively regulates CXCL8 in GC. Furthermore, CXCL8's negative regulation of KLF4 in vivo and in vitro, indicates that CagA may downregulate KLF4 by inducing CXCL8 expression, low expression of KLF4 further promotes that of CXCL8, forming a vicious circle in GC. Targeted KLF4 activation might improve the immunosuppressive microenvironment through direct negative regulation of CXCL8, providing a new potential target to strengthen the efficacy of immunotherapy in GC patients.

Arginine methyltransferase PRMT5 methylates and stabilizes KLF5 via decreasing its phosphorylation and ubiquitination to promote basal-like breast cancer.

Krüppel-like factor 5 (KLF5) is an oncogenic factor that is highly expressed in basal-like breast cancer (BLBC) and promotes cell proliferation, survival, migration, stemness, and tumor growth; however, its posttranslational modifications are poorly defined. Protein arginine methyltransferase 5 (PRMT5) is also an oncogene implicated in various carcinomas, including breast cancer. In this study, we found that PRMT5 interacts with KLF5 and catalyzes the di-methylation of KLF5 at Arginine 57 (R57) in a methyltransferase activity-dependent manner in BLBC cells. Depletion or pharmaceutical inhibition (using PJ-68) of PRMT5 decreased the expression of KLF5 and its downstream target genes in vitro and in vivo. PRMT5-induced KLF5R57me2 antagonizes GSK3β-mediated KLF5 phosphorylation and subsequently Fbw7-mediated KLF5 ubiquitination and coupled degradation. Functionally, PRMT5 promotes breast cancer stem cell maintenance and proliferation, at least partially, by stabilizing KLF5. PRMT5 and KLF5 protein levels were positively correlated in clinical BLBCs. Taken together, PRMT5 methylates KLF5 to prevent its phosphorylation, ubiquitination, and degradation, and thus promotes breast cancer stem cell maintenance and proliferation. These findings suggest that PRMT5 is a potential therapeutic target for BLBC.

LncRNA MEG3 Protects Chondrocytes From IL-1β-Induced Inflammation via Regulating miR-9-5p/KLF4 Axis.

BackgroundOsteoarthritis (OA) is a chronic degenerative disease of the joints characterized by articular cartilage damage, subchondral bone remodeling, osteophyte formation, and inflammatory changes. This work aims to investigate the protective role of long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) against the apoptosis of chondrocytes.MethodsChondrocyte cell lines, CHON-001, and ATDC5 were treated with different doses of interleukin-1β (IL-1β) to mimic the inflammatory response during OA pathogenesis. Quantitative real-time polymerase chain reaction was performed to measure MEG3, miR-9-5p, and Krüppel-like factor 4 (KLF4) mRNA expression levels. MEG3 and KLF4 overexpression plasmids, MEG3 shRNA, miR-9-5p mimics, and miR-9-5p inhibitors were transfected into the cells. Cell counting kit-8, wound healing assay, and flow cytometry were conducted to determine cell viability, migration, and apoptotic rate. Dual-luciferase reporter assay was adopted to verify the targeting relationships among MEG3, miR-9-5p, and KLF4. Western blot was used to detect KLF4 protein expression. Enzyme-linked immunosorbent assay was employed to measure the levels of inflammatory factors.ResultsMEG3 expression in chondrocytes was down-regulated by the stimulation of IL-1β, and MEG3 negatively regulated miR-9-5p expression but positively regulated KLF4 expression. MEG3 overexpression strengthened the viability and migration of CHON-001 and ATDC5 cells but restrained the apoptosis and inflammatory response, while MEG3 knockdown had opposite effects. miR-9-5p inhibition or KLF4 overexpression could counteract the effects of MEG3 knockdown on chondrocytes. Besides that, MEG3 was proved to be a molecular sponge for miR-9-5p, and KLF4 was verified as the target of miR-9-5p.ConclusionMEG3 can promote chondrocyte proliferation and migration and inhibit apoptosis and inflammation by sponging miR-9-5p to induce KLF4 expression, which provides a promising therapy target for OA treatment.

Prognostic Value and Function of KLF5 in Papillary Thyroid Cancer.

Simple SummaryThis study was conducted to investigate the clinical significance and prognostic value of KLF5 in a large cohort of Middle Eastern PTC patients and explore its functional role and mechanism in PTC cell lines in vitro and in vivo. We found KLF5 over-expression in PTC patient cases and this was significantly associated with aggressive clinico-pathological parameters and worse outcome. We also found a significant association between KLF5 and HIF-1α in PTC patients and cell lines. Functionally, KLF5 promoted cell growth, stemness, invasion, migration, and angiogenesis, while its inhibition reverses its action in PTC cell lines. Finally, the depletion of KLF5 regressed PTC tumor growth in nude mice. These data suggest that KLF5 may potentially be a suitable therapeutic target in PTC, and pharmacological inhibition of KLF5 might be a viable therapeutic option for the treatment of patients with an aggressive subtype of PTC.The Krüppel-like factor 5 (KLF5), a zinc-finger transcriptional factor, is highly expressed in several solid tumors, but its role in PTC remains unclear. We investigated the expression of KLF5 protein in a large cohort of PTC patient samples and explored its functional role and mechanism in PTC cell lines in vitro and in vivo. KLF5 overexpression was observed in 65.1% of all PTC cases and it was significantly associated with aggressive clinico-pathological parameters and poor outcome. Given the significant association between KLF5 and HIF-1α overexpression in PTC patients, we investigated the functional correlation between KLF5 and HIF-1α in PTC cells. Indeed, the analysis revealed the co-immunoprecipitation of KLF5 with HIF-1α in PTC cells. We also identified KLF5-binding sites in the HIF-1α promoter that specifically bound to KLF5 protein. Mechanistically, KLF5 promoted PTC cell growth, invasion, migration, and angiogenesis, while KLF5 downregulation via specific inhibitor or siRNA reverses its action in vitro. Importantly, the silencing of KLF5 decreases the self-renewal ability of spheroids generated from PTC cells. In addition, the depletion of KLF5 reduces PTC xenograft growth in vivo. These findings suggest KLF5 can be a possible new molecular therapeutic target for a subset of PTC.

Regulation of adipocyte differentiation by clusterin-mediated Krüppel-like factor 5 stabilization.

Clusterin (CLU) is a heterodimeric glycoprotein involved in a range of biological processes. We investigated the function of CLU as a novel regulator of adipogenesis. CLU expression increased during 3T3-L1 preadipocyte differentiation. CLU overexpression promoted adipogenic differentiation of preadipocytes and increased the mRNA levels of adipogenic markers including peroxisome proliferator-activated receptor γ (Pparg) and CCAAT enhancer-binding protein α (Cebpa). Conversely, knockdown of CLU attenuated adipogenesis and reduced transcript levels of Pparg and Cebpa. However, the promoter activities of both the Pparg and the Cebpa gene were not affected by alteration of CLU expression on its own. Additionally, the protein level of Krüppel-like factor 5 (KLF5), an upstream transcription factor of Pparg and Cebpa involved in adipogenic differentiation, was upregulated by CLU overexpression, although the mRNA level of Klf5 was not altered by changes in the expression level of CLU. Cycloheximide chase assay showed that the increased level of KLF5 by CLU overexpression was due to decreased degradation of KLF5 protein. Interestingly, CLU increased the stability of KLF5 by decreasing KLF5 ubiquitination. CLU inhibited the interaction between KLF5 and F-box/WD repeat-containing protein 7, which is an E3 ubiquitin ligase that targets KLF5. The adipogenic role of CLU was also addressed in mesenchymal stem cells (MSCs) and Clu-/- mouse embryonic fibroblasts (MEFs). Furthermore, CLU enhanced KLF5-mediated transcriptional activation of both the Cebpa and the Pparg promoter. Taken together, these results suggest that CLU is a novel regulator of adipocyte differentiation by modulating the protein stability of the adipogenic transcription factor KLF5.

Can Racial Differences In Endothelial Dysfunction Be Explained By Mirnas?

MicroRNAs (miRs) are short noncoding RNAs that regulate gene expression post-transcriptionally. It is well documented that exercise improves endothelial function, possibly, by modifying flow-responsive miR expression (e.g. miRs-126*, -92-a and -21). MiR-126* is an anti-atherogenic miR that regulates vascular integrity, angiogenesis, and inflammation. Whereas, miR-92a and -21 are pro-atherogenic miRs that result in a reduction in endothelial nitic oxide synthase (eNOS) activity. MiR-92a targets the transcription factor Kruppel-like factor 4 (KLF4) leading to lower eNOS expression. Whereas miR-21 targets phosphatase and tensin homologue (PTEN) to inhibit activation of the PI3K/Akt signaling pathway involved in activating eNOS. We have shown that African Americans (AA) exhibit a low grade of chronic systemic inflammation, exaggerated response to inflammatory cytokines, and a higher prevalence of endothelial dysfunction compared to Caucasians (CA). PURPOSE: To investigate potential racial differences in miRs expression and downstream target proteins in activated endothelial cells (EC). METHODS: Human Umbilical Vein Endothelial Cells (n=6 cell lines; 3 CA & 3 AA) were cultured under two conditions: Control or TNF-α (10 ng/mL) for 4 hours. Protein expression was measured for PTEN, KLF4, p-eNOSser-1117 and eNOS. Total RNA was harvested to measure miR-21, miR-126*, and miR92a. A two-way ANOVA was used to assess the effects of race and condition. RESULTS: No racial differences were found in protein expression of PTEN, KLF4, and eNOS at baseline or with TNF-α stimulation. AA had lower p-eNOS expression (p=0.008) compared to CA ECs under both conditions. No racial differences were found in miR-126 and miR-21 in either condition. However, AA were trending towards higher miR-92a expression (p=0.07) compared to CA ECs under both conditions. CONCLUSIONS: Although we show significantly lower levels of p-eNOS and higher miR-92a expression in activated ECs of AA compared to CA, KLF4 protein levels were not significantly different. Therefore, the specific miRs studied do not explain the racial differences observed in endothelial dysfunction in an inflammation model. Future research should investigate potential racial differences in how miRs respond to high laminar shear stress, as an exercise memetic.

A new Schiff base copper(II) complex induces cancer cell growth inhibition and apoptosis by multiple mechanisms

A new Schiff base copper(II) complex [N,N′-bis(2′-hydroxyphenylacetone)-o-ethanediamine] copper (II) (M1) has been synthesized and characterized by single X-ray crystallography. The cytotoxicity of complex M1 was evaluated against HeLa, LoVo, A549, A549/cis cancer cell lines, and the normal cell lines LO2 and HUVEC, by MTT (3-(4,5-dimethylthiazoyl-2-yl)2,5-diphenyltetrazoliumbromide) assays. The IC50 (50% inhibition concentrations) is in the range of 5.13–11.68 μM, which is somewhat lower than cisplatin on the basis of platinum molar concentration. Furthermore, anticancer mechanistic studies showed that the complex M1 inhibited cell proliferation by blocking DNA synthesis and then acted on nuclear division of HeLa cells over time. Moreover, M1 increased intracellular ROS (Reactive oxygen species) levels in a dose-dependent manner. Western blot analysis indicated M1 dramatically decrease c-Myc transcription factor and KLF5 (Krüppel-like factor 5) protein expression levels in HeLa. M1 did not inhibit proteasomal activity. Finally, M1 induced DNA damages and activated the DNA damage repair pathways.

Off the back burner: diverse and gender-inclusive decision-making for COVID-19 response and recovery.

<h3>Objectives</h3> The mechanism of transformation to intestinal metaplasia in Barrett9s oesophagus has not been clarified. We previously reported that bile acids activate the Cdx2 promoter via nuclear factor kappa B (NF-κB) and stimulate production of Cdx2 protein in oesophageal keratinocytes, resulting in production of intestinal-type mucin. Krüppel-like factor 4 (KLF4) is an important transcription factor in the development of intestinal mucosa and has similar functions as Cdx2. In the present study, we investigated the direct effects of bile acids on KLF4 expression as well as the precise mechanisms of expression in cultured oesophageal squamous epithelial cells. <h3>Methods</h3> We investigated the expression of KLF4 in rat and human Barrett9s epithelium specimens, while the response of that expression to bile acids was studied using a KLF4 promoter luciferase assay. In addition, oesophageal squamous epithelial cells were transfected with a KLF4 expression vector, after which their possible transformation into intestinal-type epithelial cells was investigated. <h3>Results</h3> In both rat and human tissues, Barrett9s epithelium strongly expressed KLF4. Furthermore, a bile acids mixture increased KLF4 promoter activity, and mRNA and protein expression in oesophageal epithelial cells. Results from mutation analysis of the KLF4 promoter suggested that the NF-κB binding site is responsible for bile acid-induced activation of the KLF4 promoter. In addition, KLF4 and Cdx2 stimulated each other by directly binding to the promoter of the other, while transfection of the KLF4 expression vector in oesophageal epithelial cells induced production of MUC2 protein. <h3>Conclusion</h3> Bile acid-induced sequential expression of KLF4 followed by MUC2 production may have an important role in the development of Barrett9s epithelium.