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Background: Escaping from ER stress-induced apoptosis plays an important role in the progression of many tumours. However, its molecular mechanism in osteosarcoma remains incompletely understood. Methods: The molecular mechanism was investigated using RNA sequencing, qRT–PCR and Western blot assays. The relationship between LINC00629 and KLF4 was investigated using RNA pulldown and ubiquitylation assays. The transcriptional regulation of laminin subunit alpha 4 (LAMA4) by KLF4 was identified using bioinformatic analysis, a luciferase assay, and a chromatin immunoprecipitation assay. Results: Here, we demonstrated that LINC00629 was increased under ER stress treatment. Elevated LINC00629 inhibited ER stress-induced osteosarcoma cell apoptosis and promoted clonogenicity and migration in vitro and in vivo. Further mechanistic studies indicated that LINC00629 interacted with KLF4 and suppressed its degradation, which led to a KLF4 increase in osteosarcoma. In addition, we also found that KLF4 upregulated LAMA4 expression by directly binding to its promoter and that LINC00629 inhibited ER stress-induced apoptosis and facilitated osteosarcoma cell clonogenicity and metastasis by activating the KLF4-LAMA4 pathway. Conclusion: Collectively, our data indicate that LINC00629 is a critical long noncoding RNA (lncRNA) induced by ER stress and plays an oncogenic role in osteosarcoma cell by activating the KLF4-LAMA4 axis. [ABSTRACT FROM AUTHOR]

The interplay between microRNAs (miRNAs) and pluripotency transcription factors (TFs) orchestrates the acquisition of cancer stem cell (CSC) features during the course of malignant transformation, rendering them essential cancer cell dependencies and therapeutic vulnerabilities. In this review, we discuss emerging themes in tumor heterogeneity, including the clonal evolution and the CSC models and their implications in resistance to cancer therapies, and then provide thorough coverage on the roles played by key TFs in maintaining normal and malignant stem cell pluripotency and plasticity. In addition, we discuss the reciprocal interactions between miRNAs and MYC, OCT4, NANOG, SOX2, and KLF4 pluripotency TFs and their contributions to tumorigenesis. We provide our view on the potential to interfere with key miRNA-TF networks through the use of RNA-based therapeutics as single agents or in combination with other therapeutic strategies, to abrogate the CSC state and render tumor cells more responsive to standard and targeted therapies. [ABSTRACT FROM AUTHOR]

Background: Stomach adenocarcinoma (STAD) is the most common type of gastric cancer. In this study, the functions and potential mechanisms of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB) in STAD were explored. Methods: Different bioinformatics analyses were performed to confirm HADHB expression in STAD. HADHB expression in STAD tissues and cells was also evaluated using western blot, qRT-PCR, and immunohistochemistry. Further, the viability, proliferation, colony formation, cell cycle determination, migration, and wound healing capacity were assessed, and the effects of HADHB on tumour growth, cell apoptosis, and proliferation in nude mice were determined. The upstream effector of HADHB was examined using bioinformatics analysis and dual luciferase reporter assay. GSEA was also employed for pathway enrichment analysis and the expression of Hippo-YAP pathway-related proteins was detected. Results: The expression of HADHB was found to be low in STAD tissues and cells. The upregulation of HADHB distinctly repressed the viability, proliferation, colony formation, cell cycle progression, migration, invasion, and wound healing of HGC27 cells, while knockdown of HADHB led to opposite effects. HADHB upregulation impeded tumour growth and cell proliferation, and enhanced apoptosis in nude mice. KLF4, whose expression was low in STAD, was identified as an upstream regulator of HADHB. KLF4 upregulation abolished the HADHB knockdown-induced tumour promoting effects in AGS cells. Further, HADHB regulates the Hippo-YAP pathway, which was validated using a pathway rescue assay. Low expression of KLF4 led to HADHB downregulation in STAD. Conclusion: HADHB might function as a tumour suppressor gene in STAD by regulation the Hippo-YAP pathway. [ABSTRACT FROM AUTHOR]

Acute kidney injury (AKI) can progress to renal fibrosis and chronic kidney disease (CKD), which reduces quality of life and increases the economic burden on patients. However, the molecular mechanisms underlying renal fibrosis following AKI remain unclear. This study tested the hypothesis that the Krüppel-like factor 4 (KLF4)/miR-101/Collagen alpha-1X (COL10A1) axis could inhibit epithelial-mesenchymal transition (EMT) and renal fibrosis after AKI in a mouse model of ischemia-reperfusion (I/R)-induced renal fibrosis and HK-2 cells by gene silencing, overexpression, immunofluorescence, immunohistochemistry, real-time quantitative PCR, Western blotting, dual-luciferase reporter assay, fluorescence in situ hybridization (FISH) and ELISA. Compared with the Sham group, I/R induced renal tubular and glomerular injury and fibrosis, and increased the levels of BUN, serum Scr and neutrophil gelatinase-associated lipocalin (NGAL), Col10a1 and Vimentin expression, but decreased E-cadherin expression in the kidney tissues of mice at 42 days post-I/R. Similarly, hypoxia promoted fibroblastic morphological changes in HK-2 cells and enhanced NGAL, COL10A1, Vimentin, and α-SMA expression, but reduced E-cadherin expression in HK-2 cells. These pathological changes were significantly mitigated in COL10A1-silenced renal tissues and HK-2 cells. KLF4 induces miR-101 transcription. More importantly, hypoxia upregulated Vimentin and COL10A1 expression, but decreased miR-101, KLF4, and E-cadherin expression in HK-2 cells. These hypoxic effects were significantly mitigated or abrogated by KLF4 over-expression in the HK-2 cells. Our data indicate that KLF4 up-regulates miR-101 expression, leading to the downregulation of COL10A1 expression, inhibition of EMT and renal fibrosis during the pathogenic process of I/R-related renal fibrosis.

Repressor elements significantly influence economically relevant phenotypes in pigs; however, their precise roles and characteristics are inadequately understood. In the present study, we employed H3K27me3 profiling, assay for transposase-accessible chromatin with highthroughput sequencing (ATAC-seq), and RNA sequencing (RNA-seq) data across six tissues derived from three embryonic layers to identify and map 2 034 super repressor elements (SREs) and 22 223 typical repressor elements (TREs) in the pig genome. Notably, many repressor elements were conserved across mesodermal and ectodermal tissues. SREs exhibited tight regulation of their target genes, affecting a limited number of genes within a specific genomic region with pronounced effects, while TREs exerted broader but weaker regulation over a wider range of target genes. Furthermore, in neuronal tissues, genes regulated by repressor elements started to be repressed during the differentiation of stem cells into progenitor cells. Notably, analysis showed that many repressor elements exhibited cooperative and additive effects on the modulation of KLF4 expression. This research provides the first comprehensive map of pig repressor elements, serving as an essential reference for future studies on repressor elements.

This study examines KLF4's role in endothelial cells (ECs), emphasizing its effects on nitric oxide (NO) production, microvascular formation, and oxidative stress regulation following ischemic stroke. Through high-throughput sequencing, we identified eight cell subpopulations in carotid artery tissues post-stroke, with KLF4 notably elevated in ECs. KLF4 overexpression in ECs promoted NO synthesis, enhanced endothelial tube formation, mitigated oxidative stress, and improved smooth muscle cells (SMCs) function, collectively boosting blood flow in ischemic regions. These findings highlight KLF4 as pivotal in vascular regeneration and oxidative stress reduction, positioning it as a promising target for cardiovascular and cerebrovascular therapies., Competing Interests: Declaration of Competing Interest The author declares no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Stress is an independent risk factor for cognitive impairment, with elevated plasma homocysteine (HCY) levels playing a crucial role in stress-induced cognitive decline. While the rise in plasma HCY levels is linked to abnormal peripheral catabolism, the impact of stress on HCY catabolism in the brain remains unclear. This study investigated the effect of stress on HCY metabolism in the brain by analyzing HCY and its metabolic enzymes in the hippocampus and prefrontal cortex. The results showed a significant decrease in enzymes MS (methionine-synthase), CBS (cystathionineβ-synthase), and CSE (cystathionine γ-lyase) in these brain regions of mice subjected to 3 weeks of restraint stress, leading to HCY accumulation. Additionally, the enzyme MTHFR (methylenetetrahydrofolate reductase) remained unchanged. Immunofluorescence double-labeling revealed the downregulation of HCY metabolic enzymes in neurons of stressed mice. The transcription factor KLF4 (Kruppel-likefactor4), known for its inhibitory role, increased after stress or glucocorticoid treatment and suppressed the expression of MS, CBS, and CSE, contributing to elevated HCY levels in the brain. These findings offer new insights into the impairment of HCY catabolism in the stressed brain, suggesting that the downregulation of HCY metabolic enzymes may underlie HCY accumulation and exacerbate stress-induced cognitive dysfunction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 International Brain Research Organization (IBRO). Published by Elsevier Inc. All rights reserved.)

Selenium nanoparticles (SeNPs) have limited bioavailability because of their poor stability in aqueous solutions. AAGL, a naturally active protein, extracted from Agrocybe aegerita has strong antitumor activity. However, whether AAGL can been used to stabilize SeNPs, and exerts anti-lung cancer effects remains unknown. In this study, a novel nanocomposite, AAGL-SeNPs, was prepared using AAGL-encapsulated SeNPs. The particle size of the AAGL-SeNPs was approximately 206.1 nm, which was uniform and well dispersed in aqueous solution and showed satisfactory stability. AAGL-SeNPs was non-toxic and reduced the hepatotoxicity of AAGL in mice. Importantly, AAGL-SeNPs inhibited the proliferation of lung cancer cells and suppressed tumor growth in tumor-bearing mice. AAGL-SeNPs enhanced the cytotoxic effects on lung cancer cells by stimulating immune cells. In addition, the combination of AAGL-SeNPs and osimertinib inhibited lung cancer, and AAGL-SeNPs reversed osimertinib resistance in H1975 cells. Mechanistically, Krüppel-like transcriptional factor 4 (KLF4) was identified by data-independent acquisition mass spectrometry (DIA-MS), and its expression levels in lung cancer increased after AAGL-SeNPs treatment. This study demonstrated that nanocomposite AAGL-SeNPs is stable, safe, and has excellent antitumor efficacy, which will be a potential therapeutic drug for lung cancer treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In Parkinson's disease (PD), exogenous ghrelin protects dopaminergic neurons through its receptor, growth hormone secretagogue receptor (GHSR). However, in contrast to the strikingly low levels of ghrelin, GHSR is highly expressed in the substantia nigra (SN). What role does GHSR play in dopaminergic neurons is unknown. In this study, using GHSR knockout mice (Ghsr -/- mice) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model, we found that GHSR deletion aggravated dopaminergic neurons degeneration, and the expression and activity of GHSR were significantly reduced in PD. Furthermore, we explored the potential mechanism that GHSR deficiency aggregated PD-related neurodegeneration. We showed that DEPTOR, a subunit of mTORC1, was overexpressed in Ghsr -/- mice, positively regulating autophagy and enhancing autophagy initiation. The expression of lysosomal markers was abnormal, implying lysosomal dysfunction. As a result, the damaged mitochondria could not be effectively eliminated, which ultimately exacerbated the injury of nigral dopaminergic neurons. In particular, we demonstrated that DEPTOR could be transcriptionally regulated by KLF4. Specific knockdown of KLF4 in dopaminergic neurons effectively alleviated neurodegeneration in Ghsr -/- mice. In summary, our results suggested that endogenous GHSR deletion-compromised autophagy by impairing lysosomal function, is a key contributor to PD, which provided ideas for therapeutic approaches involving the manipulation of GHSR., Competing Interests: Declaration of competing interest The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Zhenjun Xu,1,* Kai Zhong,2,* Guanjun Guo,1,* Can Xu,1 Zhizhao Song,1 Dongjin Wang,1 Jun Pan1 1Department of Thoracic and Cardiovascular Surgery, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, 210008, People’s Republic of China; 2Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjing, 210008, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jun Pan; Zhenjun XuDepartment of Thoracic and Cardiovascular Surgery, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, 321 Zhongshan Road, Nanjing, 210008, People’s Republic of ChinaTel +86 25 68182222-60721Fax +86 25 83105117Email pj791028@163.com; xzj881225@163.comBackground: Aortic dissection (AD) is a threatening and catastrophic vascular disease with high mortality rate and limited therapeutic strategies. There is emerging evidence showing that circular RNAs play crucial role in regulating various cardiovascular diseases. However, the biological functions and molecular mechanisms of circRNAs in AD still remains elusive. The purpose of this study was to illustrate the potential functional roles and mechanisms of hsa_circ_TGFBR2 in vitro and in vivo.Methods: The vascular smooth muscle cells (VSMCs) and AD-VSMCs were isolated from normal aorta and AD tissues. The expression of circ_TGFBR2, miR-29a and KLF4 were detected by realtime polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH). Cell proliferation was assessed by CCK-8 assay, colony formation and EDU assay. Cell migration was evaluated through transwell assay. Dual-luciferase reporter assay and RNA pulldown were performed to identify the interaction between circ_TGFBR2 and miR-29a or between miR-29a and KLF4. The wild-type sequence of circ_TGFBR2 or KLF4 were cloned into the luciferase reporter plasmid, and the activity was measured using dual-luciferase reporter assay system. And for RNA pulldown, the relative RNA enrichment of circ_TGFBR2 and miR-29a were confirmed using RT-PCR. Western Blot measured the expression of phenotype switch-related proteins. AD rat model induced by β-aminopropionitrile monofumarate (BAPN) was used to verify the role and mechanism of circ_TGFBR2.Results: Circ_TGFBR2 inhibited cell proliferation and migration of AD-VSMCs cells. Overexpression of circ_TGFBR2 promoted the expression of contractile markers (α-SMA, SM22α) and inhibited the expression of synthetic markers (MGP, OPN) in AD-VSMCs cells. Circ_TGFBR2 served as a sponge for miR-29a targeting KLF4. MiR-29a mimics rescued biological roles induced by circ_TGFBR2 overexpression. The in vivo experiments revealed that overexpression of TGFBR2 suppressed the progression of AD and increased the expression of contractile markers while inhibited the expression of synthetic markers.Conclusion: Our study revealed that circ_TGFBR2 regulated VSMCs phenotype switch and suppressed the progression of AD.Keywords: aortic dissection, circ_TGFBR2, miR-29a, KLF4, vascular smooth muscle cells

Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow with 5-year overall survival of less than 10% in patients over the age of 65. Limited progress has been made in the patient outcome because of the inability to selectively eradicate the leukemic stem cells (LSC) driving the refractory and relapsed disease. Herein, we investigated the role of the reprogramming factor KLF4 in AML because of its critical role in the self-renewal and stemness of embryonic and cancer stem cells. Using a conditional Cre-lox Klf4 deletion system and the MLL-AF9 retroviral mouse model, we demonstrated that loss-of-KLF4 does not significantly affect the induction of leukemia but markedly decreased the frequency of LSCs evaluated in limiting-dose transplantation studies. Loss of KLF4 in leukemic granulocyte-macrophage progenitors (L-GMP), a population enriched for AML LSCs, showed lessened clonogenicity and percentage in the G2/M phase of the cell cycle. RNAseq analysis of purified L-GMPs revealed decreased expression of stemness genes and MLL-target genes and upregulation of the RNA sensing helicase DDX58. However, silencing of DDX58 in KLF4 knockout leukemia indicated that DDX58 is not mediating this phenotype. CRISPR/Cas9 deletion of KLF4 in MOLM13 cell line and AML patient-derived xenograft cells showed impaired expansion in vitro and in vivo associated with a defective G2/M checkpoint. Collectively, our data suggest a mechanism in which KLF4 promotes leukemia progression by establishing a gene expression profile in AML LSCs supporting cell division and stemness. KLF4 promotes leukemic stem cell survival in MLL-AF9 acute myeloid leukemia by regulating genes in the leukemic stem cell signature, MLL targets, and cell cycle. [ABSTRACT FROM AUTHOR]

Although apoptotic cells (ACs) contain nucleic acids that can be recognized by Toll-like receptors (TLRs), engulfment of ACs does not initiate inflammation in healthy organisms. Here we identified macrophage populations that continually engulf ACs in distinct tissues and found that these macrophages share characteristics compatible with immunologically silent clearance of ACs; such characteristics include high expression of AC recognition receptors, low expression of TLR9, and reduced TLR responsiveness to nucleic acids. Removal of the macrophages from tissues resulted in loss of many of these characteristics and the ability to generate inflammatory responses to AC-derived nucleic acids, suggesting that cues from the tissue microenvironment program macrophages for silent AC clearance. The transcription factors KLF2 and KLF4 control the expression of many genes within this AC clearance program. The coordinated expression of AC receptors with genes that limit responses to nucleic acids might ensure maintenance of homeostasis and thus represent a central feature of tissue macrophages.

Oct4, Sox2, Klf4, and cMyc (OSKM) reprogram somatic cells to pluripotency. To gain a mechanistic understanding of their function, we mapped OSKM-binding, stage-specific transcription factors (TFs), and chromatin states in discrete reprogramming stages and performed loss- and gain-of-function experiments. We found that OSK predominantly bind active somatic enhancers early in reprogramming and immediately initiate their inactivation genome-wide by inducing the redistribution of somatic TFs away from somatic enhancers to sites elsewhere engaged by OSK, recruiting Hdac1, and repressing the somatic TF Fra1. Pluripotency enhancer selection is a stepwise process that also begins early in reprogramming through collaborative binding of OSK at sites with high OSK-motif density. Most pluripotency enhancers are selected later in the process and require OS and other pluripotency TFs. Somatic and pluripotency TFs modulate reprogramming efficiency when overexpressed by altering OSK targeting, somatic-enhancer inactivation, and pluripotency enhancer selection. Together, our data indicate that collaborative interactions among OSK and with stage-specific TFs direct both somatic-enhancer inactivation and pluripotency-enhancer selection to drive reprogramming.

ABSTRACT The transmembrane protein heart of glass1 (HEG1) directly binds to and recruits Krev interaction trapped protein 1 (KRIT1) to endothelial junctions to form the HEG1–KRIT1 protein complex that establishes and maintains junctional integrity. Genetic inactivation or knockdown of endothelial HEG1 or KRIT1 leads to the upregulation of transcription factors Krüppel‐like factors 4 and 2 (KLF4 and KLF2), which are implicated in endothelial vascular homeostasis; however, the effect of acute inhibition of the HEG1–KRIT1 interaction remains incompletely understood. Here, we report a high‐throughput screening assay and molecular design of a small‐molecule HEG1–KRIT1 inhibitor to uncover acute changes in signaling pathways downstream of the HEG1–KRIT1 protein complex disruption. The small‐molecule HEG1–KRIT1 inhibitor 2 (HKi2) was demonstrated to be a bona fide inhibitor of the interaction between HEG1 and KRIT1 proteins, by competing orthosterically with HEG1 through covalent reversible interactions with the FERM (4.1, ezrin, radixin, and moesin) domain of KRIT1. The crystal structure of HKi2 bound to KRIT1 FERM revealed that it occupies the same binding pocket on KRIT1 as the HEG1 cytoplasmic tail. In human endothelial cells (ECs), acute inhibition of the HEG1–KRIT1 interaction by HKi2 increased KLF4 and KLF2 mRNA and protein levels, whereas a structurally similar inactive compound failed to do so. In zebrafish, HKi2 induced expression of klf2a in arterial and venous endothelium. Furthermore, genome‐wide RNA transcriptome analysis of HKi2‐treated ECs under static conditions revealed that, in addition to elevating KLF4 and KLF2 expression, inhibition of the HEG1–KRIT1 interaction mimics many of the transcriptional effects of laminar blood flow. Furthermore, HKi2‐treated ECs also triggered Akt signaling in a phosphoinositide 3‐kinase (PI3K)‐dependent manner, as blocking PI3K activity blunted the Akt phosphorylation induced by HKi2. Finally, using an in vitro colocalization assay, we show that HKi6, an improved derivative of HKi2 with higher affinity for KRIT1, significantly impedes recruitment of KRIT1 to mitochondria‐localized HEG1 in CHO cells, indicating a direct inhibition of the HEG1–KRIT1 interaction. Thus, our results demonstrate that early events of the acute inhibition of HEG1–KRIT1 interaction with HKi small‐molecule inhibitors lead to: (i) elevated KLF4 and KLF2 gene expression; and (ii) increased Akt phosphorylation. Thus, HKi’s provide new pharmacologic tools to study acute inhibition of the HEG1–KRIT1 protein complex and may provide insights to dissect early signaling events that regulate vascular homeostasis.

Background: In recent years, the role of micro-RNAs in the cancer pathophysiology has attracted a great deal of scientific attention. MiRNAs regulate a variety of cellular functions, such as apoptosis, differentiation and migration by targeting oncogenic or tumor suppressor genes. We conducted the current study to assess the expression of miR-107, Krüppel-like factor 4 (KLF4) and death-associated protein kinase (DAPK1) genes in malignant and normal colon tissues and also colorectal cancer (CRC) model cells exposed to oxaliplatin and 5-FU chemotherapy agents. Method: In this case-control study, the tissue samples from CRC patients were collected during colonoscopy process in 2013 -2016 at Imam Reza hospital. Subsequently, the expression levels of miR-107, KLF4, and DAPK1 were detected with quantitative Real-Time PCR. Furthermore, in the in vitro phase of this study, we investigated the changes in the expression level of miR-107, KLF4 and DAPK1 transcripts after oxaliplatin and 5-FU treatment. Results: Unlike miR-107, the expression levels of KLF4 and DAPK1 genes decreased in the tumor samples compared to those in the marginal specimens. In addition, both oxaliplatin and 5-FU significantly increased the expression level of miR-107. There were significant correlations between the expression levels of miR-107, KLF4, and DAPK1genes and clinicopathological features, for instance lymph node metastasis and cell differentiation. Conclusion: The current study suggested a tumor suppressor role for KLF4 and DAPK1 in CRC. The altered expression of miR-107, KLF-4, and DAPK1 genes in CRC tumors and healthy tissues could be utilized for CRC diagnosis and prognosis. Furthermore, the studied genes could be considered as potential therapeutic targets in CRC.

Inflammation is the response of the human body to injury, infection, or other abnormal states, which is involved in the development of many diseases. As a member of the Krüppel-like transcription factors (KLFs) family, KLF4 plays a crucial regulatory role in physiological and pathological processes due to its unique dual domain of transcriptional activation and inhibition. A growing body of evidence has demonstrated that KLF4 plays a pivotal role in the pathogenesis of various inflammatory disorders, including inflammatory bowel disease, osteoarthritis, renal inflammation, pneumonia, neuroinflammation, and so on. Consequently, KLF4 has emerged as a promising new therapeutic target for inflammatory diseases. This review systematically generalizes the molecular regulatory network, specific functions, and mechanisms of KLF4 to elucidate its complex roles in inflammatory diseases. An in-depth study on the biological function of KLF4 is anticipated to offer a novel research perspective and potential intervention strategies for inflammatory diseases. [Display omitted] • A variety of factors regulate KLF4 expression at diverse levels. • KLF4 can control cell proliferation, differentiation, and apoptosis, which is involved in the process of inflammation. • KLF4 represents a promising therapeutic target for the treatment of inflammation and related diseases. [ABSTRACT FROM AUTHOR]

Krüppel‐like factor 4 (KLF4) is a zinc‐finger transcription factor involved in inflammation, cancer development, and progression. However, the relationship between KLF4, inflammation, and prognosis in oral cancer is not fully understood. KLF4 expression levels were examined in a multicenter cohort of 128 oral squamous cell carcinoma (OSCC) specimens from the tongue (OTSCC) using immunohistochemistry. In two external KLF4 mRNA datasets (The Cancer Genome Atlas/The Genotype‐Tissue Expression Portal), lower KLF4 mRNA expression was found in OSCC and head and neck squamous cell carcinomas (HNSCC) than in control oral epithelium. These data indicate that down‐regulation of KLF4 mRNA is linked to OSCC/HNSCC progression. Using Cox‐multivariate analysis, a significantly favorable 5‐year disease‐specific survival rate was observed for a subgroup of patients with a combination of high levels of KLF4 expression and inflammation. OSCC cell lines exposed to IFN‐γ showed a significant upregulation of nuclear KLF4 expression, indicating a link between inflammation and KLF4 expression in OSCC. Overall, the current data suggest a functional link between KLF4 and inflammation. The combination of high KLF4 nuclear expression and marked/moderate stromal inflammation might be useful as a favorable prognostic marker for a subgroup of OTSCC patients. [ABSTRACT FROM AUTHOR]

Background: This study aimed to explore the effect and mechanism of iguratimod (IGT) on M1 macrophage polarization and antibody-mediated rejection (ABMR) after renal transplant. Methods: Bioinformatics analysis was performed using three public databases derived from the GEO database. Sprague–Dawley (SD) rats were pre-sensitized with donors of Wistar rats in skin transplantation and a rat renal transplant ABMR model was established from the donors to skin pre-sensitized recipients. Subsequently, IGT was treated on the ABMR model. Routine staining and immunofluorescence (IF) staining were performed to observe the pathological changes in each group and flow cytometry was performed to detect the changes of DSA titers in peripheral blood. In addition, bone-marrow-derived macrophage (BMDM) was extracted and interfered with IGT to explore the effect of IGT in vivo. PCR, IF staining, and Western blot were used to detect the expression of related genes and proteins. Results: Bioinformatics analysis revealed that several immune cells were significantly infiltrated in the ABMR allograft, while M1 macrophage was noticed with the most significance. Results of IF staining and PCR proved the findings of the bioinformatics analysis. Based on this, IGT was observed to significantly attenuate the degree of peritubular capillary vasculitis and arteriolitis in the rat renal transplant ABMR model, whereas it decreases the expression of C4d and reduces the titer of DSA. Results in vitro suggested that M1 macrophage-related transcripts and proteins were significantly reduced by the treatment of IGT in a dose- and time-dependent manner. Furthermore, IGT intervention could remarkably decrease the expression of KLF4. Conclusion: Polarization of M1 macrophages may aggravate ABMR after renal transplant by promoting DSA-mediated endothelial cell injury, and IGT may attenuate the pathogenesis of ABMR by targeting KLF4. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is a very common life-threatening malignancy. Transcription factor-like 5 (TCFL5) has been suggested to be involved in CRC. Here, we describe the expression of four alternative transcripts of TCFL5 and their relevance in CRC. Complete deletion of all isoforms drastically decreased pro-tumoural properties such as spheroids formation and in vivo tumour growth, although increased migration in CRC cell lines. Overexpression of the two main isoforms, TCFL5_E8 and CHA, had opposite effects: TCFL5_E8 reduced proliferation and spheroids formation, while CHA increased them. TCFL5_E8 reduced in vivo tumour formation, while CHA had no effect. In addition, TCFL5_E8 and CHA have different roles in the regulation of the pluripotency-related genes SOX2 and KLF4. Both isoforms bind directly to their promoters; however, TCFL5_E8 induced SOX2 and reduced KLF4 mRNA levels, whereas CHA did the opposite. Together, our results show that TCFL5 plays an important role in the development of CRC, being however isoform-specific. This work also points to the need to analyse separately TCFL5 isoforms in cancer, due to their different and opposite functions. [ABSTRACT FROM AUTHOR]

Vascular smooth muscle cell (VSMC) proliferation and migration play key roles in the progression of atherosclerosis and restenosis. A variety of ginsenosides exert various cardiovascular benefits. However, whether and how ginsenoside Rh1 (Rh1) inhibits VSMC dysfunction remain unclear. Here, we investigated the inhibitory effects of Rh1 on rat aortic smooth muscle cell (RASMC) migration and proliferation induced by angiotensin II (Ang II) and the underlying mechanisms. Cell proliferation and migration were evaluated using sulforhodamine B and wound-healing assay. The molecular mechanisms were investigated using Western blotting, quantitative reverse-transcription polymerase chain reaction analysis, immunofluorescence staining, and luciferase assay. Reactive oxygen species (ROS) production was measured using dihydroethidium and MitoSOX staining. We found that Rh1 dose-dependently suppressed Ang II-induced cell proliferation and migration. Concomitantly, Ang II increased protein levels of osteopontin, vimentin, MMP2, MMP9, PCNA, and cyclin D1, while these were reduced by Rh1 pretreatment. Notably, Ang II enhanced both the protein expression and promoter activity of KLF4, a key regulator of phenotypic switching, whereas pretreatment with Rh1 reversed these effects. Mechanistically, the effects of Rh1 on VSMC proliferation and migration were found to be associated with inhibition of ERK1/2/p90RSK signaling. Furthermore, the inhibitory effects of Rh1 were accompanied by inhibition of ROS production. In conclusion, Rh1 inhibited the Ang II-induced migration and proliferation of RASMCs by suppressing the ROS-mediated ERK1/2/p90RSK signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract Background Ovarian cancer stem cells (OCSC), endowed with tumor-initiating and self-renewal capacity, would account not only for the tumor growth, the peritoneal metastasis, and the relapse, but also for the acquisition of chemotherapy resistance. Nevertheless, figuring out their phenotypical and functional traits has proven quite challenging, mainly because of the heterogeneity of ovarian cancer. A deeper understanding of OCSC mechanisms will shed light on the development of the disease. Therefore, we aim to explore it for the design of innovative treatment regimens which aim at the eradication of ovarian cancer through the elimination of the CSC component. Methods In this study, immunohistochemistry assay and western blot assay were used to detect protein expression in the primary tumor and peritoneal multi-cellular aggregates/spheroids (MCAs/MCSs). OCSCs induced from cell line SKOV3 and HO-8910 were enriched in a serum-free medium (SFM). The effect of EIF5A2 on CSC-like properties was detected by sphere-forming assays, re-differentiation assays, quantitative real-time polymerase chain reaction, western blotting, flow cytometry, cell viability assays, immunofluorescence staining, and in vivo xenograft experiments. RNA-sequencing (RNA-seq) was used to reveal the mechanism by which EIF5A2 positively modulates the stem-like properties of ovarian cancer cells. Results Expression of EIF5A2 was significantly higher in peritoneal MCAs/MCSs compared to matched primary tumors, and EIF5A2 was also unregulated in ovarian cancer cell line-derived spheroids. Knockdown of EIF5A2 reduced the expression of the stem-related markers (ALDH1A1 and OCT-4), inhibited self-renewal ability, improved the sensitivity to chemotherapeutic drugs, and inhibited tumorigenesis in vivo. Mechanistic studies revealed that EIF5A2 knockdown reduced the expression of KLF4, which could partially rescue stem-like properties abolished by EIF5A2 knockdown or strengthened by EIF5A2 overexpression, through the transcription factor E2F1, which directly bind to KLF4 promoter. Conclusion Our results imply that EIF5A2 positively regulates stemness in ovarian cancer cells via E2F1/KLF4 pathway and may serve as a potential target in CSCs-targeted therapy for ovarian cancer.

Objective To investigate the role of LncRNAs-MEG3 in the carcinogenesis and progression of nasopharyngeal carcinoma (NPC) and the possible molecular mechanism. Methods qRT-PCR was used to detect the content of MEG3 and miR-543 in NPC cells. Luciferase reporter method was used to study the relation between MEG3 and miR-543, and the changes of cell proliferation and apoptosis induced by MEG3 or KLF4 were analyzed. Western blot was used to detect the expression of KLF4, Bcl-2 and Bax proteins. Results Compared with the control group, the expression of miR-543 in NPC cell line was significantly increased (P < 0.05), while the expression of MEG3 was decreased (P < 0.05). Luciferase report and Western blot showed that MEG3 could regulate the expression of KLF4 by adsorbing miR-543 to inhibit cell proliferation, promote cell apoptosis and affect the expression levels of Bcl-2 and Bax proteins. Conclusion LncRNA-MEG3 could regulate the expression of KLF4 by adsorbing miR-543 and then plays a role in inhibiting the occurrence and development of NPC. It may be a new biomarker for NPC targeted therapy.

Abstract Background Previous research has highlighted the ability of Homeobox A10 (HOXA10) to the promote proliferation, migration, and epithelial-mesenchymal transformation of various cancers, including lung adenocarcinoma (LAD), which is characterized by an aggressive disease course that exhibits rapid proliferation and migration, with studies suggesting histone deacetylase 1 (HDAC1) to be a downstream mediator of HOXA10. The current study aimed to investigate the mechanism by which HOXA10-mediated HDAC1 influences the development of LAD. Methods The expression patterns of HOXA10, HDAC1, DNA methyltransferase 1 (DNMT1), and Kruppel-like factor 4 (KLF4) were determined. Additionally, the effect of HOXA10, HDAC1, or DNMT1 on invasive phenotypes of LAD was analyzed using depletion experiments. The interactions among HOXA10, HDAC1, DNMT1, and KLF4 were evaluated via chromatin immunoprecipitation, dual luciferase assay or co-immunoprecipitation. Furthermore, the tumorigenic ability of the LAD cells following HOXA10 silencing and/or HDAC1 overexpression in vivo was also investigated. Results In the LAD tissues and cells, HOXA10, HDAC1, and DNMT1 all exhibited high levels of expression, while KLF4 was poorly expressed. HOXA10 silencing inhibited the expression of HDAC1, reduced LAD cell proliferation, migration, and invasion, and promoted the apoptosis. HDAC1 promoted DNMT1 expression through deacetylation, and DNMT1 inhibited the KLF4 expression through DNA methyltransferase. The in vitro findings were further attested through the use of in vivo assays. Conclusion Taken together, the key observations of the current study highlight the role of HOXA10 and HDAC1 in promoting the proliferation and migration of LAD cells. HOXA10-induced upregulation of HDAC1 interacts with DNMT1-KLF4 axis, while the inhibition of HOXA10 or HDAC1 represents a promising anti-tumor therapy target for LAD.

Abstract Background Numerous signaling pathways have been demonstrated experimentally to affect the pathogenesis of cerebral cavernous malformations (CCM), a disease that can be caused by CCM3 deficiency. However, the understanding of the CCM progression is still limited. The objective of the present work was to elucidate the role of CCM3 by RNA-seq screening of CCM3 knockout mice. Results We found that ATPIF1 was decreased in siCCM3-treated Human Umbilical Vein Endothelial Cells (HUVECs), and the overexpression of ATPIF1 attenuated the changes in cell proliferation, adhesion and migration caused by siCCM3. The probable mechanism involved the conserved ATP concentration in mitochondria and the elongated morphology of the organelles. By using the CRISPR-cas9 system, we generated CCM3-KO Endothelial Progenitor Cells (EPCs) and found that the knockout of CCM3 destroyed the morphology of mitochondria, impaired the mitochondrial membrane potential and increased mitophagy. Overexpression of ATPIF1 contributed to the maintenance of normal structure of mitochondria, inhibiting activation of mitophagy and other signaling proteins (e.g., KLF4 and Tie2). The expression of KLF4 returned to normal in CCM3-KO EPCs after 2 days of re-overexpression of CCM3, but not other signaling proteins. Conclusion ATPIF1 maintains the normal structure of mitochondria, inhibiting the activation of mitophagy and other signaling pathway in endothelial cells. Loss of CCM3 leads to the destruction of mitochondria and activation of signaling pathways, which can be regulated by KLF4.

Differential expression of non-coding RNA after traumatic spinal cord injury (TSCI) is closely related to the pathophysiological process. The purposes of this study were to systematically profile and characterize expression of circular RNA (circRNA) in the lesion epicenter of spinal tissues after TSCI, and predict the structure and potential function of the regulatory circRNA/miRNA network. Forty-eight C57BL/6 mice were randomly and equally assigned to two groups: one subjected to TSCI at T8–10 with an Allen’s drop impactor, and a second subjected to laminectomy without TSCI. Spinal cord samples were stained with hematoxylin and eosin, sequenced, and validated. RNA-Seq, Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, and network analyses (Targetscan and miRanda) were used to predict and annotate the circRNA/miRNA/mRNA network. Luciferase reporter, quantitative reverse transcription polymerase chain reaction, and western blot assays were used to profile expression and regulation patterns of the network in mouse models of TSCI. Hematoxylin-eosin staining revealed severe damage to the blood-spinal cord barrier after TSCI. Differentially expressed circRNA and miRNA profiles were obtained after TSCI; differentially expressed circRNAs, which were abundant in the cytoplasm, were involved in positive regulation of transcription and protein phosphorylation. miR-135b-5p was the most significantly downregulated miRNA after TSCI; circRNAAbca1 and KLF4 were predicted to be its target circRNA and mRNA, respectively. Subsequently, the circAbca1/miR-135b-5P/KLF4 regulatory axis was predicted and constructed, and its targeted binding was verified. After inhibiting circAbca1, GAP43 expression was upregulated. Differential expression of circRNAs might play an important role after TSCI. circAbca1 plays a neuroinhibitory role by targeted binding of the miR-135b-5P/KLF4 axis. The identified circRNA/miRNA/mRNA network could provide the basis for understanding pathophysiological mechanisms underlying TSCI, as well as guide the formulation of related therapeutic strategies. All animal protocols were approved by the Research Ethics Committee of West China Hospital of China (approval No. 2017128) on May 16, 2017.

Axonal regeneration plays an important role in functional recovery after nervous system damage. However, after axonal injury in mammals, regeneration is often poor. The deletion of Krüppel-like factor-4 (Klf4) has been shown to promote axonal regeneration in retinal ganglion cells. However, the effects of Klf4 deletion on the corticospinal tract and peripheral nervous system are unknown. In this study, using a mouse model of sciatic nerve injury, we show that the expression of Klf4 in dorsal root ganglion sensory neurons was significantly reduced after peripheral axotomy, suggesting that the regeneration of the sciatic nerve is associated with Klf4. In vitro, dorsal root ganglion sensory neurons with Klf4 knockout exhibited significantly enhanced axonal regeneration. Furthermore, the regeneration of the sciatic nerve was enhanced in vivo following Klf4 knockout. Finally, AAV-Cre virus was used to knockout the Klf4 gene in the cortex. The deletion of Klf4 enhanced regeneration of the corticospinal tract in mice with spinal cord injury. Together, our findings suggest that regulating KLF4 activity in neurons is a potential strategy for promoting axonal regeneration and functional recovery after nervous system injury. This study was approved by the Animal Ethics Committee at Soochow University, China (approval No. SUDA20200316A01).

Sorafenib, a multikinase inhibitor, has been widely used as a first-line anticancer drug for advanced hepatocellular carcinoma (HCC). However, the development of drug resistance to sorafenib is frequently observed in clinical applications. Potential nonkinase targets of sorafenib have not been well documented and may provide insights into reversing drug resistance and enhancing drug efficacy. Herein, we report that sorafenib exerts its anticancer effects by activating metallothionein 1 G (MT1G) expression. MT1G is a novel marker in HCC that correlates well with patient survival. MT1G overexpression suppressed the cellular proliferation, migration, invasion, and tumour formation of HCC and sensitised cells to sorafenib treatment. However, the disruption of MT1G attenuated the anticancer effects of sorafenib. Mechanistically, sorafenib upregulated MT1G expression via hypomethylation of its promoter region by binding and inhibiting DNA methyltransferase 1 (DNMT1) and increasing its promoter accessibility in HCC cells. Activation of MT1G also inhibited CA9 transcription through the suppression of HIF1A as mediated by KLF4. Our collective data revealed that sorafenib exerts its anticancer effects through epigenetic regulation of the DNMT1/MT1G/KLF4/CA9 axis in HCC and the activation of MT1G might constitute a strategy for enhancing the effect of sorafenib to suppress HCC cells.

Background: This study aimed to explore the effect and mechanism of iguratimod (IGT) on M1 macrophage polarization and antibody-mediated rejection (ABMR) after renal transplant.Methods: Bioinformatics analysis was performed using three public databases derived from the GEO database. Sprague–Dawley (SD) rats were pre-sensitized with donors of Wistar rats in skin transplantation and a rat renal transplant ABMR model was established from the donors to skin pre-sensitized recipients. Subsequently, IGT was treated on the ABMR model. Routine staining and immunofluorescence (IF) staining were performed to observe the pathological changes in each group and flow cytometry was performed to detect the changes of DSA titers in peripheral blood. In addition, bone-marrow-derived macrophage (BMDM) was extracted and interfered with IGT to explore the effect of IGT in vivo. PCR, IF staining, and Western blot were used to detect the expression of related genes and proteins.Results: Bioinformatics analysis revealed that several immune cells were significantly infiltrated in the ABMR allograft, while M1 macrophage was noticed with the most significance. Results of IF staining and PCR proved the findings of the bioinformatics analysis. Based on this, IGT was observed to significantly attenuate the degree of peritubular capillary vasculitis and arteriolitis in the rat renal transplant ABMR model, whereas it decreases the expression of C4d and reduces the titer of DSA. Results in vitro suggested that M1 macrophage-related transcripts and proteins were significantly reduced by the treatment of IGT in a dose- and time-dependent manner. Furthermore, IGT intervention could remarkably decrease the expression of KLF4.Conclusion: Polarization of M1 macrophages may aggravate ABMR after renal transplant by promoting DSA-mediated endothelial cell injury, and IGT may attenuate the pathogenesis of ABMR by targeting KLF4.

Objectives: Long non-coding RNAs (lncRNAs) are key regulators involved in the progression of glioma, and many functional lncRNAs are yet to be identified. This study aimed to explore the function of CHRM3-AS2, a rarely reported lncRNA, in glioma, as well as the underlying mechanisms involving miR-370-5p/KLF4. Methods: Differentially expressed RNAs (DERs) were screened from two gene expression profiles of glioblastoma (GBM). Fluorescence in situ hybridisation was performed to determine the subcellular localisation of CHRM3-AS2. Cell viability, colony formation, apoptosis, migration, and invasion were evaluated using cell counting kit-8, colony counts, flow cytometry, wound healing, and Transwell assays, respectively. mRNA and protein expression of specific genes were measured using quantitative real-time polymerase chain reaction and western blotting, respectively. Dual luciferase reporter gene, RNA immunoprecipitation, and RNA pull-down assays were performed to identify the target relationships. A mouse xenograft model was established for in vivo validation. Results: CHRM3-AS2 was screened as a prognosis-associated DER in GBM. CHRM3-AS2 expression was up-regulated in glioma cells, and CHRM3-AS2 was localised in the cytoplasm. Silencing of CHRM3-AS2 expression inhibited cell viability, colony formation, migration, and invasion and promoted apoptosis of U251 and SHG-44 cells. In addition, CHRM3-AS2 targeted miR-370-5p/KLF4 in glioma cells. The anti-tumour effect of CHRM3-AS2 silencing was weakened by miR-370-5p silencing or KLF4 overexpression. In vivo , silencing of CHRM3-AS2 expression inhibited tumour growth and Ki67 expression in mice. Overexpression of KLF4 also weakened the anti-tumour effect of CHRM3-AS2 silencing in mice. Conclusions: Silencing of CHRM3-AS2 expression inhibited the malignant progression of glioma by regulating miR-370-5p/KLF4 expression. [ABSTRACT FROM AUTHOR]

Background: Lung cancer results in the highest mortality associated with cancer worldwide. Non‐small cell cancer (NSCLC) is the leading subtype of lung cancer. Ovarian tumor protease (OTU) domain‐containing protein 1 (OTUD1) is a member of the OTU subfamily of DUBs, and its function in NSCLC remains unclear. Methods: GEPIA database was employed to reveal the expression level of OTUD1 in addition to Krüppel‐ like factor 4 (KLF4) in NSCLC tissue samples and prove the correlation between OTUD1 and KLF4. The protein level was estimated using western blot. Cell counting kit‐8 (CCK‐8) assay was used to detect cell viability and transwell assay was utilized to observe cell migration and invasion. Cycloheximide (CHX) was introduced to measure half‐lives of KLF4 and deubiquitination assay was used to detect deubiquitination ability of OTUD1. Results: OTUD1 expression was downregulated in NSCLC tissues and cells. Overexpression of OTUD1 inhibited NSCLC cell progression and it was promoted by knockdown of OTUD1. OTUD1 was positively correlated with KLF4 and stabilized KLF4 at protein level by deubiquitinating KLF4. Overexpressing KLF4 dramatically eliminated the effects of OTUD1 on the development of NSCLC cells. Conclusions: Our study revealed that OTUD1 suppresses NSCLC progression by mediating KLF4 stabilization, which suggests a potential gene target for the future treatment of NSCLC. [ABSTRACT FROM AUTHOR]

• MiR-92 inhibition promoted macrophage polarization to M2 state in vitro and in vivo. • MiR-92 knockdown alleviated plaque size and increased stability. • KLF4 was required for miR-92 silencing-mediated macrophage polarization. Atherosclerosis is a chronic inflammatory disease in which macrophage polarization plays an important role in contribution to atherosclerotic plaque formation and stability. Here we tested the effect of miR-92 regulation on the development of atherosclerosis beyond tumorigenesis and explored the potential mechanism. In the present study, bone marrow derived macrophages (BMDMs), mouse peritoneal macrophages (MPMs), and human macrophages were used to test the expression of miR-92. Here we noticed miR-92 levels were enhanced in classic M1 macrophage but decreased in alternative M2 macrophage, respectively. In vitro, we demonstrated that macrophages transfected with miR-92 inhibitor attenuated proinflammatory cytokine secretion represented by polarized M1 markers but promoted anti-inflammatory state that was indicative of an M2 phenotype. Mechanistically, miR-92 was found to directly interact with KLF4 and we further identified a requirement role of KLF4 in mediating the effect of miR-92 silencing macrophage polarization. Concomitantly, miR-92 inhibition treated ApoE−/− mice promoted macrophage polarization toward alternative M2 macrophage, thus protecting against atherosclerotic plaque formation and preventing a vulnerable phenotype. miR-92 inhibition promoted alternative macrophage activation and attenuated atherosclerosis regression partially regulated in a KLF4-dependent manner, which indicated that miR-92/KLF4 axis may serve as a promising strategy for prevention of atherosclerotic diseases. We showed herein that miR-92 contributed to the development of atherosclerosis by directly controlling macrophage polarization. Inhibition of miR-92 downregulated markers that represent classical activated macrophages but increased alternative activated macrophages markers. Moreover, miR-92 inhibitor treated ApoE-/- mice fed with HFD leaded to alleviated atherosclerotic plaque size and increased stability characterizes. Mechanistically, miR-92 can directly target KLF4 which was required for the macrophage polarization mediated by miR-92 silencing. [Display omitted] [ABSTRACT FROM AUTHOR]

Background and Aims: High glucose and its byproducts are important factors causing dysfunction of endothelial cells. Autophagy is critical for endothelial cellular homeostasis. However, the specific molecular mechanism of how autophagy is regulated in endothelial cells under high-glucose condition remains unknown. We aim to explore the role Sirt6 plays in regulating autophagy in AGE-treated endothelial cells and how this function is exerted via KLF4.Methods and Results: Our results indicate that autophagy level increased in AGE-treated endothelial cells alongside with higher Sirt6 and KLF4 expression level. What's more, knock-in of Sirt6 by adenovirus led to augmented autophagy level while knockdown of Sirt6 led to the opposite. We also verified that Sirt6 affected KLF4 expression positively but KLF4 didn't influence Sirt6 expression level while knocking out of KLF4 impaired Sirt6-enhanced autophagy. Finally we found that STZ-induced diabetic mice showed more autophagosomes in endothelium and Sirt6 knockdown by adeno-associated virus reduced the number of autophagosomes. Knockdown of Sirt6 also caused impaired endothelium integrity but echocardiography indicated there were no significant functional differences.Conclusion: Our research reveals more about how Sirt6 regulates autophagy in endothelial cells under high-glucose simulated condition and provides further insight into the relationships between Sirt6 and KLF4. [ABSTRACT FROM AUTHOR]

Background: Kruppel-like factor 4 (KLF4) is a zinc finger-containing transcription factor predominantly expressed in terminally differentiated epithelial tissues. Many studies have shown that KLF4 has various mechanisms in different tumours; however, the prognostic role of KLF4 remains unclear.Methods and Results: We searched the relevant literature that evaluated the prognostic value of KLF4 in different cancers, and the original survival data were obtained from the text, tables or Kaplan-Meier curves for both comparative groups. Thirty studies were included in this meta-analysis, and a total of 10 malignant tumours were involved. The expression of KLF4 was not associated with the prognosis for overall survival (hazard ratio(HR)0.86, 95% confidence interval (CI): 0.65-1.13, P = 0.28), disease-free survival/recurrence-free survival/metastasis-free survival (HR 0.87, 95% CI: 0.52-1.44, P = 0.58) or disease-specific survival (HR 1.13, 95% CI: 0.44-2.87, P = 0.8).Conclusion: This study showed that the expression of KLF4 was not related to the prognosis of the tumours that were included in the study. [ABSTRACT FROM AUTHOR]

circular RNAs (circRNAs) is a broad and diverse endogenous subfamily of non-coding RNAs, regulating the gene expression by acting as a microRNA (miRNA) sponge. However, the biological functions of circRNAs in odontoblast differentiation remain largely unknown. Our preliminary study identified an unknown mouse circRNA by circRNA sequencing generated from mouse dental papilla and we termed it circKLF4. In this study, quantitative real-time PCR and in situ hybridization were used and demonstrated that circKLF4 was upregulated during odontoblastic differentiation. Gene knockdown and overexpression assays indicated that circKLF4 promoted odontoblastic differentiation of mouse dental papilla cells (mDPCs). Mechanistically, we found that circKLF4 increased the linear KLF4 expression in a microRNA-dependent manner. By mutating the binding sites of microRNA and circKLF4, we further confirmed that circKLF4 acted as sponge of miRNA-1895 and miRNA-5046 to promote the expression of KLF4. We then also found that ENDOGLIN was also up-regulated by circKLF4 by transfection of circKLF4 overexpression plasmids with or without microRNA inhibitor. In conclusion, circKLF4 increases the expression of KLF4 and ENDOGLIN to promote odontoblastic differentiation via sponging miRNA-1895 and miRNA-5046. [ABSTRACT FROM AUTHOR]

Recent studies have confirmed the existence and key roles of microRNA (miRNAs) in cancer drug resistance, including cervical cancer (CC). The present study aims to establish a novel role for miR-92a-3p and its associated gene networks in cisplatin (DDP) resistance of CC. First, the disparities in miRNA expression between CC tissues and adjacent normal tissues were screened based on GSE19611 microarray data that retrieved from Gene Expression Omnibus (GEO), and we identified several miRs that were significantly downregulated or upregulated in CC tissues including miR-92a-3p. Moreover, miR-92a-3p was significantly up-regulated in DDP-resistant cells and was the most differently expressed miRNA. Functionally, knockdown of miR-92a-3p increased the sensitivity of DDP-resistant cells to DDP via inhibiting cell proliferation, migration and invasion, and promoting apoptosis. Conversely, overexpression of miR-92a-3p significantly induced DDP resistance in CC parental cells including HeLa and SiHa cells. Moreover, Krüppel-like factor 4 (KLF4) was identified as a direct target of miR-92a-3p, and an obvious inverse correlation was observed between the expression of miR-92a-3p and KLF4 in 40 pairs of cancer tissues. Furthermore, KLF4 knockdown reversed the promoting effect of miR-92a-3p inhibition on DDP sensitivity in DDP-resistant CC cells. Besides, high expression of miR-92a-3p was associated with DDP resistance, as well as a short overall survival in clinic. Taken together, these findings provide important evidence that miR-92a-3p targets KLF4 and is significant in DDP resistance in CC, indicating that miR-92a-3p may be an attractive target to increase DDP sensitivity in clinical CC treatment. [ABSTRACT FROM AUTHOR]

During reprogramming of somatic cells, heightened proliferation is one of the earliest changes observed. While other early events such as mesenchymal-to-epithelial transition have been well studied, the mechanisms by which the cell cycle switches from a slow cycling state to a faster cycling state are still incompletely understood. To investigate the role of Oct-3/4 in this early transition, we created a 4-Hydroxytamoxifen (OHT) dependent Oct-3/4 Estrogen Receptor fusion (OctER). We confirmed that OctER can substitute for Oct-3/4 to reprogram mouse embryonic fibroblasts to a pluripotent state. During the early stages of reprograming, Oct-3/4 and Klf4 individually did not affect cell proliferation but in combination hastened the cell cycle. Using OctER + Klf4, we found that proliferative enhancement is OHT dose-dependent, suggesting that OctER is the driver of this transition. We identified Cyclin A2 as a likely target of Oct-3/4 + Klf4. In mESC, Klf4 and Oct-3/4 bind ∼100bp upstream of Cyclin A2 CCRE, suggesting a potential regulatory role. Using inducible OctER, we show a dose-dependent induction of Cyclin A2 promoter-reporter activity. Taken together, our results suggest that Cyclin A2 is a key early target during reprogramming, and support the view that a rapid cell cycle assists the transition to pluripotency. • Created a functional tamoxifen-inducible Oct-3/4 estrogen receptor fusion protein (OctER). • OctER along with Klf4 enhances MEF proliferation during early reprogramming. • Increase in MEF proliferation is due to dose-dependent induction of Cyclin A2. • Oct-3/4 and Klf4 occupy the Cyclin A2 promoter in mESC. • Suggests early enhancement of cell cycle in reprogramming MEFs is linked with Oct-3/4 and Klf4 targeting of Cyclin A2. [ABSTRACT FROM AUTHOR]

Interleukin-6 (IL-6) overproduction has been considered to contribute to inflammatory damage of glomerular mesangial cells (GMCs) in human mesangial proliferative glomerulonephritis (MsPGN) and its rat model called Thy-1 nephritis (Thy-1N). However, the regulatory mechanisms of IL-6 expression in GMCs upon sublytic C5b-9 timulation remain poorly understood. We found that Krüppel-like factor 4 (KLF4) bound to the IL-6 promoter (−618 to −126 nt) and activated IL-6 gene transcription. Furthermore, lysine residue 224 of KLF4 was acetylated by p300/CBP-associated factor (PCAF), which was important for KLF4-mediated transactivation. Moreover, lysine residue 5 on histone H2B and lysine residue 9 on histone H3 at the IL-6 promoter were also acetylated by PCAF, which resulted in an increase in IL-6 transcription. Besides, NF-κB activation promoted IL-6 expression by elevating the expression of PCAF. Overall, these findings suggest that sublytic C5b-9-induced the expression of IL-6 involves KLF4-mediated transactivation, PCAF-mediated acetylation of KLF4 and histones, and NF-κB activation in GMCs. [ABSTRACT FROM AUTHOR]

Abstract KLF4 plays a critical role in determining cell fate responding to various stresses or oncogenic signaling. Here, we demonstrated that KLF4 is tightly regulated by poly(ADP‐ribosyl)ation (PARylation). We revealed the subcellular compartmentation for KLF4 is orchestrated by PARP1‐mediated PARylation. We identified that PARylation of KLF4 is critical to govern KLF4 transcriptional activity through recruiting KLF4 from soluble nucleus to the chromatin. We mapped molecular motifs on KLF4 and PARP1 that facilitate their interaction and unveiled the pivotal role of the PBZ domain YYR motif (Y430, Y451 and R452) on KLF4 in enabling PARP1‐mediated PARylation of KLF4. Disruption of KLF4 PARylation results in failure in DNA damage response. Depletion of KLF4 by RNA interference or interference with PARP1 function by KLF4YYR/AAA (a PARylation‐deficient mutant) significantly sensitizes breast cancer cells to PARP inhibitors. We further demonstrated the role of KLF4 in modulating homologous recombination through regulating BRCA1 transcription. Our work points to the synergism between KLF4 and PARP1 in tumorigenesis and cancer therapy, which provides a potential new therapeutic strategy for killing BRCA1‐proficient triple‐negative breast cancer cells.

Accumulating evidence suggests that dysregulation of long non-coding RNAs is closely associated with various human diseases, including Parkinson’s disease (PD). However, the role of nuclear-enriched abundant transcript 1 (NEAT1) in the PD process remains unclear. The number of TH+ cells was reduced, and the expression levels of NEAT1 and Krüppel-like factor 4 (KLF4) were increased in the midbrain of MPTP-HCl-treated mice. In addition, the expression of cleaved-caspase-3 (cleaved-casp-3) and Bax (apoptosis-related proteins) was increased, while the expression of Bcl-2 (anti-apoptotic protein) was reduced in MPTP-HCl-treated mice. The expression levels of NEAT1 and KLF4 were increased in MPP+-treated SH-SY5Y cells. Knockdown of NEAT1 promoted cell viability and decreased apoptosis in MPP+-treated SH-SY5Y cells, which could be reversed by upregulating KLF4. KLF4 was verified as a direct target of miR-124, and miR-124 could particularly bind to NEAT1. Downregulation of NEAT1 significantly increased cell viability and decreased apoptosis by regulating miR-124 expression in MPP+-treated SH-SY5Y cells. Additionally, interference of NEAT1 increased the number of TH+ cells and miR-124 expression, while reduced apoptosis and expression of KLF4 in vivo. NEAT1 knockdown increased cell viability and suppressed apoptosis in PD via regulating the miR-124/KLF4 axis, providing a promising avenue for the treatment of PD.

Abstract Background Clinically relevant methods are not available that prioritize and validate potential therapeutic targets for individual tumors, from the vast amount of tumor descriptive expression data. Methods We established inducible transgene expression in clinically relevant patient-derived xenograft (PDX) models in vivo to fill this gap. Results With this technique at hand, we analyzed the role of the transcription factor Krüppel-like factor 4 (KLF4) in B-cell acute lymphoblastic leukemia (B-ALL) PDX models at different disease stages. In competitive preclinical in vivo trials, we found that re-expression of wild type KLF4 reduced the leukemia load in PDX models of B-ALL, with the strongest effects being observed after conventional chemotherapy in minimal residual disease (MRD). A nonfunctional KLF4 mutant had no effect on this model. The re-expression of KLF4 sensitized tumor cells in the PDX model towards systemic chemotherapy in vivo. It is of major translational relevance that azacitidine upregulated KLF4 levels in the PDX model and a KLF4 knockout reduced azacitidine-induced cell death, suggesting that azacitidine can regulate KLF4 re-expression. These results support the application of azacitidine in patients with B-ALL as a therapeutic option to regulate KLF4. Conclusion Genetic engineering of PDX models allows the examination of the function of dysregulated genes like KLF4 in a highly clinically relevant translational context, and it also enables the selection of therapeutic targets in individual tumors and links their functions to clinically available drugs, which will facilitate personalized treatment in the future.

Background: Anthocyanins (ACNs) are capable of suppressing breast cancer growth; however, investigation on the effect and mechanism of ACNs on epithelial-to-mesenchymal transition (EMT), and cell migration and invasion in breast cancer cells is limited. A complete understanding of those properties may provide useful information on of how to use these natural compounds for cancer prevention and treatment. Objectives: The aim of this work was to investigate the role of cyanidin-3-O-glucoside (Cy3G), one of the most widely distributed ACNs in edible fruits, in the EMT process, and cell migration and invasion of breast cancer cells, and its underlying molecular mechanisms of how Cy3G establishes these functional roles in these cells. Methods: MDA-MB-231 and MDA-MB-468 breast cancer cells were treated with Cy3G (20 μM) for 24 h, and then the cells were used for cell migration and invasion assay. Western blotting, luciferase assay, ubiquitination assay, gene knockdown, and cycloheximide chase assay were performed to analyze the molecular mechanisms of Cy3G in suppressing EMT, and cell migration and invasion. Results: Cy3G inhibited the EMT process in these cells and significantly suppressed the migration and invasion of breast cancer cells (P ≤ 0.05) by upregulating Krüppel-like factor 4 (KLF4) expression at protein level. KLF4 knockdown in MDA-MB-231 cells did not reveal any change in EMT marker expression, and cell migration and invasion upon treatment with Cy3G (P ≥ 0.05), which strongly indicated that the effects of Cy3G were mediated by KLF4. Furthermore, we determined that Cy3G indirectly upregulated KLF4 expression by downregulating FBXO32, which is the E3 ligase of KLF4. Conclusion: Cy3G is a potential anticancer reagent as it can inhibit EMT and breast cancer cell migration and invasion by upregulating KLF4.

Abstract 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.

In vertebrates, the four transcription factors Sox2, c-Myc, Pou5f1 and Klf4 are involved in the differentiation of several tissues during vertebrate embryogenesis; moreover, they are normally co-expressed in embryonic stem cells and play roles in pluripotency, self-renewal, and maintenance of the undifferentiated state in adult cells. The in vitro forced co-expression of these factors, named Yamanaka factors (YFs), induces pluripotency in human or mouse fibroblasts. Botryllus schlosseri is a colonial tunicate undergoing continuous stem cell-mediated asexual development, providing a valuable model system for the study of pluripotency in the closest living relatives of vertebrates. In this study, we identified B. schlosseri orthologs of human Sox2 and c-Myc genes, as well as the closest homologs of the vertebrate-specific Pou5f1 gene, through an in-depth evolutionary analysis of the YF gene families in tunicates and other deuterostomes. Then, we studied the expression of these genes during the asexual cycle of B. schlosseri using in situ hybridization in order to investigate their possible involvement in tissue differentiation and in pluripotency maintenance. Our results show a shared spatio-temporal expression pattern consistent with the reported functions of these genes in invertebrate and vertebrate embryogenesis. Moreover, Myc, SoxB1 and Pou3 were expressed in candidate stem cells residing in their niches, while Pou2 was found expressed exclusively in the immature previtellogenic oocytes, both in gonads and circulating in the colonial vascular system. Our data suggest that Myc, SoxB1 and Pou3 may be individually involved in the differentiation of the same territories seen in other chordates, and that, together, they may play a role in stemness even in this colonial ascidian.

ObjectivesLong non-coding RNAs (lncRNAs) are key regulators involved in the progression of glioma, and many functional lncRNAs are yet to be identified. This study aimed to explore the function of CHRM3-AS2, a rarely reported lncRNA, in glioma, as well as the underlying mechanisms involving miR-370-5p/KLF4.MethodsDifferentially expressed RNAs (DERs) were screened from two gene expression profiles of glioblastoma (GBM). Fluorescence in situ hybridisation was performed to determine the subcellular localisation of CHRM3-AS2. Cell viability, colony formation, apoptosis, migration, and invasion were evaluated using cell counting kit-8, colony counts, flow cytometry, wound healing, and Transwell assays, respectively. mRNA and protein expression of specific genes were measured using quantitative real-time polymerase chain reaction and western blotting, respectively. Dual luciferase reporter gene, RNA immunoprecipitation, and RNA pull-down assays were performed to identify the target relationships. A mouse xenograft model was established for in vivo validation.ResultsCHRM3-AS2 was screened as a prognosis-associated DER in GBM. CHRM3-AS2 expression was up-regulated in glioma cells, and CHRM3-AS2 was localised in the cytoplasm. Silencing of CHRM3-AS2 expression inhibited cell viability, colony formation, migration, and invasion and promoted apoptosis of U251 and SHG-44 cells. In addition, CHRM3-AS2 targeted miR-370-5p/KLF4 in glioma cells. The anti-tumour effect of CHRM3-AS2 silencing was weakened by miR-370-5p silencing or KLF4 overexpression. In vivo, silencing of CHRM3-AS2 expression inhibited tumour growth and Ki67 expression in mice. Overexpression of KLF4 also weakened the anti-tumour effect of CHRM3-AS2 silencing in mice.ConclusionsSilencing of CHRM3-AS2 expression inhibited the malignant progression of glioma by regulating miR-370-5p/KLF4 expression.

circular RNAs (circRNAs) is a broad and diverse endogenous subfamily of non-coding RNAs, regulating the gene expression by acting as a microRNA (miRNA) sponge. However, the biological functions of circRNAs in odontoblast differentiation remain largely unknown. Our preliminary study identified an unknown mouse circRNA by circRNA sequencing generated from mouse dental papilla and we termed it circKLF4. In this study, quantitative real-time PCR and in situ hybridization were used and demonstrated that circKLF4 was upregulated during odontoblastic differentiation. Gene knockdown and overexpression assays indicated that circKLF4 promoted odontoblastic differentiation of mouse dental papilla cells (mDPCs). Mechanistically, we found that circKLF4 increased the linear KLF4 expression in a microRNA-dependent manner. By mutating the binding sites of microRNA and circKLF4, we further confirmed that circKLF4 acted as sponge of miRNA-1895 and miRNA-5046 to promote the expression of KLF4. We then also found that ENDOGLIN was also up-regulated by circKLF4 by transfection of circKLF4 overexpression plasmids with or without microRNA inhibitor. In conclusion, circKLF4 increases the expression of KLF4 and ENDOGLIN to promote odontoblastic differentiation via sponging miRNA-1895 and miRNA-5046.

Pulmonary Arterial Hypertension (PAH) is a rare disease caused by the obliteration of the pulmonary arterioles, increasing pulmonary vascular resistance and eventually causing right heart failure. Endothelin-1 (EDN1) is a vasoconstrictor peptide whose levels are indicators of disease progression and its pathway is one of the most common targeted by current treatments. We sequenced the EDN1 untranslated regions of a small subset of patients with PAH, predicted the effect in silico, and used a luciferase assay with the different genotypes to analyze its influence on gene expression. Finally, we used siRNAs against the major transcription factors (TFs) predicted for these regions [peroxisome proliferator-activated receptor γ (PPARγ), Krüppel-Like Factor 4 (KLF4), and vitamin D receptor (VDR)] to assess EDN1 expression in cell culture and validate the binding sites. First, we detected a single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR; rs397751713) and another in the 3'regulatory region (rs2859338) that altered luciferase activity in vitro depending on their genotype. We determined in silico that KLF4/PPARγ could bind to the rs397751713 and VDR to rs2859338. By using siRNAs and luciferase assays, we determined that PPARγ binds differentially to rs397751713. PPARγ and VDR Knock-Down (KD) increased the EDN1 mRNA levels and EDN1 production in porcine aortic endothelial cells (PAECs), while PPARγ and KLF4 KD increased the EDN1 production in HeLa. In conclusion, common variants in EDN1 regulatory regions could alter EDN1 levels. We were able to validate that PPARγ binds in rs397751713 and is a key regulator of EDN1. In addition, KLF4 and VDR regulate EDN1 production in a cell-dependent manner, but VDR does not bind directly to the regions we studied.

Recent studies have confirmed the existence and key roles of microRNA (miRNAs) in cancer drug resistance, including cervical cancer (CC). The present study aims to establish a novel role for miR-92a-3p and its associated gene networks in cisplatin (DDP) resistance of CC. First, the disparities in miRNA expression between CC tissues and adjacent normal tissues were screened based on GSE19611 microarray data that retrieved from Gene Expression Omnibus (GEO), and we identified several miRs that were significantly downregulated or upregulated in CC tissues including miR-92a-3p. Moreover, miR-92a-3p was significantly up-regulated in DDP-resistant cells and was the most differently expressed miRNA. Functionally, knockdown of miR-92a-3p increased the sensitivity of DDP-resistant cells to DDP via inhibiting cell proliferation, migration and invasion, and promoting apoptosis. Conversely, overexpression of miR-92a-3p significantly induced DDP resistance in CC parental cells including HeLa and SiHa cells. Moreover, Krüppel-like factor 4 (KLF4) was identified as a direct target of miR-92a-3p, and an obvious inverse correlation was observed between the expression of miR-92a-3p and KLF4 in 40 pairs of cancer tissues. Furthermore, KLF4 knockdown reversed the promoting effect of miR-92a-3p inhibition on DDP sensitivity in DDP-resistant CC cells. Besides, high expression of miR-92a-3p was associated with DDP resistance, as well as a short overall survival in clinic. Taken together, these findings provide important evidence that miR-92a-3p targets KLF4 and is significant in DDP resistance in CC, indicating that miR-92a-3p may be an attractive target to increase DDP sensitivity in clinical CC treatment.

Background: The role and molecular mechanisms of collagen type VII (COL7A1) in cholangiocarcinoma (CCA) remain unknown., Methods: We analyzed the expression of COL7A1 in CCA and its relationship with patient prognosis using bioinformatic techniques. Expression levels of COL7A1 in CCA cells and tissues were detected using reverse transcription-quantitative PCR, western blotting, and immunohistochemistry. The effects of COL7A1 expression on the proliferation, migration, and invasion of CCA cells were assessed using CCK-8, colony formation, and Transwell assays. Bioinformatics and luciferase reporter gene assays were performed to examine the binding of KLF4 to COL7A1, and cytological experiments further verified the role of KLF4 in regulating the CCA phenotype through COL7A1. Xenograft mouse models were established to investigate the effects of COL7A1 on CCA tumor growth in vivo ., Results: CCA tissues exhibited higher COL7A1 expression than normal bile duct tissues. There was no significant correlation between high or low COL7A1 expression and the survival time of patients with CCA. COL7A1 knockdown inhibited CCA cell proliferation, migration, and invasion. Furthermore, COL7A1 knockdown suppressed the activation of the PI3K/AKT signaling pathway. KLF4 can bind to COL7A1 and regulate COL7A1 expression, which in turn regulates the PI3K/AKT signaling pathway and impacts the proliferation and metastasis of CCA cells., Conclusion: Our findings suggest that KLF4 regulates CCA cell proliferation, migration, and invasion via the COL7A1/PI3K/AKT axis., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)