The zinc-finger transcription factor KLF6 regulates cardiac fibrosis.

AIMS: Heart failure (HF) is one of the most devastating consequences of cardiovascular diseases. Regardless of etiology, cardiac fibrosis is present and promotes the loss of heart function in HF patients. Cardiac resident fibroblasts, in response to a host of pro-fibrogenic stimuli, trans-differentiate into myofibroblasts to mediate cardiac fibrosis, the underlying mechanism of which remains incompletely understood. METHODS: Fibroblast-myofibroblast transition was induced in vitro by exposure to transforming growth factor (TGF-ß). Cardiac fibrosis was induced in mice by either transverse aortic constriction (TAC) or by chronic infusion with angiotensin II (Ang II). RESULTS: Through bioinformatic screening, we identified Kruppel-like factor 6 (KLF6) as a transcription factor preferentially up-regulated in cardiac fibroblasts from individuals with non-ischemic cardiomyopathy (NICM) compared to the healthy donors. Further analysis showed that nuclear factor kappa B (NF-κB) bound to the KLF6 promoter and mediated KLF6 trans-activation by pro-fibrogenic stimuli. KLF6 knockdown attenuated whereas KLF6 over-expression enhanced TGF-ß induced fibroblast-myofibroblast transition in vitro. More importantly, myofibroblast-specific KLF6 depletion ameliorated cardiac fibrosis and rescued heart function in mice subjected to the TAC procedure or chronic Ang II infusion. SIGNIFICANCE: In conclusion, our data support a role for KLF6 in cardiac fibrosis.

Decreased expression of KLF6 in ectopic endometrial stromal cells contributes to endometriosis progression by targeting CTNNB1.

Despite decades of research, endometriosis remains a mysterious gynecological disease with unknown etiology and pathogenesis. Krüppel-like Factor 6 (KLF6), a transcription factor, has a wide expression profile and regulates a variety of biological processes. Here, we investigated the expression and function of KLF6 and its possible regulatory mechanisms in endometriosis. To determine the function of KLF6, knockdown and overexpression experiments were performed in eutopic endometrial stromal cells (EU-ESCs) and ectopic endometrial stromal cells (EC-ESCs), respectively. Cell viability, apoptosis, migration, invasion, and angiogenesis assays were conducted in ESCs. ChIP-sequencing and mRNA-sequencing were performed to investigate the functional mechanism of KLF6 in regulating ESCs. We found that KLF6 was highly expressed in eutopic endometrium of endometriosis patients, compared with ectopic endometrium. Similarly, the same was true in EU-ESCs, which was compared with EC-ESCs. Overexpression of KLF6 significantly suppressed EC-ESC proliferation, migration and invasion and induced cell apoptosis, while knockdown of KLF6 resulted in the opposite effects on EU-ESCs. Overexpression of KLF6 significantly inhibited EC-ESC angiogenesis. Mechanistically, the results of ChIP sequencing and mRNA sequencing revealed that CTNNB1 may be a transcriptional target regulated by KLF6. Reintroduction of KLF6 reversed the effects of KLF6 knockdown on EU-ESCs. KLF6 inhibited the proliferation, migration and angiogenesis of EC-ESCs by inhibiting the expression of CTNNB1. Our findings provided a new perspective on the role of KLF6 in endometriosis progression and inspire potential targeted therapeutic strategies.

Investigating the Role of KLF6 in the Growth of Bovine Preadipocytes: Using Transcriptomic Analyses to Understand Beef Quality.

Intramuscular fat is a crucial determinant of carcass quality traits like tenderness and taste, which in turn is influenced by the proliferation of intramuscular preadipocytes. This study aimed to investigate the Krüppel-like factor 6 (KLF6)-mediated proliferation of bovine preadipocytes and identify underlying molecular mechanisms. Down-regulation of KLF6 by siKLF6 resulted in a significant (p < 0.01) suppression of cell cycle-related genes including CDK1, MCM6, ZNF4, PCNA, CDK2, CCNB1, and CDK6. Conversely, the expression level of p27 was significantly (p < 0.01) increased. Moreover, EdU (5-ethynyl-20-deoxyuridine) staining revealed a significant decrease in EdU-labeled cells due to KLF6 down-regulation. Collectively, these findings indicate that KLF6 down-regulation inhibits adipocyte proliferation. Furthermore, RNA sequencing of preadipocytes transfected with siKLF6 and NC, followed by differential gene expression analysis, identified 100 up-regulated and 70 down-regulated genes. Additionally, the differentially expressed genes also significantly influenced various Gene Ontology (GO) terms related to cell cycle, nuclear chromosomes, and catalytic activity on DNA. Furthermore, the top 20 pathways enriched in these DEGs included cell cycle, DNA replication, cellular senescence, and homologous recombination. These GO terms and KEGG pathways play key roles in bovine preadipocyte proliferation. In conclusion, the results of this study suggest that KLF6 positively regulates the proliferation of bovine preadipocytes.

Ropinirole suppresses LPS-induced periodontal inflammation by inhibiting the NAT10 in an ac4C-dependent manner.

BACKGROUND: Periodontitis is a chronic osteolytic inflammatory disease, where anti-inflammatory intervention is critical for restricting periodontal damage and regenerating alveolar bone. Ropinirole, a dopamine D2 receptor agonist, has previously shown therapeutic potential for periodontitis but the underlying mechanism is still unclear. METHODS: Human gingival fibroblasts (HGFs) treated with LPS were considered to mimic periodontitis in vitro. The dosage of Ropinirole was selected through the cell viability of HGFs evaluation. The protective effects of Ropinirole on HGFs were evaluated by detecting cell viability, cell apoptosis, and pro-inflammatory factor levels. The molecular docking between NAT10 and Ropinirole was performed. The interaction relationship between NAT10 and KLF6 was verified by ac4C Acetylated RNA Immunoprecipitation followed by qPCR (acRIP-qPCR) and dual-luciferase reporter assay. RESULTS: Ropinirole alleviates LPS-induced damage of HGFs by promoting cell viability, inhibiting cell apoptosis and the levels of IL-1ß, IL-18, and TNF-α. Overexpression of NAT10 weakens the effects of Ropinirole on protecting HGFs. Meanwhile, NAT10-mediated ac4C RNA acetylation promotes KLF6 mRNA stability. Upregulation of KLF6 reversed the effects of NAT10 inhibition on HGFs. CONCLUSIONS: Taken together, Ropinirole protected HGFs through inhibiting the NAT10 ac4C RNA acetylation to decrease the KLF6 mRNA stability from LPS injury. The discovery of this pharmacological and molecular mechanism of Ropinirole further strengthens its therapeutic potential for periodontitis.

WTAP-MEDIATED M6A MODIFICATION OF KLF6 AGGRAVATES HYPOXIA/REOXYGENATION-INDUCED HUMAN CARDIOMYOCYTE INJURY.

ABSTRACT: Background: Myocardial infarction (MI) is a severe condition that typically results from the ischemia and necrosis of heart muscle. Kruppel-like factor 6 (KLF6) can aggravate myocardial ischemia/reperfusion injury. This work aims to reveal its role and mechanism in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury. Methods: Human cardiomyocyte (AC16) was exposed to hypoxic treatment to mimic MI-like cell injury. mRNA expression levels of KLF6 and WT1-associated protein (WTAP) were detected by quantitative real-time polymerase chain reaction. Protein expression was detected by western blotting assay. Cell viability was assessed by CCK-8 assay. Cell apoptosis and cell cycle were investigated by flow cytometry. Enzyme-linked immunosorbent assays were conducted to detect IL-1ß, TNF-α and IL-6 levels. Fe 2+ colorimetric assay kit was used to detect Fe 2+ level. MDA Content Assay Kit was used to detect MDA level. Cellular ROS Assay kit was applied to assess ROS level. The association of KLF6 and WTAP was identified by RNA immunoprecipitation assay and dual-luciferase reporter assay. Results: KLF6 and WTAP expression at mRNA and protein levels were significantly upregulated in serum samples of MI patients and H/R-induced AC16 cells when compared with control groups. KLF6 silencing attenuated H/R-induced AC16 cell apoptosis, inflammatory response, oxidative stress, and ferroptosis. Additionally, WTAP stabilized KLF6 mRNA by regulating its m6A modification. Furthermore, WTAP knockdown rescued H/R-induced AC16 cell apoptosis, inflammatory response, oxidative stress, and ferroptosis by decreasing KLF6 expression. Conclusion: WTAP-mediated m6A modification of KLF6 aggravated hypoxia/reoxygenation-induced apoptosis, inflammatory response, oxidative stress, and ferroptosis of human cardiomyocytes, providing a therapeutic strategy for MI.

Acquired immune resistance is associated with interferon signature and modulation of KLF6/c-MYB transcription factors in myeloid leukemia.

Resistance to immunity is associated with the selection of cancer cells with superior capacities to survive inflammatory reactions. Here, we tailored an ex vivo immune selection model for acute myeloid leukemia (AML) and isolated the residual subpopulations as "immune-experienced" AML (ieAML) cells. We confirmed that upon surviving the immune reactions, the malignant blasts frequently decelerated proliferation, displayed features of myeloid differentiation and activation, and lost immunogenicity. Transcriptomic analyses revealed a limited number of commonly altered pathways and differentially expressed genes in all ieAML cells derived from distinct parental cell lines. Molecular signatures predominantly associated with interferon and inflammatory cytokine signaling were enriched in the AML cells resisting the T-cell-mediated immune reactions. Moreover, the expression and nuclear localization of the transcription factors c-MYB and KLF6 were noted as the putative markers for immune resistance and identified in subpopulations of AML blasts in the patients' bone marrow aspirates. The immune modulatory capacities of ieAML cells lasted for a restricted period when the immune selection pressure was omitted. In conclusion, myeloid leukemia cells harbor subpopulations that can adapt to the harsh conditions established by immune reactions, and a previous "immune experience" is marked with IFN signature and may pave the way for susceptibility to immune intervention therapies.

miRNA­21 promotes the progression of acute liver failure via the KLF6/autophagy/IL­23 signaling pathway.

Acute liver failure (ALF) is a complex syndrome characterized by overactivation of innate immunity, and the recruitment and differentiation of immune cells at inflammatory sites. The present study aimed to explore the role of microRNA (miRNA/miR)­21 and its potential mechanisms underlying inflammatory responses in ALF. Baseline serum miR­21 was analyzed in patients with ALF and healthy controls. In addition, miR­21 antagomir was injected via the tail vein into C57BL/6 mice, and lipopolysaccharide/D­galactosamine (LPS/GalN) was injected into mice after 48 h. The expression levels of miR­21, Krüppel­like­factor­6 (KLF6), autophagy­related proteins and interleukin (IL)­23, and hepatic pathology were then assessed in the liver tissue. Furthermore, THP­1­derived macrophages were transfected with a miRNA negative control, miR­21 inhibitor, miR­21 mimics or KLF6 overexpression plasmid, followed by treatment with or without rapamycin, and the expression levels of miR­21, KLF6, autophagy­related proteins and IL­23 were evaluated. The results revealed that baseline serum miR­21 levels were significantly upregulated in patients with ALF. In addition, LPS/GalN­induced ALF was attenuated in the antagomir­21 mouse group. KLF6 was identified as a target of miR­21­5p with one putative seed match site identified by TargetScan. A subsequent luciferase activity assay demonstrated a direct interaction between miR­21­5p and the 3'­UTR of KLF6 mRNA. Further experiments suggested that miR­21 promoted the expression of IL­23 via inhibiting KLF6, which regulated autophagy. In conclusion, in the present study, baseline serum miR­21 levels were highly upregulated in patients with ALF, antagomir­21 attenuated LPS/GalN­induced ALF in a mouse model, and miR­21 could promote the expression of IL­23 via inhibiting KLF6.

Silencing KLF6 Alleviates Cigarette Smoke Extract-Induced Mitochondrial Dysfunction in Bronchial Epithelial Cells by SIRT4 Upregulation.

Background: The incidence of chronic obstructive pulmonary disease (COPD) is increasing year by year. Kruppel-like factor 6 (KLF6) plays an important role in inflammatory diseases. However, the regulatory role of KLF6 in COPD has not been reported so far. Methods: The viability of human bronchial epithelial cells BEAS-2B induced by cigarette smoke extract (CSE) was detected by CCK-8 assay. The protein expression of KLF6 and sirtuin 4 (SIRT4) was appraised with Western blot. RT-qPCR and Western blot were applied to examine the transfection efficacy of sh-KLF6 and Oe-KLF6. Cell apoptosis was detected using flow cytometry. The levels of inflammatory factors IL-6, TNF-α and IL-1ß were assessed with ELISA assay. DCFH-DA staining was employed for the detection of ROS activity and the levels of oxidative stress markers SOD, CAT and MDA were estimated with corresponding assay kits. The mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) content and Complex I activity were evaluated with JC-1 staining, ATP colorimetric/fluorometric assay kit and Complex I enzyme activity microplate assay kit. With the application of mitochondrial permeability transition pore detection kit, mPTP opening was measured. Luciferase report assay was employed to evaluate the activity of SIRT4 promoter and chromatin immunoprecipitation (ChIP) to verify the binding ability of KLF6 and SIRT4 promoter. Results: KLF6 expression was significantly elevated in CSE-induced cells. KLF6 was confirmed to suppress SIRT4 transcription. Interference with KLF6 expression significantly inhibited cell viability damage, cell apoptosis, inflammatory response, oxidative stress and mitochondrial dysfunction in CSE-induced BEAS-2B cells, which were all reversed by SIRT4 overexpression. Conclusion: Silencing KLF6 alleviated CSE-induced mitochondrial dysfunction in bronchial epithelial cells by SIRT4 upregulation.

TNF-α stimulated exosome derived from fibroblast-like synoviocytes isolated from rheumatoid arthritis patients promotes HUVEC migration, invasion and angiogenesis by targeting the miR-200a-3p/KLF6/VEGFA axis.

The pathogenesis of rheumatoid arthritis (RA) is heavily impacted by the inflammation and activation of fibroblast-like synoviocytes (FLS). The objective of this investigation is to clarify the involvement of exosomes derived from FLS stimulated by tumour necrosis factor α (TNF-α) in angiogenesis and the underlying mechanisms. FLS cells were obtained from synovial fluid of RA patients and exosomes were obtained from FLS cell supernatant with TNF-α stimulation by ultracentrifugation. Exosomes were subsequently analysed using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. The functional effects of exosomes with TNF-α stimulation on human umbilical vein endothelial cells (HUVEC) migration, invasion, and angiogenesis was evaluated using wound scratch healing test, transwell invasion assay, and tube formation assay. DNA nanoball-seq (DNBSEQ) sequencing platform was utilised to analysis different expression miRNA from exosomes, miRNA and mRNA from HUVEC. The expression level of miR-200a-3p was determined through quantitative real-time polymerase chain reaction (qRT-PCR). The quantification of KLF6 and VEGFA expression levels were performed by qRT-PCR and western blot analysis. The validation of the association between miR-200a-3p and KLF6 was established through a fluorescence enzyme reporting assay. In comparison to exosome induced by PBS, exosome induced by TNF-α exhibited a substantial exacerbation of invasion, migration, and angiogenesis in HUVEC. 4 miRNAs in exosomes and HUVEC cells, namely miR-1246, miR-200a-3p, miR-30a-3p, and miR-99b-3p was obtained. MiR-200a-3p maintained high consistency with the sequencing results. We obtained 5 gene symbols, and KLF6 was chose for further investigation. The expression of miR-200a-3p in exosomes induced by TNF-α and in HUVEC treated with these exosomes demonstrated a significantly increase. Additionally, HUVEC cells displayed a notable decrease in KLF6 expression and a significant elevation in VEGFA expression. This was further confirmed by the fluorescence enzyme report assay, which provided evidence of the direct targeting of KLF6 by miR-200a-3p. Exosomes induced by TNF-α have the ability to enhance the migration, invasion, and angiogenesis of HUVEC cells via the miR-200a-3p/KLF6/VEGFA axis.

The Krüppel-like factor 6 represses proliferation and invasion of cutaneous malignant melanoma.

Cutaneous carcinoma is one of the most common neoplasm tumors in the West. Its incidence rate is one of the fastest growing tumors in China. The Krüppel-like factor 6 (KLF6) is a latent tumor suppressor. Decreased KLF6 is related to the occurrence and progression of many cancers in human. Our previous studies have demonstrated that KLF6 was down-regulation in cutaneous malignant melanoma (CMM), and was significant correlated with ulcer, lymph node metastasis and clinical stage, suggesting that KLF6 loss is expected to become a biological indicator of poor prognosis in CMM patients. In this research, we would further study the features of KLF6 in the malignant progression of CMM. The expression of KLF6 was up-regulated by lentivirus infection containing KLF6, and short hairpin RNA (shRNA) was used for knockdown of KLF6 in CMM cells. Western blot, RT-qpcr, CCK8 assay, transwell migration assays, wound healing assay and flow cytometry were used to test the role of KLF6 in the CMM. We found that reduced expression of KLF6 significantly enhanced proliferation, migration and invasion. Moreover, KLF6 induced CMM cell apoptosis and G1 cycle arrest. The decreased KLF6 expression is expected to be a biological indicator of poor prognosis in CMM patients.

A genome-wide genetic screen uncovers determinants of human pigmentation.

Skin color, one of the most diverse human traits, is determined by the quantity, type, and distribution of melanin. In this study, we leveraged the light-scattering properties of melanin to conduct a genome-wide screen for regulators of melanogenesis. We identified 169 functionally diverse genes that converge on melanosome biogenesis, endosomal transport, and gene regulation, of which 135 represented previously unknown associations with pigmentation. In agreement with their melanin-promoting function, the majority of screen hits were up-regulated in melanocytes from darkly pigmented individuals. We further unraveled functions of KLF6 as a transcription factor that regulates melanosome maturation and pigmentation in vivo, and of the endosomal trafficking protein COMMD3 in modulating melanosomal pH. Our study reveals a plethora of melanin-promoting genes, with broad implications for human variation, cell biology, and medicine.

Nicotine induces macrophage pyroptosis via LINC01272/miR-515/KLF6 axis.

Nicotine contributes to the causation of atherosclerosis, which the prominent cellular components are macrophages. Long non-coding RNAs (lncRNAs) play an important role in regulating cell functions such as cell proliferation, differentiation and programmed death. However, the function and mechanism of lncRNAs in nicotine-induced macrophage pyroptosis has not been reported. We screened the deferentially expressed lncRNAs of human carotid artery plaque (GSE97210) and verified them in nicotine-induced pyroptosis of macrophages. Results showed only LINC01272 was up-regulated in a dose-dependent manner in macrophages. The immunofluorescence staining result confirmed that interfering LINC01272 inhibited nicotine-induced macrophage pyroptosis. Through bioinformatics analysis, dual luciferase reporter gene assay and qPCR, we identified miR-515 was significantly negatively correlated with the expression of LINC01272, and KLF6 is the target gene of miR-515. Furthermore, our results demonstrated that LINC01272/miR-515/KLF6 axis meditated nicotine-induced macrophage pyroptosis. In addition, in human peripheral blood mononuclear cells of smoking populations, the expression of GSDMD-N, NLRP3, LINC01272 and KLF6 was significantly increased, while the level of miR-515 was reduced. This study confirmed that nicotine increases the expression of LINC01272 to competitively bind with miR-515 in macrophages, reducing the inhibitory effect of miR-515 on its target gene KLF6, which ultimately induces macrophage pyroptosis.

SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6.

The transcription factor SREBP2 is the main regulator of cholesterol homeostasis and is central to the mechanism of action of lipid-lowering drugs, such as statins, which are responsible for the largest overall reduction in cardiovascular risk and mortality in humans with atherosclerotic disease. Recently, SREBP2 has been implicated in leukocyte innate and adaptive immune responses by upregulation of cholesterol flux or direct transcriptional activation of pro-inflammatory genes. Here, we investigate the role of SREBP2 in endothelial cells (ECs), since ECs are at the interface of circulating lipids with tissues and crucial to the pathogenesis of cardiovascular disease. Loss of SREBF2 inhibits the production of pro-inflammatory chemokines but amplifies type I interferon response genes in response to inflammatory stimulus. Furthermore, SREBP2 regulates chemokine expression not through enhancement of endogenous cholesterol synthesis or lipoprotein uptake but partially through direct transcriptional activation. Chromatin immunoprecipitation sequencing of endogenous SREBP2 reveals that SREBP2 bound to the promoter regions of two nonclassical sterol responsive genes involved in immune modulation, BHLHE40 and KLF6. SREBP2 upregulation of KLF6 was responsible for the downstream amplification of chemokine expression, highlighting a novel relationship between cholesterol homeostasis and inflammatory phenotypes in ECs.

Roles of Krüppel-Like Transcription Factors KLF6 and KLF15 in Proximal Tubular Metabolism.

Members of the Krüppel-like family of transcription factors are widely expressed, including in the kidney. Expression of some KLFs changes in acute kidney injury, and this may be adaptive or maladaptive, and result in effects on various cellular pathways. This mini-review will highlight the roles of KLF6 and KLF15 in control of proximal tubular cell metabolism.

MicroRNA-22-3p in human umbilical cord mesenchymal stem cell-secreted exosomes inhibits granulosa cell apoptosis by targeting KLF6 and ATF4-ATF3-CHOP pathway in POF mice.

BACKGROUND: Human umbilical cord mesenchymal stem cells (hUCMSCs)-derived exosomes carrying microRNAs (miRNAs) have promising therapeutic potential in various disorders, including premature ovarian failure (POF). Previous evidence has revealed the low plasma level of miR-22-3p in POF patients. Nevertheless, exosomal miR-22-3p specific functions underlying POF progression are unclarified. METHODS: A cisplatin induced POF mouse model and in vitro murine ovarian granulosa cell (mOGC) model were established. Exosomes derived from miR-22-3p-overexpressed hUCMSCs (Exos-miR-22-3p) were isolated. CCK-8 assay and flow cytometry were utilized for measuring mOGC cell viability and apoptosis. RT-qPCR and western blotting were utilized for determining RNA and protein levels. The binding ability between exosomal miR-22-3p and Kruppel-like factor 6 (KLF6) was verified using luciferase reporter assay. Hematoxylin-eosin staining, ELISA, and TUNEL staining were performed for examining the alteration of ovarian function in POF mice. RESULTS: Exos-miR-22-3p enhanced mOGC viability and attenuated mOGC apoptosis under cisplatin treatment. miR-22-3p targeted KLF6 in mOGCs. Overexpressing KLF6 reversed the above effects of Exos-miR-22-3p. Exos-miR-22-3p ameliorated cisplatin-triggered ovarian injury in POF mice. Exos-miR-22-3p repressed ATF4-ATF3-CHOP pathway in POF mice and cisplatin-treated mOGCs. CONCLUSION: Exosomal miR-22-3p from hUCMSCs alleviates OGC apoptosis and improves ovarian function in POF mouse models by targeting KLF6 and ATF4-ATF3-CHOP pathway.

MiRNA-21-5p Accelerates EMT and Inhibits Apoptosis of Laryngeal Carcinoma via Inhibiting KLF6 Expression.

The incidence of laryngeal carcinoma accounts for 1 to 5% of systemic malignancies and ranks second among head and neck malignancies. Screening more effective targets are meaningful for the treatment of laryngeal carcinoma. The purpose was to research the action of miR-21-5p in the occurrence of laryngeal carcinoma. Genecards combined with g:profiler was used for cluster analysis to predict gene-related miRNAs. Q-PCR assay was performed for measuring the level of miR-21-5p and Kruppel-like factor 6 (KLF6). miR-21-5p-mimic, miR-21-5p-inhibitor and sh-KLF6 were transfected using LipofectamineTM 2000. Both CCK-8 and EdU experiments were undertaken to detect cell proliferation ability. Western blot was used to detect apoptosis and epithelial-mesenchymal transition (EMT) related proteins. Wound healing assay and transwell assay were undertaken for migration and invasion, respectively. Three online software (ENCORI, miRWalk, and miRDB) were applied to screen the downstream of miR-21-5p. At the same time, a dual-luciferase reporter experiment was processed to verify the binding. Finally, a rescue experiment was applied to reveal the mediating role of miR-21-5p and KLF6. MiR-21-5p expressed highly in laryngeal carcinoma tissues and cell lines. Knockdown of miR-21-5p reduced the EMT, while enhancing apoptosis of laryngeal carcinoma cell lines. MiR-21-5p targeted KLF6 with negative relationships. The rescue assay results confirmed that sh-KLF6 rescued the action of miR-21-5p knockdown in developing laryngeal carcinoma cells. MiR-21-5p promotes the occurrence and development of laryngeal cancer by targeting KLF6. This finding may provide new insights into miRNA as a biomarker for diagnosing and treating laryngeal carcinoma in the future.

[SPARC Overexpression Enhances the Sensitivity of SKM-1 Cells to Ara-C by Regulating CPBP/MLKL].

OBJECTIVE: To investigate the effect of SPARC gene overexpression on the chemotherapeutic sensitivity of AML-MDS cell line SKM-1 to Ara-C and to further explore its mechanism. METHODS: Subjects were divided into 6 groups: SKM-1 cells (Control), Negative control (LV-NC), SPARC overexpression (LV-SPARC), SKM-1 cells+30 ng/ml Ara-C (30 ng/ml Ara-C), LV-NC+30 ng/ml Ara-C and LV-SPARC+30 ng/ml Ara-C. Cell activity was detected by CCK-8 assay, cell cycle distribution and apoptosis were detected by flow cytometry, mRNA expression levels of SPARC, CPBP and MLKL were detected by RT-qPCR, and the expression levels of related protein were detected by Western blot. RESULTS: After co-treatment with SPARC overexpression and Ara-C, the cell viability decreased and apoptosis increased significantly, with obvious up-regulation of Bax and down-regulation of BCL-2 (P<0.05). Compared with the control group, the cell cycle of LV-SPARC+30 ng/ml Ara-C group was significantly arrested in S phase with obvious down-regulation of CDK2 and up-regulation of p27KIP1 (P<0.05). Compared with LV-SPARC group and 30 ng/ml Ara-C group, the mRNA and protein expression levels of CPBP and MLKL (p-MLKL) were significantly elevated in LV-SPARC+30 ng/ml Ara-C group (P<0.05). In addition, after co-treatment with SPARC overexpression and Ara-C, the protein expression level of p-AKT decreased and the protein expression level of p53 increased (P<0.05). CONCLUSION: SPARC overexpression enhanced the sensitivity of SKM-1 cells to Ara-C and promoted cell cycle arrest and apoptosis, the mechanism of which may be related to the regulation of CPBP/MLKL pathway.

Deciphering the Key Regulatory Roles of KLF6 and Bta-miR-148a on Milk Fat Metabolism in Bovine Mammary Epithelial Cells.

MicroRNAs (miRNAs) are non-coding RNAs that regulate the expression of their target genes involved in many cellular functions at the post-transcriptional level. Previously, bta-miR-148a showed significantly high expression in bovine mammary epithelial cells (BMECs) of Chinese Holstein cows producing high milk fat compared to those with low milk fat content. Here, we investigated the role of bta-miR-148a through targeting Krüppel-like factor 6 (KLF6) and further analyzed the role of KLF6 in regulating fat metabolism through targeting PPARA, AMPK/mTOR/PPARG, and other fat marker genes in BMECs of Chinese Holstein. The bioinformatics analysis showed that the 3' UTR of KLF6 mRNA possesses the binding sites for bta-miR-148a, which was further verified through dual-luciferase reporter assay. The BMECs were transfected with bta-miR-148a-mimic, inhibitor, and shNC, and the expression of KLF6 was found to be negatively regulated by bta-miR-148a. Moreover, the contents of triglyceride (TG), and cholesterol (CHO) in BMECs transfected with bta-miR-148a-mimic were significantly lower than the contents in BMECs transfected with bta-miR-148a-shNC. Meanwhile, the TG and CHO contents were significantly increased in BMECs transfected with bta-miR-148a-inhibitor than in BMECs transfected with bta-miR-148a-shNC. In addition, the TG and CHO contents were significantly decreased in BMECs upon the down-regulation of KLF6 through transfection with pb7sk-KLF6-siRNA1 compared to the control group. Contrarily, when KLF6 was overexpressed in BMECs through transfection with pBI-CMV3-KLF6, the TG and CHO contents were significantly increased compared to the control group. Whereas, the qPCR and Western blot evaluation of PPARA, AMPK/mTOR/PPARG, and other fat marker genes revealed that all of the genes were considerably down-regulated in the KLF6-KO-BMECs compared to the normal BMECs. Taking advantage of deploying new molecular markers and regulators for increasing the production of better-quality milk with tailored fat contents would be the hallmark in dairy sector. Hence, bta-miR-148a and KLF6 are potential candidates for increased milk synthesis and the production of valuable milk components in dairy cattle through marker-assisted selection in molecular breeding. Furthermore, this study hints at the extrapolation of a myriad of functions of other KLF family members in milk fat synthesis.

Inhibition of KLF6 reduces the inflammation and apoptosis of type II alveolar epithelial cells in acute lung injury.

BACKGROUND: The development of acute lung injury (ALI) into a severe stage leads to acute respiratory distress syndrome (ARDS). The morbidity and mortality of ALI and ARDS are very high. Objective: This study is aimed to explore the effect of Krüppel-like factor 6 (KLF6) on lipopolysaccharide (LPS)-induced type II alveolar epithelial cells in ALI by interacting with cysteine-rich angiogenic inducer 61 (CYR61). MATERIAL AND METHODS: ALI mice model and LPS-induced type II alveolar epithelial cells were conducted to simulate ALI in vivo and in vitro. The messenger RNA (mRNA) and protein expression of KLF6 in lung tissues were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. Pathological changes in lung tissues were observed by hematoxylin and eosin (H&E) staining. The viability and KLF6 expression of A549 cells treated with different concentrations of LPS were detected by cell counting kit-8 (CCK-8) assay, RT-qPCR, and Western blot analysis. After indicated treatment, the viability and apoptosis of A549 cells were analyzed by CCK-8 and TUNEL assays, and the inflammation factors of A549 cells were detected by Enzyme-linked-immunosorbent serologic assay, RT-qPCR, and Western blot analysis. The combination of KLF6 and CYR61 was determined by chromatin immunoprecipitation (ChIP)-PCR and dual-luciferase reporter assay. RESULTS: KLF6 expression was increased in lung tissues of ALI mice and LPS-induced A549 cells. Interference with KLF6 improved the viability, reduced the inflammatory damage, and promoted the apoptosis of LPS-induced A549 cells. In addition, KLF6 could bind to CYR61. Interference with KLF6 could decrease CYR61 expression in LPS-induced A549 cells. LPS also enhanced the TLR4/MYD88 signaling pathway, which was reversed by KLF6 interference. The above phenomena in LPS-induced A549 cells transfected with Si-KLF6 could be reversed by overexpression of CYR61. CONCLUSION: Inhibition of KLF6 promoted the viability and reduced the inflammation and apoptosis of LPS-induced A549 cells, which was reversed by CYR61.

Downregulation of krüppel-like factor 6 expression modulates extravillous trophoblast cell behavior by increasing reactive oxygen species.

INTRODUCTION: Placental extravillous trophoblasts play a crucial role in the establishment of a healthy pregnancy. Reactive oxygen species (ROS) may contribute to their differentiation and function as mediators in signaling processes or might cause oxidative stress resulting in trophoblast dysfunction. The krüppel-like transcription factor 6 (KLF6) regulates many genes involved in essential cell processes where ROS are also involved. However, whether KLF6 regulates ROS levels has not been previously investigated. MATERIALS AND METHODS: KLF6 was silenced by siRNAs in HTR8-SV/neo cells, an extravillous trophoblast model. Total and mitochondrial ROS levels, as well as mitochondrial membrane potential and apoptosis were analyzed by flow cytometry. The expression of genes and proteins of interest were analyzed by qRT-PCR and Western blot, respectively. Cell response to oxidative stress, proliferation, viability, morphology, and migration were evaluated. RESULTS: KLF6 downregulation led to an increase in ROS and NOX4 mRNA levels, accompanied by reduced cell proliferation and increased p21 protein expression. Catalase activity, 2-Cys peroxiredoxin protein levels, Nrf2 cytoplasmic localization and hemoxygenase 1 expression, as well as mitochondrial membrane potential and cell apoptosis were not altered suggesting that ROS increase is not associated with cellular damage. Instead, KLF6 silencing induced cytoskeleton modifications and increased cell migration in a ROS-dependent manner. DISCUSSION: Present data reveal a novel role of KLF6 on ROS balance and signaling demonstrating that KLF6 downregulation induces an increase in ROS levels that contribute to extravillous trophoblast cell migration.