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.

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.

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.

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.

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

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

KLF6 activates Sp1-mediated prolidase transcription during TGF-ß1 signaling.

Prolidase (PEPD) is the only hydrolase that cleaves the dipeptides containing C-terminal proline or hydroxyproline-the rate-limiting step in collagen biosynthesis. However, the molecular regulation of prolidase expression remains largely unknown. In this study, we have identified overlapping binding sites for the transcription factors Krüppel-like factor 6 (KLF6) and Specificity protein 1 (Sp1) in the PEPD promoter and demonstrate that KLF6/Sp1 transcriptionally regulate prolidase expression. By cloning the PEPD promoter into a luciferase reporter and through site-directed deletion, we pinpointed the minimal sequences required for KLF6 and Sp1-mediated PEPD promoter-driven transcription. Interestingly, Sp1 inhibition abrogated KLF6-mediated PEPD promoter activity, suggesting that Sp1 is required for the basal expression of prolidase. We further studied the regulation of PEPD by KLF6 and Sp1 during transforming growth factor ß1 (TGF-ß1) signaling, since both KLF6 and Sp1 are key players in TGF-ß1 mediated collagen biosynthesis. Mouse and human fibroblasts exposed to TGF-ß1 resulted in the induction of PEPD transcription and prolidase expression. Inhibition of TGF-ß1 signaling abrogated PEPD promoter-driven transcriptional activity of KLF6 and Sp1. Knock-down of KLF6 as well as Sp1 inhibition also reduced prolidase expression. Chromatin immunoprecipitation assay supported direct binding of KLF6 and Sp1 to the PEPD promoter and this binding was enriched by TGF-ß1 treatment. Finally, immunofluorescence studies showed that KLF6 co-operates with Sp1 in the nucleus to activate prolidase expression and enhance collagen biosynthesis. Collectively, our results identify functional elements of the PEPD promoter for KLF6 and Sp1-mediated transcriptional activation and describe the molecular mechanism of prolidase expression.

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.

MiR-191-5p inhibits KLF6 to promote epithelial-mesenchymal transition in breast cancer.

BACKGROUND: MicroRNAs (miRNAs) exert certain functions in the development of several cancers and can be a potential hallmark for cancer diagnosis and prognosis. MiR-191-5p has been proven to have high expression in breast cancer (BC), while its biological role and potential regulatory mechanisms in BC remain an open issue. OBJECTIVE: Bioinformatics was utilized to assay miR-191-5p level in BC tissues and predict its downstream target gene as well as the enriched signaling pathways of the target gene. METHODS: qRT-PCR was carried out to assay miR-191-5p and KLF6 levels in BC cells as well as miR-191-5p level in blood-derived exosomes from BC patients. Western blot was to examine the expression of proteins linked with cell adhesion, epithelial-mesenchymal transition (EMT), and exosome markers. A dual luciferase reporter assay was utilized to verify the interaction between miR-191-5p and KLF6. Abilities of cell phenotypes of BC cells were detected by CCK8, Transwell, and cell adhesion assay, separately. RESULTS: Upregulated miR-191-5p expression and downregulated KLF6 expression were observed in BC cells. There was a targeting relationship between miR-191-5p and KLF6. MiR-191-5p negatively regulated KLF6 to promote EMT and malignant progression of BC cells. Additionally, we described a dramatically high level of miR-191-5p in the blood exosomes of BC patients. CONCLUSION: MiR-191-5p advances the EMT of BC by targeting KLF6, indicating that miR-191-5p and KLF6 may be new biomarkers for BC.

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.

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.

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 Regulates the Apoptosis of Lens Epithelial Cells Through Targeting KLF6 in Diabetic Cataracts.

Purpose: The purpose of this study was to identify novel abnormally expressed microRNAs (miRNAs) and their downstream target in diabetic cataract (DC). Methods: General feature, fasting blood glucose, glycosylated hemoglobin, and type A1c (HbA1c) expression level of patients were collected. DC capsular tissues were obtained from patients and the lens cells (HLE-B3) exposed to different concentrations of glucose were used to simulate the model in vitro. Both mimic and inhibitor of miR-22-3p were transferred into HLE-B3 to up- and downregulate miR-22-3p expression, respectively. The cellular apoptosis was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence. The downstream target gene of miR-22-3p was identified by dual luciferase reporter. Results: In DC capsules and HLE-B3 under hyperglycemia, miR-22-3p showed a significant downward trend. The expression of BAX was upregulated and the BCL-2 was downregulated following high glucose. The expression of BAX was significantly down- or upregulated in HLE-B3 cells following transfection of mimic or inhibitor of miR-22-3p, respectively. Conversely, BCL-2 was significantly increased or decreased. Dual luciferase reporter assay showed that miR-22-3p directly targeted Krüppel Like Factor 6 (KLF6) to regulate cell apoptosis. In addition, the expression of KLF6 were significantly up- or downregulated following transfection of inhibitor or mimic of miR-22-3p. Conclusions: This study suggested that miR-22-3p could inhibit lens apoptosis by targeting KLF6 directly under high glucose condition. The miR-22-3p/KLF6 signal axis may provide novel insights into the pathogenesis of DC. Translational Relevance: Differential expression of miR-22-3p may account for the pathogenesis of DC and lead to a new therapeutic strategy for DC.

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.

MUTYH and KLF6 gene expression fluctuations in tumor tissue and tumor margins tissues of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most important cancers in the world, and its prevalence varies depending on the geographical area. Genetically, tumor regeneration in CRC as a multi-step process involves activating mutations in protocogenes and losing the function of tumor suppressor genes as well as DNA repair and recovery genes. Occur in this way, our goal was to investigate the expression of KLF6 genes as a tumor suppressor and MUTYH involved in the DNA repair process in colorectal cancer. METHODS: This research was conducted during the years 2019-2018 in Razi Hospital, Rasht. The subjects included 30 tumoral and 30 non-tumoral tissues of colorectal cancer and 20 healthy controls. The real-time PCR method was used to investigate the gene expression. For data analysis by SPSS, parametric statistical tests ANOVA and T test and regression analysis were used and p value values less than 0.05 were considered significant. RESULTS: The expression of KLF6 gene in tumoral tissues showed a significant decrease compared to non-tumoral tissues (P = 0.04). Also, the expression of MUTYH gene in tumor tissue showed a significant decrease compared to non-tumoral (P = 0.02) and this decrease in MUTYH gene expression had a significant relationship with increasing tumor stage (P = 0.01). CONCLUSION: These findings suggest that decreased expression of KLF6 and MUTYH genes in the study population has a significant relationship with colorectal cancer and can be considered as tumor marker in diagnostic purpose.

Circ_FOXO3 regulates KLF6 through sponge adsorption of miR-122-5p to repress H2O2-induced HBVSMC proliferation, thus promoting IA development in vitro model.

PURPOSE: The phenotypic transformation of human brain vascular smooth muscle cells (HBVSMC) is widely involved in the appearance and progression of intracranial aneurysms (IA). Aneurysm (IA) Circular RNA circ_FOXO3 functions pivotally in vascular diseases and tumors, but its regulatory role as well as its molecular mechanism in IA is still uncertain. This research was to explore how circ_FOXO3 works and its mechanism in vitro model of HBVSMC IA induced by H2O2. METHODS: Thirty-eight patients with IA and their normal tissues were clinically collected. Examination of endothelin-1, vascular hematoma factor, circ_FOXO3, microRNA (miR)-122-5p and KLF6 and the correlation of circ_FOXO3 with clinical case information were ensured. Establishment of an in vitro IA model was through HBVSMC induced by H2O2 and transfection with circ_FOXO3, miR-122-5p and KLF6 related plasmids was to figure out their roles in cell growth. The relationship among circ_FOXO3, miR-122-5p with KLF6 was detected. RESULTS: Up-regulated circ_FOXO3 and KLF6 and reduced miR-122-5p were in IA tissues; Circ_FOXO3 was associated with smoking history, Hunt-Hess grading and endothelial injury degree. Repressive circ_FOXO3 or KLF6 and strengthening miR-122-5p facilitated H2O2-induced proliferation and repressed HBVSMC apoptosis, while elevation of circ_FOXO3 or depressive miR-122-5p was opposite. circ_FOXO3 bound to miR-122-5p, whose target was KLF6, which participated in controlling IA by mediating the circ_FOXO3/miR-122-5p axis. CONCLUSION: In summary, the findings suggest that circ_FOXO3 suppresses H2O2-induced proliferation of HBVSMC but promotes apoptosis via modulation of miR-122-5p/KLF6 axis. Targeted therapy of circ_FOXO3/miR-122-5p/KLF6 axis is supposed to be a promising treatment approach for IA patients.

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.

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 respi-ratory 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 lipopoly-saccharide (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 expres-sion 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 fac-tors 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 (AU)

Circular RNA circ-ABCB10 Promotes Proliferation and Inhibits Apoptosis of Laryngeal Carcinoma by Inhibiting KLF6.

Objective: To explore the effect of circular RNA circ-ABCB10 on the proliferation and apoptosis of laryngeal carcinoma via inhibiting KLF6. Methods: RT-qPCR assay was adopted to detect the expression of circ-ABCB10 and KFL6 in laryngeal carcinoma tissues and cell lines. Cell counting kit-8 (CCK-8) and clone formation assay were employed to detect laryngeal cancer cell viability and proliferation when circ-ABCB10 was silenced or upregulated. In this study, the apoptosis rate was detected by flow cytometry and the protein expression was detected by Western blotting. Wound healing and cross-hole invasion were used to study the migration and invasion of laryngeal cancer cells when circ-ABCB10 was silenced or upregulated. Results: The results of RT-qPCR detection indicated that the expression of circ-ABCB10 in all three laryngeal carcinoma cells was downregulated by 3.2 times compared with that of HaCat cells. There is low expression of circ-ABCB10 in most laryngeal carcinoma tissues, the diagnostic cutoff value of circ-ABCB10 is 0.0008, the area under the curve is 0.718, the sensitivity is 0.981, and the specificity is 0.556. The expression level of KLF6 in laryngeal carcinoma is on the rise, which is significantly higher compared to healthy tissues (P < 0.05); 48 hours after transfection, RT-qPCR analysis confirmed the transfection efficiency, and upregulation of circ-ABCB10 could significantly promote cell proliferation. Compared with the control group, silencing circ-MTCL1 could inhibit cell proliferation, overexpression of circ-ABCB10 promoted cell migration, and downregulation of circ-ABCB10 significantly inhibited cell movement (P < 0.001). Upregulation of circ-ABCB10 significantly enhanced the invasiveness and motility of laryngeal cancer cells, while downregulation of circ-ABCB10 was the opposite. Compared with the KLF6 NC group, KLF6 level increased significantly in the KLF6 group, while cell viability, colony formation, scratch healing rate, invasive cell number, and Bcl-2 expression level decreased significantly in the KLF6 group, while apoptosis rate and Bax expression level increased significantly (P < 0.05). KLF6 level in the si-circ-ABCB10+anti-KLF6 group was significantly lower than that in the si-circ-ABCB10+anti-KLF6-NC group (P < 0.05). Meanwhile, the cell activity, colony formation number, cell scratch healing rate, number of invaded cells, and Bcl-2 all indicated an upward trend, while the cell apoptosis rate and Bax expression indicated a downward trend (P < 0.05). Conclusion: The expression of circ-ABCB10 in laryngeal carcinoma was significantly higher compared to that in paracancerous tissues. Silencing circ-ABCB10 could significantly inhibit the growth and proliferation of laryngeal adenocarcinoma cells, while overexpression of circ-ABCB10 could significantly promote the growth of laryngeal adenocarcinoma cells, probably by inhibiting KLF6 to enhance the proliferation of laryngeal carcinoma and inhibit apoptosis.