Krüppel-like Factor 6 Research Articles

Anti-apoptosis and anti-inflammation activity of circ_0097010 downregulation in lipopolysaccharide-stimulated periodontal ligament cells by miR-769-5p/Krüppel like factor 6 axis

Background/purposePeriodontitis is a prevalent infectious inflammatory disease. Growing evidence has revealed important roles for circular RNAs (circRNAs) and circRNA sponge activity in periodontitis. Here, we elucidated the precise part of circ_0097010 in periodontitis pathogenesis. Materials and methodsHuman periodontal ligament cells (hPDLCs) were exposed to lipopolysaccharide (LPS). Cell viability, proliferation and apoptosis were evaluated by CCK-8 assay, EdU incorporation assay and flow cytometry, respectively. Circ_0097010, microRNA (miR)-769-5p and Krüppel like factor 6 (KLF6) were quantified by qRT-PCR and Western blot. Interleukin 6 (IL-6) level, tumor necrosis factor-α (TNF-α) secretion, superoxide dismutase (SOD) activity and malondialdehyde (MDA) level were detected by enzyme-linked immunosorbent assay (ELISA). Dual-luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays were used to confirm the direct relationship between miR-769-5p and circ_0097010 or KLF6. ResultsOur data showed that LPS repressed cell proliferation and induced cell apoptosis and inflammation in hPDLCs. Circ_0097010 was upregulated in periodontitis samples and LPS-exposed hPDLCs. Downregulation of circ_0097010 exerted anti-apoptosis and anti-inflammation functions in LPS-exposed hPDLCs. Mechanistically, circ_0097010 acted as a miR-769-5p sponge, and reduced abundance of miR-769-5p reversed the anti-apoptosis and anti-inflammation effects of circ_0097010 suppression. KLF6 was a direct miR-769-5p target, and miR-769-5p-mediated inhibition of KLF6 possessed anti-apoptosis and anti-inflammation functions in LPS-induced hPDLCs. Moreover, circ_0097010 controlled KLF6 expression by miR-769-5p. ConclusionThese data identify circ_0097010 as a key regulator of LPS-induced inflammation and apoptosis in hPDLCs and highlight a novel mechanism of circ_0097010 regulation through miR-769-5p/KLF6 axis.

Bone marrow mesenchymal stem cell-derived extracellular vesicles containing miR-181d protect rats against renal fibrosis by inhibiting KLF6 and the NF-κB signaling pathway

Recent studies have investigated the ability of extracellular vesicles (EVs) in regulating neighboring cells by transferring signaling molecules, such as microRNAs (miRs) in renal fibrosis. EVs released by bone marrow mesenchymal stem cells (BMSCs) contain miR-181d, which may represent a potential therapy for renal fibrosis. miR-181d has been speculated to regulate Krüppel-like factor 6 (KLF6), which activates the nuclear factor-kappa B (NF-κB) signaling pathway. Luciferase assays were performed to confirm the relationship between miR-181d and KLF6. Gain- and loss-of-function studies in vivo and in vitro were performed to assess the effect of BMSC-derived EVs (BMSC-EVs), which contained miR-181d, on KLF6, NF-κB, and renal fibrosis. Transforming growth factor-β (TGF-β)-induced renal tubular epithelial HK-2 cells were treated with EVs derived from BMSCs followed by evaluation of collagen type IV α1 (Col4α1), Collagen I and α-smooth muscle actin (α-SMA) as indicators of the extent of renal fibrosis. Renal fibrosis was induced in rats by unilateral ureteral obstruction (UUO) followed by the subsequent analysis of fibrotic markers. BMSC-EVs had higher miR-181d expression. Overexpression of miR-181d correlated with a decrease in KLF6 expression as well as the levels of IκBα phosphorylation, α-SMA, Col4α1, TGF-βR1 and collagen I in HK-2 cells. In vivo, treatment with miR-181d-containing BMSC-derived EVs was able to restrict the progression of fibrosis in UUO-induced rats. Together, BMSC-EVs suppress fibrosis in vitro and in vivo by delivering miR-181d to neighboring cells, where it targets KLF6 and inhibits the NF-κB signaling pathway.

Circular RNA_0078767 upregulates Kruppel-like factor 9 expression by targeting microRNA-889, thereby inhibiting the progression of osteosarcoma

ABSTRACT Among kids and juveniles, osteosarcoma (OS) is a common bone malignancy. Circular RNAs (circs, circRNAs) play important roles in multiple malignancies including OS, yet circ_0078767ʹs biological functions in OS are far from well elucidated. This study is targeted at understanding circ_0078767ʹs biological functions in OS and its molecular mechanisms. This study confirmed that circ_0078767 expression was reduced in OS cell lines and tissues. Circ_0078767 overexpression remarkably inhibited OS cell growth, migration, invasion, epithelial-mesenchymal transition (EMT), and promoted apoptosis, whereas circ_0078767 knockdown resulted in the opposite effects. MicroRNA-889 (miR-889) was targeted and regulated by circ_0078767, and miR-889 could negatively modulate Kruppel-like factor 9 (KLF9) expression. Besides, circ_0078767 positively regulated KLF9 expression in OS cells via repressing miR-889. In conclusion, circ_0078767 enhances KLF9 expression by targeting miR-889 to inhibit OS progression.

VEGF-A controls the expression of its regulator of angiogenic functions, dopamine D2 receptor, on endothelial cells.

We have previously demonstrated significant upregulation of dopamine D2 (DAD2) receptor (DRD2) expression on tumor endothelial cells. The dopamine D2 receptors, upon activation, inhibit the proangiogenic actions of vascular endothelial growth factor-A (VEGF-A, also known as vascular permeability factor). Interestingly, unlike tumor endothelial cells, normal endothelial cells exhibit very low to no expression of dopamine D2 receptors. Here, for the first time, we demonstrate that through paracrine signaling, VEGF-A can control the expression of dopamine D2 receptors on endothelial cells via Krüppel-like factor 11 (KLF11)-extracellular signal-regulated kinase (ERK) 1/2 pathway. These results thus reveal a novel bidirectional communication between VEGF-A and DAD2 receptors.

CircCDK14 ameliorates interleukin-1β-induced chondrocyte damage by the miR-1183/KLF5 pathway in osteoarthritis

Background The pathogenesis of osteoarthritis (OA), an endemic and debilitating disease, remains unclear. The study aimed to reveal the role of circular RNA cyclin dependent kinase 14 (circCDK14) in OA development and the underlying mechanism. Methods Human chondrocytes were stimulated by 10 ng/mL interleukin-1β (IL-1β) to mimic OA cell model. The RNA expression of circCDK14, microRNA-1183 (miR-1183) and kruppel like factor 5 (KLF5) was checked through quantitative real-time polymerase chain reaction. Western blot was employed to detect protein expression. Cell viability, proliferation and apoptosis were investigated by cell counting kit-8, 5-Ethynyl-29-deoxyuridine and flow cytometry analysis, respectively. Starbase online database was performed to identify the interaction between miR-1183 and circCDK14 or KLF5. Exosomes were isolated by differential centrifugation and identified by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. Results CircCDK14 and KLF5 expression were significantly decreased, while miR-1183 was increased in OA cartilage tissues and IL-1β-treated chondrocytes in comparison with controls. CircCDK14 overexpression attenuated the inhibitory effect of IL-1β treatment on cell proliferation and the promoting effects on cell apoptosis and extracellular matrix degradation. Additionally, miR-1183 was targeted by circCDK14, and miR-1183 mimics reversed circCDK14-mediated actions in IL-1β-treated chondrocytes. The knockdown of KLF5, a target mRNA of miR-1183, also rescued the effects of miR-1183 inhibitors in IL-1β-induced chondrocytes. Moreover, circCDK14 could induce KLF5 expression by interacting with miR-1183. Further, exosomal circCDK14 had a high diagnostic value in OA. Conclusion CircCDK14 reintroduction assuaged IL-1β-caused chondrocyte damage by the miR-1183/KLF5 pathway, providing a diagnostic biomarker for OA.

Krüppel-like factor 6 participates in extravillous trophoblast cell differentiation and its expression is reduced in abnormally invasive placenta.

Trophoblast cell differentiation is of paramount importance for successful pregnancy. Krüppel-like factor 6 (KLF6), a transcription factor with diverse roles in cell physiology and tumor biology, is required for trophoblast differentiation through the syncytial pathway. Herein, we demonstrate that extravillous trophoblast (EVT) cell migration and mesenchymal phenotype are increased upon KLF6 downregulation or the expression of a deletion mutant lacking its transcriptional regulatory domain (KΔac). Raman spectroscopy revealed molecular modifications compatible with increased differentiation in cells stably expressing the KΔac mutant. Moreover, abnormally invasive placenta showed lower KLF6 immunostaining compared with the normal placenta. Thus, impaired KLF6 expression or function stimulates EVT migration and differentiation in vitro and may contribute to the physiopathology of the abnormally invasive placenta.

MiR-600 promotes ovarian cancer cells stemness, proliferation and metastasis via targeting KLF9

Previous studies have revealed that miRNAs participate in the pathogenesis of ovarian cancer; however, whether miR-600 is also involved remains unclear. In this study, we aimed to investigated the role of miR-600 in ovarian cancer progression. Here, miR-600 expression was significantly upregulated in ovarian cancer tissues and stem cells. Functional studies showed that miR-600 promoted ovarian cancer cell stemness, proliferation and metastasis. Mechanistic studies revealed that Kruppel like factor 9 (KLF9) was indicated as the target of miR-600. The luciferase reporter assay suggested that miR-600 directly bound to the 3′-untranslated region of KLF9. Additionally, miR-600 expression was negatively associated with KLF9 expression in human ovarian cancer tissues. Si-KLF9 partially abolished the discrepancy of self-renewal, growth and metastasis capacity between miR-600 knockdown ovarian cancer cells and control cells. In conclusion, our results suggest that miR-600 promotes ovarian cancer cell stemness, proliferation and metastasis via directly downregulating KLF9, and impairing miR-600 levels may be a new treatment strategy for ovarian cancer in the future.

Stearoyl-CoA desaturase 1 regulates malignant progression of cervical cancer cells

ABSTRACT The primary regulatory gene for fatty acid synthesis, stearoyl-CoA desaturase 1 (SCD1), has been linked to the progression of several malignancies. Its role in cervical cancer remains unclear till now. This paper aimed to explore the role and mechanism of SCD1 in cervical cancer. The GEPIA database was used to perform a bioinformatics analysis of the role of SCD1 in cervical cancer staging and prognosis. The influences of SCD1 knockdown on cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) progress were then investigated. Following transcription factor Kruppel like factor 9 (KLF9) was discovered to be negatively correlated with SCD1, the regulatory role of KLF9 in the effects of SCD1 on cervical cancer cells and the signaling pathway was evaluated. According to the GEPIA database, SCD1 level was associated with the cervical cancer stage, the overall survival level, and the disease-free survival level. Cell proliferation, migration, invasion, and EMT progress were all hindered when its expression was knocked down. Novelty, KLF9 reversed the effects of SCD1 on cells, as well as the Akt/glycogen synthase kinase 3β (GSK3β) signaling pathway. Together, SCD1 was negatively regulated by KLF9 and it activated the Akt/GSK3β signaling pathway to promote the malignant progression of cervical cancer cells. Developing SCD1 inhibitors offers novel ideas for the biological treatment of cervical cancer.

Bone mesenchymal stem cells (BMSCs)-derived exosomal microRNA-21-5p regulates Kruppel-like factor 3 (KLF3) to promote osteoblast proliferation in vitro

ABSTRACT Bone mesenchymal stem cells (BMSCs)-derived exosomes (Exos) play important roles in osteoporosis, while the regulation of microRNA (miR)-21-5p remains unclear. The BMSCs-derived exosomes were isolated from femoral bone marrow of trauma patients, which were then used to stimulate human osteoblasts (hFOB1.19 cells). The miR-21-5p mimic or inhibitor was transfected into BMSCs to overexpress or knockdown miR-21-5p. The functions of miR-21-5p in osteoporosis were assessed by cell counting kit-8 (CCK-8) assay, alkaline phosphatase (ALP) staining and alizarin red staining assays. We found that BMSCs-derived exosomes could enhance proliferation, osteoblastic differentiation and ALP activity of hFOB1.19 cells. BMSCs-derived exosomes with upregulated miR-21-5p could further enhance these protective impacts compared with that in BMSCs-derived exosomes, while BMSCs-derived exosomes with downregulated miR-21-5p reduced these cell phenotypes. MiR-21-5p could directly bind to the 3’-untranslated region (UTR) of Kruppel-like factor 3 (KLF3), and knockdown of KLF3 obviously attenuated these inhibitory effects of BMSCs-derived exosomes with downregulated miR-21-5p on osteoblastic differentiation and ALP activity of hFOB1.19 cells. In summary, BMSCs-derived exosomal miR-21-5p improved osteoporosis through regulating KLF3, providing a potential therapeutic strategy for osteoporosis.

KLF8 and OGT/O‐GlcNAcylation regulate breast cancer stem‐like cells

One of the main challenges in treating breast cancer is the intra‐tumor heterogeneity, which, partly, is maintained and promoted by a sub‐population of breast cancer cells called breast cancer stem‐like cells (BCSCs). BCSCs shares traits of mammary stem cells, capable of self‐renewing and differentiating, while promotes invasion, metastasis, drug resistance and relapse. BCSCs are highly adaptive, capable of reprogramming metabolism and signaling activity in response to tumor microenvironment. One key component of cell nutrient sensing mechanism, linking alteration in metabolism to cell signaling, is O‐GlcNAc transferase (OGT). OGT is responsible to adding O‐GlcNAc moieties to target nuclear, cytoplasmic and mitochondrial proteins from UDP‐GlcNAc – final product of the hexosamine biosynthetic pathway. This modification is termed O‐GlcNAcylation and both OGT and O‐GlcNAcylation are upregulated in many cancers. Recently, we showed that OGT/O‐GlcNAcylation is enriched in BCSCs, promoting the stemness and tumorigenesis of breast cancer cells in vitro and in vivo. RNA‐seq analysis of BCSC‐enriched mammospheres with OGT overexpression revealed Kruppel‐like‐factor 8 (KLF8) as a downstream target of OGT in promoting BCSCs. Here, we show that KLF8 expression in breast cancer cells is regulated by OGT at the mRNA and protein level. Knockdown of KLF8 reduced mammosphere forming efficiency (MFE) and NANOG expression and ALDH activity in breast cancer cells, while overexpressing KLF8 increased MFE, NANOG+ and ALDH+BCSC population. Knockdown of KLF8 also reduced breast tumor growth in vivo. Importantly, KLF8 knockdown abolished the effect of elevated OGT and O‐GlcNAcylation in breast cancer cell MFE and BCSC population, while KLF8 overexpression rescued OGT inhibition in MFE and BCSC population. suggesting that KLF8 is required in promoting BCSCs. Consistently, KLF8 knockdown in breast cancer cells significantly reduced expression of stem cells markers at both mRNA and protein level. Interestingly, KLF8 knockdown in breast cancer cell reduced OGT expression, while KLF8 overexpression increased OGT and O‐GlcNAcylation, suggesting a possible feed‐forward loop between OGT and KLF8 in promoting BCSCs. Supporting the idea that KLF8 and OGT are critical for breast cancer progression, high expression of KLF8 and OGT is correlated with poor breast cancer patient outcome. Furthermore, overexpression of KLF8 showed increase in resistance to paclitaxel in triple‐negative breast cancer cells in vitro, suggesting a possible role of KLF8 in promoting chemotherapy resistance in breast cancer. Together, our results suggested that KLF8 and OGT may work in concert in promoting breast cancer stem‐like cells population.

KLF6 facilitates differentiation of odontoblasts through modulating the expression of P21 in vitro

Multiple signaling pathways are involved in the regulation of cell proliferation and differentiation in odontogenesis and dental tissue renewal, but the details of these mechanisms remain unknown. Here, we investigated the expression patterns of a transcription factor, Krüppel-like factor 6 (KLF6), during the development of murine tooth germ and its function in odontoblastic differentiation. KLF6 was almost ubiquitously expressed in odontoblasts at various stages, and it was co-expressed with P21 (to varying degrees) in mouse dental germ. To determine the function of Klf6, overexpression and knockdown experiments were performed in a mouse dental papilla cell line (iMDP-3). Klf6 functioned as a promoter of odontoblastic differentiation and inhibited the proliferation and cell cycle progression of iMDP-3 through p21 upregulation. Dual-luciferase reporter assay and chromatin immunoprecipitation showed that Klf6 directly activates p21 transcription. Additionally, the in vivo study showed that KLF6 and P21 were also co-expressed in odontoblasts around the reparative dentin. In conclusion, Klf6 regulates the transcriptional activity of p21, thus promoting the cell proliferation to odontoblastic differentiation transition in vitro. This study provides a theoretical basis for odontoblast differentiation and the formation of reparative dentine regeneration.

KLF5 inhibition overcomes oxaliplatin resistance in patient-derived colorectal cancer organoids by restoring apoptotic response

Oxaliplatin resistance is a major challenge in the treatment of colorectal cancer (CRC). Many molecular targeted drugs for refractory CRC have been developed to solve CRC drug resistance, but their effectiveness and roles in the progression of CRC and oxaliplatin resistance remain unclear. Here, we successfully constructed CRC PDOs and selected the Kruppel-like factor 5 (KLF5) inhibitor ML264 as the research object based on the results of the in vitro drug screening assay. ML264 significantly restored oxaliplatin sensitivity in CRC PDOs by restoring the apoptotic response, and this effect was achieved by inhibiting the KLF5/Bcl-2/caspase3 signaling pathway. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays verified that KLF5 promoted the transcription of Bcl-2 in CRC cells. KLF5 inhibition also overcame oxaliplatin resistance in xenograft tumors. Taken together, our study demonstrated that ML264 can restore oxaliplatin sensitivity in CRC PDOs by restoring the apoptotic response. KLF5 may be a potential therapeutic target for oxaliplatin-resistant CRC. PDOs have a strong potential for evaluating inhibitors and drug combination therapy in a preclinical environment.

Krüppel-like Factor 9 (KLF9) Suppresses Hepatocellular Carcinoma (HCC)-Promoting Oxidative Stress and Inflammation in Mice Fed High-Fat Diet.

Simple SummaryThe nuclear protein Krüppel-like factor 9 (KLF9) suppresses development of cancers in multiple tissues including the liver. However, a mechanistic basis for how KLF9 decreases cancer initiation/development has not been elucidated. Here, we used a mouse model in which the Klf9 gene was ablated and placed wild-type and mutant animals of both sexes on a high-fat, obesogenic diet. We then examined how the presence or absence of KLF9 affected adiposity, as well as indices of liver oxidative stress and inflammation, and systemic oxidative stress. Results demonstrate that KLF9 is a suppressor of liver oxidative stress and inflammation which may underlie its tumor suppressive actions in liver and other tissues.Obesity, oxidative stress, and inflammation are risk factors for hepatocellular carcinoma (HCC). We examined, in mice, the effects of Krüppel-like factor 9 (KLF9) knockout on: adiposity, hepatic and systemic oxidative stress, and hepatic expression of pro-inflammatory and NOX/DUOX family genes, in a high-fat diet (HFD) context. Male and female Klf9+/+ (wild type, WT) and Klf9−/− (knockout, KO) mice were fed HFD (beginning at age 35 days) for 12 weeks, after which liver and adipose tissues were obtained, and serum adiponectin and leptin levels, liver fat content, and markers of oxidative stress evaluated. Klf9−/− mice of either sex did not exhibit significant alterations in weight gain, adipocyte size, adipokine levels, or liver fat content when compared to WT counterparts. However, Klf9−/− mice of both sexes had increased liver weight/size (hepatomegaly). This was accompanied by increased hepatic oxidative stress as indicated by decreased GSH/GSSG ratio and increased homocysteine, 3-nitrotyrosine, 3-chlorotyrosine, and 4HNE content. Decreased GSH to GSSG ratio and a trend toward increased homocysteine levels were observed in the corresponding Klf9−/− mouse serum. Gene expression analysis showed a heightened pro-inflammatory state in livers from Klf9−/− mice. KLF9 suppresses hepatic oxidative stress and inflammation, thus identifying potential mechanisms for KLF9 suppression of HCC and perhaps cancers of other tissues.

Circular RNA circ_0130438 suppresses TNF-α-induced proliferation, migration, invasion and inflammation in human fibroblast-like MH7A synoviocytes by regulating miR-130a-3p/KLF9 axis

BackgroundCircular RNAs (circRNAs) can play a critical role in rheumatoid arthritis (RA) pathogenesis by involving gene regulation by competing for shared microRNAs (miRNAs), a family of small noncoding RNAs. MiR-130a-3p is a disease-related miRNA and Kruppel-like factor 9 (KLF9) is a zinc finger transcription factor, which are involved in RA pathogenesis. Here, we identified the action of circRNA circ_0130438 in regulating fibroblast-like synoviocytes (FLSs) stimulated by tumor necrosis factor α (TNF-α). MethodsThe direct relationship between miR-130a-3p and circRNA circ_0130438 or KLF9 was predicted by bioinformatics analysis and examined by a dual-luciferase reporter or RNA immunoprecipitation (RIP) assay. CircRNA circ_0130438, miR-130a-3p and KLF9 factor expression levels were gauged by a quantitative real-time PCR (qRT-PCR) or a western blot method. Cell proliferation ability was analyzed by a 5-Ethynyl-2′-Deoxyuridine (EdU) staining assay. The transwell assay was used to evaluate cell migration and invasion capacities. The production levels of interleukin-1β (IL)-1β, IL-6 and IL-8 were assessed by enzyme-linked immunosorbent assay (ELISA). ResultsThe level of circRNA circ_0130438 was reduced in RA tissues (P = 0.0001) and FLSs isolated from RA tissues (P = 0.0001) compared with corresponding normal controls. Exposure of human fibroblast-like MH7A synoviocytes to TNF-α suppressed circRNA circ_0130438 expression (P < 0.0001). In contrast, the elevated expression of circRNA circ_0130438 suppressed the TNF-α-induced proliferation (P = 0.0047) and migration (P = 0.0023) of MH7A cells, as well as their pro-inflammatory cytokines (IL-1β, IL-6 and IL-8) production (P < 0.0001, P < 0.0001 and P < 0.0001). The circRNA circ_0130438 contained a miR-130a-3p binding site. Furthermore, the increase of miR-130-3p in TNF-α-stimulated MH7A cells reversed the effects of circRNA circ_0130438 elevation on cell proliferation (P = 0.0006), migration (P = 0.0406) and pro-inflammatory cytokines (IL-1β, IL-6 and IL-8) production (P = 0.0036, P < 0.0001 and P = 0.0004), indicating that miR-130a-3p was a functional mediator of circRNA circ_0130438 regulation. We also documented that KLF9 was a direct target and downstream effector of miR-130a-3p. Importantly, circRNA circ_0130438 enhanced KLF9 expression (P < 0.0001) in TNF-α-stimulated MH7A cells by functioning as a competing endogenous RNA (ceRNA) for miR-130a-3p (P = 0.0004). ConclusionOur findings demonstrate that the elevated expression of circRNA circ_0130438 suppresses TNF-α-induced migration, proliferation and pro-inflammatory cytokines (IL-1β, IL-6 and IL-8) production of human MH7A cells by enhancing KLF9 expression by operating as a ceRNA for miR-130a-3p.

MiR-181 Enhances Proliferative and Migratory Potentials of Retinal Endothelial Cells in Diabetic Retinopathy by Targeting KLF6

Purpose We aimed to uncover the role of microRNA-181 (miR-181) in the disease onset of diabetic retinopathy (DR) and its underlying mechanism. Methods MiR-181 levels in plasma and aqueous humor samples of non-proliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR) and healthy subjects were analyzed by microarray and quantitative real-time polymerase chain reaction (qRT-PCR). Proliferative and migrative capacities of human retinal endothelial cells (hRECs) regulated by miR-181 were assessed. The binding between miR-181 and Kruppel-like factor 6 (KLF6) was verified by dual-luciferase reporter assay. Results MiR-181 was upregulated in plasma and aqueous humor samples of NPDR and PDR patients. Overexpression of miR-181 stimulated hRECs to proliferate and migrate. KLF6 was the downstream gene binding miR-181, which was involved in the regulation of hRECs by miR-181. Conclusions MiR-181 is upregulated in plasma and aqueous humor of DR patients. It enhances proliferative and migratory potentials of retinal endothelial cells by targeting KLF6.

Fat mass and obesity associated (FTO)-mediated N6-methyladenosine modification of Krüppel-like factor 3 (KLF3) promotes osteosarcoma progression

ARSTRACT N6-methyladenosine (m6A) methylation is the most common and abundant methylation modification of eukaryotic mRNAs, which is involved in tumor initiation and progression. The study aims to explore the potential role and the regulatory mechanism of fat mass and obesity associated (FTO) in osteosarcoma (OS) progression. In this study, we detected the expressions of Krüppel-like factor 3 (KLF3) in OS cells and tissues and found that the mRNA and protein levels of KLF3 were increased in OS cells and tissues and significantly related to tumor size, metastasis, and TNM stage and poor prognosis of OS patients. FTO promoted the proliferation and invasion and suppressed apoptosis of OS cells through cell experiments in vitro. Further mechanism dissection revealed that FTO and YTHDF2 enforced the decay of KLF3 mRNA and decreased its expression. FTO-mediated mRNA demethylation inhibited KLF3 expression in the YTHDF2-dependent manner. Moreover, KLF3 overexpression abrogated FTO-induced oncogenic effects on the proliferation and invasion of OS cells. Overall, our findings showed that FTO-mediated m6A modification of KLF3 promoted OS progression, which may provide a therapeutic target for OS.

KLF8 is activated by TGF-β1 via Smad2 and contributes to ovarian cancer progression.

Krüppel-like factor 8 (KLF8) is a transcription factor expressed abnormally in various cancer types and promotes oncogenic transformation. However, the role of KLF8 in ovarian cancer (OC) progression remains unclear. This study reports that transforming growth factor-β1(TGF-β1)/Smad2/KLF8 axis regulates epithelial-mesenchymal transition (EMT) and contributes to OC progression. We analyzed the KLF8 expression in OC cells and tissues, wherein a significant overexpression of KLF8 was observed. Increased KLF8 expressions were correlated with higher cell proliferation, EMT, migration, and invasion and conferred poor clinical outcomes in OC patients. Overexpressed KLF8 increases F-actin polymerization and induces cytoskeleton remodeling of OC cells. Furthermore, a dissection of the molecular mechanism defined that TGF-β1 triggers KLF8 through the Smad2 pathway and regulates EMT. Pharmacological and genetic inhibition of Smad2 followed by TGF-β1 treatment failed to activate KLF8 expression and induction of EMT. Using promoter-luciferase reporter assays, we defined that upon TGF-β1 activation, phosphorylated Smad2 binds and promotes the KLF8 promoter activity, and knockdown of Smad2 inhibits KLF8 promoter activation. Together, these results demonstrate that TGF-β1 activates KLF8 expression by the Smad2 pathway, and KLF8 contributes to OC progression and may serve as a potential therapeutic strategy for treating OC patients.

Hsa_circ_0037128 aggravates high glucose-induced podocytes injury in diabetic nephropathy through mediating miR-31-5p/KLF9

Background Circular RNA is a key regulator involved in the progression of many human diseases including diabetic nephropathy (DN). However, the role and mechanism of hsa_circ_0037128 in the occurrence and development of DN remains to be explored. Methods High glucose (HG)-induced podocytes were used to construct in vitro DN models. The expression of hsa_circ_0037128, microRNA (miR)-31-5p, and Kruppel-like factor 9 (KLF9) was determined using quantitative real-time polymerase chain reaction. The viability and apoptosis of podocytes was measured using cell counting kit 8 assay and flow cytometry. Western blot analysis was performed to examine the protein levels of apoptosis markers and KLF9 in podocytes. Inflammation factors were detected by ELISA assay, and oxidative stress markers were assessed by corresponding Assay Kits. In addition, the interaction between miR-31-5p and hsa_circ_0037128 or KLF9 was verified using dual-luciferase reporter assay and RIP assay. Results Our data suggested that hsa_circ_0037128 was highly expressed in DN patients and HG-induced podocytes. In HG-induced podocytes, hsa_circ_0037128 knockdown could alleviate HG-induced podocytes injury. In the term of mechanism, hsa_circ_0037128 could sponge miR-31-5p to upregulate KLF9. MiR-31-5p inhibitor could reverse the negative regulation of hsa_circ_0037128 silencing on HG-induced podocytes injury. Also, miR-31-5p relieved HG-induced podocytes injury, and this effect also could be reversed by KLF9 overexpression. Conclusion In summary, our data showed that hsa_circ_0037128 could promote HG-induced podocytes injury via regulating miR-31-5p/KLF9 axis, showing that hsa_circ_0037128 might be a target for DN treatment.

Circ_0004015 silencing represses cisplatin chemoresistance and tumor progression by reducing KLF8 in a miR-198-dependent manner in non-small cell lung cancer.

Circular RNA (circRNA) plays vital roles in diverse cancer progression, including non-small cell lung cancer (NSCLC). Herein, the role of circ_0004015 in regulating the sensitivity of NSCLC to cisplatin (DDP) is revealed. The RNA expression of circ_0004015, microRNA-198 (miR-198) and kruppel like factor 8 (KLF8) was detected by quantitative real-time polymerase chain reaction. Protein expression was checked by western blot. The half maximal inhibitory concentration of DDP and cell proliferation were determined by cell counting kit-8 assay. Cell colony formation ability, migration, invasion and apoptosis were investigated by colony-forming assay, transwell assay and flow cytometry analysis, respectively. The effect of circ_0004015 knockdown on DDP sensitivity in vivo was demonstrated by mouse model assay. The interactions among circ_0004015, miR-198 and KLF8 were predicted by bioinformatics methods, and identified by mechanism assays. The expression of circ_0004015 and KLF8 was apparently upregulated, while miR-198 expression was downregulated in DDP-resistant NSCLC tissues and cells compared with control groups. Additionally, circ_0004015 silencing repressed DDP resistance, cell proliferation, migration and invasion, but induced cell apoptosis in DDP-resistant NSCLC cells. Circ_0004015 knockdown promoted the effect of DDP on tumor formation in vivo. Also, miR-198 inhibitors attenuated circ_0004015 depletion-mediated action though associating with circ_0004015. MiR-198 regulated DDP sensitivity and NSCLC progression by targeting KLF8. Furthermore, circ_0004015 modulated KLF8 expression through interaction with miR-198. Circ_0004015 conferred DDP resistance and promoted NSCLC progression by miR-198/KLF8 pathway, proving a potential target for studying DDP-mediated treatment of NSCLC.

Direct Conversion of Human Endothelial Cells Into Liver Cancer-Forming Cells Using Nonintegrative Episomal Vectors.

Liver cancer is an aggressive cancer associated with a poor prognosis. Development of therapeutic strategies for liver cancer requires fundamental research using suitable experimental models. Recent progress in direct reprogramming technology has enabled the generation of many types of cells that are difficult to obtain and provide a cellular resource in experimental models of human diseases. In this study, we aimed to establish a simple one‐step method for inducing cells that can form malignant human liver tumors directly from healthy endothelial cells using nonintegrating episomal vectors. To screen for factors capable of inducing liver cancer‐forming cells (LCCs), we selected nine genes and one short hairpin RNA that suppresses tumor protein p53 (TP53) expression and introduced them into human umbilical vein endothelial cells (HUVECs), using episomal vectors. To identify the essential factors, we examined the effect of changing the amounts and withdrawing individual factors. We then analyzed the proliferation, gene and protein expression, morphologic and chromosomal abnormality, transcriptome, and tumor formation ability of the induced cells. We found that a set of six factors, forkhead box A3 (FOXA3), hepatocyte nuclear factor homeobox 1A (HNF1A), HNF1B, lin‐28 homolog B (LIN28B), MYCL proto‐oncogene, bHLH transcription factor (L‐MYC), and Kruppel‐like factor 5 (KLF5), induced direct conversion of HUVECs into LCCs. The gene expression profile of these induced LCCs (iLCCs) was similar to that of human liver cancer cells, and these cells effectively formed tumors that resembled human combined hepatocellular–cholangiocarcinoma following transplantation into immunodeficient mice. Conclusion: We succeeded in the direct induction of iLCCs from HUVECs by using nonintegrating episomal vectors. iLCCs generated from patients with cancer and healthy volunteers will be useful for further advancements in cancer research and for developing methods for the diagnosis, treatment, and prognosis of liver cancer.