miR-448-3p通过调节KLF5的表达抑制颅内动脉瘤进展

目的:明确mi R-448-3p对颅内动脉瘤发展的影响。方法:我们通过结扎左侧肾动脉和左侧颈总动脉的方法建立大鼠IA模型;qRT-PCR用于检测mi R-448-3p表达;qRT-PCR和western blot用于检测KLF5 m RNA和蛋白表达;qRT-PCR和ELISA法用于检测炎症因子水平。结果:我们发现IA诱导大鼠中mi R-448-3p表达下调,而KLF5表达上调。我们发现并鉴定出KLF5是平滑肌细胞中mi R-448-3p的直接靶点。此外,mi R-448-3p处理使得IA诱导4周后动脉瘤大小和瘤腔截面积变小。mi R-448-3p处理保护了IA诱导后的壁厚比,抑制了巨噬细胞浸润。IAs引起KLF5表达显著增加,被mi R-448-3p处理后表达显著降低。我们还发现mi R-448-3p在脂多糖诱导的RAW 264.7巨噬细胞中具有抗炎作用。脂多糖促进KLF5、MMP2、MMP9的表达水平,但却被mi R-448-3p所抑制。结论:研究结果表明,mi R-448-3p可以抑制IA的进展,其机制可能是通过下调KLF5的介导的炎症反应。     

MiR-543 regulates myoblast proliferation and differentiation of C2C12 cells by targeting KLF6

MicroRNA-543 (miR-543) has been found to play a suppressive role in various human cancers in many studies, whereas the specific functions of miR-543 in muscle development remain poorly understood. Here, we found that the expression of miR-543 was high in skeletal muscle and increased during the differentiation of C2C12 cells. Overexpression of miR-543 repressed C2C12 cell proliferation and promoted differentiation, while knockdown of miR-543 expression produced the opposite results. During myogenesis, we predicted and verified that Krüppel-like factor 6 (KLF6), a suppressor of multiple tumor cells, was a target gene of miR-543. Then, miR-543 was found to specifically target KLF6 and repress its expression. Besides this, knockdown of KLF6 promoted the differentiation but inhibited the proliferation of C2C12 cells. Si-KLF6 can rescue the influence of miR-543 inhibitor on C2C12 cell differentiation. Our results indicate a new regulatory mechanism of miR-543 on KLF6 expression and suggest the possibility of using the miR-543/KLF6 pathway as a potential target for studying myogenesis.     

Methylation of miR-19b-3p promoter exacerbates inflammatory responses in sepsis-induced ALI via targeting KLF7

Sepsis-induced acute lung injury is associated with dysregulated inflammatory reactions. MiR-19b-3p level was reported to be downregulated in patients with sepsis. To evaluate the role of miR-19b-3p in sepsis, cecum ligation and puncture-induced mouse sepsis model and lpopolysaccharide (LPS)-treated pulmonary microvascular endothelial cells (PMVECs) were used. For in vivo study, lung tissue was harvested for hematoxylin and eosin (H&E) staining, tumor necrosis factor-α, interleukin-6 (IL-6), IL-1β, and p-p65, p-IκB measuring. Cell apoptosis was assessed by TUNEL assay. For in vitro study, cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, respectively. Methylation of miR-19b-3p promoter was measured by methylation-specific PCR (MSP) assay. The target of miR-19b-3p was determined by dual-luciferase reporter gene assay. The level of miR-19b-3p was determined to be downregulated in vitro and in vivo. In addition, miR-19b-3p protected mice from inflammation injury through inhibiting NF-κB signaling pathway. Overexpression of miR-19b-3p increased cell viability, decreased apoptosis, and proinflammatory cytokines secretion in LPS-treated PMVECs. Besides these, Krüppel-like factor 7 (KLF7) was confirmed as the target of miR-19b-3p. And methylation of miR-19b-3p was the reason of decreased miR-19b-3p level. In conclusion, miR-19b-3p protected cells from sepsis-induced inflammation injury via inhibiting NF-κB signaling pathway, and KLF7 was a potential target.     

miR-146a regulates inflammatory cytokine production in Porphyromonas gingivalis lipopolysaccharide-stimulated B cells by targeting IRAK1 but not TRAF6

It has been suggested that microRNAs (miRs) are involved in the immune regulation of periodontitis. However, it is unclear whether and how miRs regulate the function of B cells in the context of periodontitis. This study is to explore the role of miR-146a on the inflammatory cytokine production of B cells challenged by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). Primary B cells were harvested from mouse spleen. Quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of inflammatory cytokines in B cells in the presence or absence of P. gingivalis LPS and/or miR-146a. Bioinformatics, luciferase reporter assay and overexpression assay were used to explore the binding target of miR-146a. Our results showed that miR-146a level in B cells was elevated by P. gingivalis LPS stimulation, and the mRNA expressions of interleukin (IL)-1β, 6 and 10, and IL-1 receptor associated kinase-1 (IRAK1), but not TNF receptor associated factor 6 (TRAF6), were also upregulated. The expression levels of IL-1β, 6, 10 and IRAK1 were reduced in the presence of miR-146a mimic, but were elevated by the addition of miR-146a inhibitor. MiR-146a could bind with IRAK1 3′ untranslated region (UTR) but not TRAF6 3′-UTR. Overexpression of IRAK1 reversed the inhibitory effects of miR-146a on IL-1β, 6 and 10. In summary, miR-146a inhibits inflammatory cytokine production in B cells through directly targeting IRAK1, suggesting a regulatory role of miR-146a in B cell-mediated periodontal inflammation.     

Circ_0026579 alleviates LPS-induced WI-38 cells inflammation injury in infantile pneumonia

Circular RNA (circRNA) represents an important regulator in infantile pneumonia progression. To clarify the role of circ_0026579 in this disease, LPS was used to treat WI-38 cells to mimic inflammation injury. The levels of inflammatory factors were determined by ELISA assay. Cell proliferation and apoptosis were measured by MTT assay, EdU staining and flow cytometry. The protein levels of cyclinD1, cleaved-caspase-3 and insulin-like growth factor 2 (IGF2) were examined using Western blot analysis. Cell oxidative stress was assessed by detecting MDA level and SOD activity. The expression of circ_0026579, miR-24-3p and IGF2 were analyzed using quantitative real-time PCR, and the interaction between miR-24-3p and circ_0026579 or IGF2 was confirmed by dual-luciferase reporter assay and RIP assay. LPS induced inflammation in WI-38 cells. Circ_0026579 expression was promoted in LPS-induced WI-38 cells, and its knockdown alleviated LPS-induced WI-38 cells inflammation. MiR-24-3p was sponged by circ_0026579, and its expression was reduced by LPS. MiR-24-3p inhibitor reversed the regulation of circ_0026579 knockdown on LPS-induced WI-38 cells inflammation. IGF2 was targeted by miR-24-3p, and its expression could be enhanced by LPS. MiR-24-3p relieved the inflammation of WI-38 cells which could be abolished by IGF2 overexpression. Circ_0026579 positively regulated IGF2 expression through sponging miR-24-3p. Circ_0026579 knockdown alleviated LPS-induced WI-38 cells inflammation by miR-24-3p/IGF2 axis, suggesting that circ_0026579 might contribute to infantile pneumonia progression.     

Exosomal microRNA-155-5p from PDLSCs regulated Th17/Treg balance by targeting sirtuin-1 in chronic periodontitis

The mechanism of local inflammation and systemic injury in chronic periodontitis is complicated, in which and exosomes play an important role. In our study, we found that T helper cell 17 (Th17)/regulatory T cell (Treg) balance is destabilized in the peripheral blood of patients with periodontitis, with upregulated Th17 or downregulated Treg, respectively. Porphyromonas gingivalis lipopolysaccharide (LPS) was used to simulate the inflammatory microenvironment of chronic periodontitis. The exosomes were extracted from periodontal ligament stem cells (PDLSCs) in LPS-induced periodontitis environment, which inversely effected on CD4+ T cells under normal and inflammatory conditions. Furthermore, compared with exosomes from normal PDLSCs, lower expression of microRNA-155-5p (miR-155-5p) and higher expression of Sirtuin-1 (SIRT1) were observed in exosomes from LPS-stimulated PDLSCs. Exosomes from PDLSCs alleviated inflammatory microenvironment through Th17/Treg/miR-155-5p/SIRT1 regulatory network. This study aimed to find the “switching” factors that affected the further deterioration of periodontitis to maximally control the multiple downstream damage signal factors to further understand periodontitis and find new targets for its treatment.     

microRNA-146a and Hey2 form a mutual negative feedback loop to regulate the inflammatory response in chronic apical periodontitis

Chronic apical periodontitis (CAP) is defined as chronic inflammation of the dental pulp and root canal system. Porphyromonas endodontalis lipopolysaccharide ( P. endodontalis LPS) plays an important role in inducing an inflammatory response in CAP. microRNA-146a (miR-146a) is a key regulator of inflammation and is induced by LPS. Hairy and enhancer-of-split related with YRPW motif 2 (Hey2) has been confirmed to be induced by the Notch signaling pathway, which is involved in tooth development, pulp regeneration, and repair after injury. Our study aimed to investigate the functional role of miR-146a via the targeting of Hey2 in CAP as well as the underlying mechanism. Compared with 13 healthy controls, miR-146a and Hey2 expressions were significantly higher in 20 patients with CAP. In addition, miR-146a, Hey2, interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α expressions were significantly increased in MC3T3-E1 cells stimulated with different concentrations (0-20 μg/mL) of P. endodontalis LPS for different amounts of time (0-48 hours). Moreover, miR-146a, which acts as an anti-inflammatory mediator, negatively regulated the expression of IL-6, IL-1β, and TNF-α, and Hey2 was confirmed as a target gene of miR-146a by a luciferase reporter assay. Hey2 also negatively regulated miR-146a, IL-6, IL-1β, and TNF-α expressions, and P. endodontalis LPS strongly induced Hey2 recruitment to the IL-6 promoter (−400 ~ −200 bp). These findings suggest that miR-146a and Hey2 form a mutual negative feedback regulatory loop, demonstrating a novel mechanism that regulates inflammatory responses in CAP.     

miR-145-5p affects the differentiation of gastric cancer by targeting KLF5 directly

Krüppel-like factor 5 (KLF5) takes part in the pathologic processes of many types of cancer; however, its expression and roles in the biological behavior of gastric cancer remain unknown. TargetScan suggested that miR-145-5p is the predicted effective and conserved microRNA (miRNA) that binds to KLF5 through its 3′-untranslated region (UTR). We investigated the expression of KLF5 and miR-145-5p messenger RNA (mRNA) in gastric cancer and then analyzed its role in the biological behavior of gastric cancer cells. Our results indicated that KLF5 expression was detected by immunohistochemistry in 39.7% of the gastric cancer cases and was increased compared with that of the corresponding noncancerous normal mucosa (0.01 < p < 0.05). The poorly differentiated subtype showed positive KLF5 expression, whereas the differentiated subtype showed negative KLF5 expression (p < 0.05). Dual-luciferase reporter assay suggested KLF5 3′-UTR was the direct target of miR-145-5p. Compared with the differentiated gastric cancer, miR-145-5p was downregulated in undifferentiated gastric cancer (p < 0.05). The downregulation of KLF5 expression and differentiation of MGC-803 and BGC-823 caused by siKLF5 or miR-145-5p mimic transfection. Our results indicated that miR-145-5p/KLF5 3′-UTR affected the differentiation of gastric cancer. miR-145-5p was able to promote gastric cancer differentiation by targeting KLF5 3′-UTR directly. Our data suggest a novel mechanism for cancer differentiation and a new facet to the role of miR-145-5p/KLF5 in gastric cancer.     

Pulmonary AngII promotes LPS-induced lung inflammation by regulating microRNA-143

Angiotensin converting enzyme 2 (ACE2) is a terminal carboxypeptidase, which cleaves single terminal residues from several bioactive peptides such as Angiotensin II (AngII). Many investigations indicated that ACE2 functions in angiotensin system and plays a crucial role in inflammatory lung diseases. However, the mechanism behind the involvement of ACE2 in inflammatory lung disease has not been fully elucidated. In this study, BEAS-2B cells were treated with gradient concentration of AngII and lipopolysaccharide (LPS) to induce inflammatory condition. Quantitative RT-PCR was performed to detect the level of ACE2 and miR-143-3p. Western blotting and immunofluorescence assays were performed to measure the expression of related proteins. The levels of inflammatory cytokines and cell viability were respectively measured by ELISA and CCK-8 kits. And ACE2 activity was detected by corresponding commercial kits. Bioinformatics methods were employed to predict the potential microRNA targeting ACE2, which was then confirmed by dual luciferase reporter assay. The results showed that ACE2 expression and activity were time-dependently decreased in LPS group for the first 12 h, after which this tendency was reversed. AngII addition enhanced these effects, compared with LPS group. Additionally, the lowest ACE2 activity level was found in both LPS and AngII + LPS groups at 6 h. The number of nuclei and the ACE2 expression were decreased in LPS groups at 6 h and further reduced by addition of AngII, detected by immunofluorescence. Moreover, ACE2 was validated to be a direct target of miR-143-3p. Pretreatment of AngII and LPS regulated the activity of ACE2, increased the expression of proinflammatory cytokines and cell apoptosis and regulated the expression of Bax, Bcl-2 and cleaved caspase-3 in BEAS-2B cells, which could be reversed by transfecting miR-143-3p inhibitor. The results collectively suggest that AngII promotes LPS-induced inflammation by regulating miR-143-3p in BEAS-2B cells. Therefore, miR-143-3p is considered a potential molecular target for the treatment of lung inflammation.     

Inhibition of Kruppel-like factor 7 attenuates cell proliferation and inflammation of fibroblast-like synoviocytes in rheumatoid arthritis through nuclear factor κB and mitogen-activated protein kinase signaling pathway

Rheumatoid arthritis (RA) is an autoimmune disease, which can lead to joint inflammation and progressive joint destruction. Kruppel-like factor 7 (KLF7) is the member of KLF family and plays an important role in multiple biological progresses. However, its precise roles in RA have not been described. Present study aimed to investigate the role of KLF7 in RA-fibroblast-like synoviocytes (FLSs). Data showed that KLF7 expression was obviously upregulated in synovial tissues of rats with adjuvant-induced arthritis. Functional studies demonstrated that the loss of KLF7 may suppress cell proliferation and the expression of pro-inflammatory factors (IL-6, IL-1β, IL-17A) and matrix metalloproteinase (MMP-1, MMP-3, MMP-13) in FLSs through the inhibition of phosphorylation of nuclear factor κB (NF-κB) p65 and JNK. We further showed that miR-9a-5p specifically interacts with KLF7 to negatively regulate the expression of KLF7 in RA-FLSs. Taken together, our results demonstrated that KLF7 which targeted by miR-9a-5p might participate in the pathogenesis of RA by promoting cell proliferation, pro-inflammatory cytokine release and MMP expression through the activation of NF-κB and JNK pathways in RA-FLSs. Hence, KLF7 could be a novel target for RA therapy.     

miR-124-3p attenuates neuropathic pain induced by chronic sciatic nerve injury in rats via targeting EZH2

Emerging evidence has suggested that microRNAs play a critical role in neuropathic pain development. However, the biological role of miRNAs in regulating neuropathic pain remains barely known. In our present study, we found that miR-124-3p was significantly downregulated in rats after chronic sciatic nerve injury (CCI). In addition, it was showed that overexpression of miR-124-3p obviously repressed mechanical allodynia and heat hyperalgesia. Meanwhile, it has been reported that neuroinflammation can contribute a lot to neuropathic pain progression. Here, we found that inflammatory cytokine (IL-6, IL-1β, and TNF-⍺) protein expression in rats after CCI greatly increased and miR-124-3p mimics depressed inflammation cytokine levels. Consistently, miR-124-3p alleviated inflammation production in lipopolysaccharide-incubated spinal microglial cells. Bioinformatics analysis revealed that EZH2 acted as a direct target of miR-124-3p, which participated in the miR-124-3p-modulated effects on neuropathic pain development and neuroinflammation. We observed that miR-124-3p was able to promote neuroinflammation and neuropathic pain through targeting EZH2. The direct correlation between them was validated in our current study using dual-luciferase reporter assays. Subsequently, it was manifested that EZH2 abrogated the inhibitory role of miR-124-3p on neuropathic pain progression in CCI rats. Taken these together, our findings highlighted a novel contribution of miR-124-3p to neuropathic pain and indicated the possibilities for developing novel therapeutic options for neuropathic pain.     

miR-214-5p targets KLF5 and suppresses proliferation of human hepatocellular carcinoma cells

MicroRNAs (miRNAs) are small endogenous conserved RNAs regulating genes expression through base pairing with the 3′-untranslated region (3′-UTR) of target messenger RNAs. MiR-214-5p is a newly identified miRNA with its biological role largely unknown. In this study, we explored miR-214-5p expression status in 78 paired tumor and nontumor tissues obtained from patients with hepatocellular carcinoma (HCC) by RT-qPCR. The effects of miR-214-5p expression on HCC cell proliferation, cell cycle progression, and cell migration were measured by CCK-8 assay, flow cytometry, and wound-healing assay. A dual-luciferase activity assay was performed to identify whether KLF5 was a target of miR-214-5p. Kaplan-Meier curve and log-rank test were used to investigate the effects of miR-214-5p and KLF5 on overall survival and disease-free survival of patients with HCC. We found miR-214-5p expression was sharply reduced in HCC tissues and cell lines compared with the normal tissues and cell lines. Functional assay revealed that miR-214-5p overexpression could downregulate cell proliferation, cell migration, and arrested cell cycle at G0/G1 phase. Further, we validated Krüppel-like factor 5 (KLF5) as a direct target of miR-214-5p, and was upregulated in HCC and inversely correlated with the expression of miR-214-5p. Moreover, we found the low expression of miR-214-5p and high expression of KLF5 were correlated with tumor size, tumor stage, and poorer 5-year overall survival and disease-free survival of patients with HCC. In conclusion, our results suggested miR-214-5p functions as a tumor suppressor through targeting KLF5 in HCC. Also, miR-214-5p and KLF5 were identified as potential prognostic markers and might be therapeutic targets in HCC.