M2 macrophages contribute to cell proliferation and migration of breast cancer

Breast cancer is a kind of malignant tumor that severely threatens women's lives and health worldwide. Tumor-associated macrophages (TAMs) have been reported to mediate tumor progression, while the mechanism still needs further identification. In this study, we found that M2 macrophages promoted increased cell proliferation and migration as well as reduced expression of interferon regulatory factor 7 (IRF7) and increased the expression of miR-1587 in breast cancer cells. Overexpression of IRF7 or miR-1587 knockdown reversed M2 macrophage-induced cell proliferation and migration as well as tumor growth in vivo. Mechanistically, miR-1587 targeted the 3ʹ-untranslated region (3ʹ-UTR) of IRF7 mRNA to regulate its protein expression leading to tumor progression. Collectively, this study revealed that the miR-1587/IRF7 axis mediates M2 macrophage-induced breast cancer progression, and this sheds light on further clinical therapy for breast cancer by targeting TAMs as well as the miR-1587/IRF7 axis.     

Nox2 Is Required for Macrophage Chemotaxis towards CSF-1

Macrophage migration and infiltration is an important first step in many pathophysiological processes, in particular inflammatory diseases. Redox modulation of the migratory signalling processes has been reported in endothelial cells, vascular smooth muscle cells and fibroblasts. However the redox modulation of the migratory process in macrophages and in particular that from the NADPH oxidase-2 (Nox2) dependent ROS has not been established. To investigate the potential role of Nox2 in the migratory response of macrophages, bone marrow derived macrophages were obtained from WT and NOX2 knockout mice (Nox2KO) and subjected to CSF-1 stimulation. We report here that loss of Nox2 expression in BMM resulted in a significant reduction in the CSF-1 induced spreading response suggesting that Nox2 can modulate cytoskeletal events. Moreover, Nox2KO BMMs were deficient in cellular displacement in the presence of CSF-1. More significantly, when challenged with a gradient of CSF-1, Nox2KO BMMs showed a complete loss of chemotaxis accompanied by a reduction in cell migration speed and directional migration persistence. These results point to a specific role for Nox2KO downstream of CSF-1 during the BMM migratory response. Indeed, we have further found that Nox2KO BMMs display a significant reduction in the levels of ERK1/2 phosphorylation following stimulation with CSF-1.Thus Nox2 is important in BMM cellular motion to CSF-1 stimulation and necessary for their directed migration towards a CSF-1 gradient, highlighting Nox2 dependent signalling as a potential anti-inflammatory target.     

Mechanisms of HuR in regulation of epithelial cell apoptosis in rat ulcerative colitis

ObjectiveTo investigate the influence of HuR on the apoptosis rate of epithelial cells in rats with ulcerative colitis (UC) and its mechanism.MethodsUC cell models were established in LPS induced Caco-2 cells. After transfection of si-HuR, pcDNA3.1-HuR, pcDNA3.1-HMGB1, miR-29a-3p mimic or miR-29a-3p inhibitor and their negative controls, apoptosis rate and apoptosis-related proteins (Bcl-2, Bax and cleaved-caspase-3) were tested by flow cytometry, qRT-PCR and Western blot. Actinomycin D treatment was applied to verify the effect of HuR in Caco-2 cells. The binding of HMGB1 to HuR/miR-29a-3p was measured by RIP and dual luciferase reporter gene assays. Experimental UC rat models were established by rectum administration of TNBS/ethanol. The colonic weight/length ratio was calculated at the day 15. The morphology of colon tissues and the apoptosis of tissues were separately detected by H&E staining and TUNEL staining. qRT-PCR and Western blot were conducted to determine the levels of HuR, miR-29a-3p and HMGB1 in colon tissues.ResultsThe apoptosis of LPS-treated Caco-2 cells was inhibited following transfection of si-HuR or miR-29a-3p mimic while facilitated following transfection of pcDNA3.1-HMGB1 or miR-29a-3p inhibitor. RIP and dual luciferase reporter gene assays showed that both HuR and miR-29a-3p can bind HMGB1. Overexpression of HuR in Caco-2 cells results in less HMGB1 that can be bind to miR-29a-3p. The degradation rate of HMGB1 mRNA was increased after transfection of si-HuR in Caco-2 cells. Additionally, miR-29a-3p overexpression can abolish the increases of HMGB1 mRNA induced by HuR, therefore consequently suppress the HMGB1 mRNA that can be bind to HuR. Knockdown of HuR can alleviate TNBS-induced UC in rats and inhibit the apoptosis of colon tissues.ConclusionHuR competitively binds HMGB1 with miR-29a-3p to promote apoptosis of colonic epithelia in rats with UC.     

MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl₄-treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation.     

miRNA-29c Suppresses Lung Cancer Cell Adhesion to Extracellular Matrix and Metastasis by Targeting Integrin β1 and Matrix Metalloproteinase2 (MMP2)

Our pilot study using miRNA arrays found that miRNA-29c (miR-29c) is differentially expressed in the paired low-metastatic lung cancer cell line 95C compared to the high-metastatic lung cancer cell line 95D. Bioinformatics analysis shows that integrin β1 and matrix metalloproteinase 2 (MMP2) could be important target genes of miR-29c. Therefore, we hypothesized that miR-29c suppresses lung cancer cell adhesion to extracellular matrix (ECM) and metastasis by targeting integrin β1 and MMP2. The gain-of-function studies that raised miR-29c expression in 95D cells by using its mimics showed reductions in cell proliferation, adhesion to ECM, invasion and migration. In contrasts, loss-of-function studies that reduced miR-29c by using its inhibitor in 95C cells promoted proliferation, adhesion to ECM, invasion and migration. Furthermore, the dual-luciferase reporter assay demonstrated that miR-29c inhibited the expression of the luciferase gene containing the 3′-UTRs of integrin β1 and MMP2 mRNA. Western blotting indicated that miR-29c downregulated the expression of integrin β1 and MMP2 at the protein level. Gelatin zymography analysis further confirmed that miR-29c decreased MMP2 enzyme activity. Nude mice with xenograft models of lung cancer cells confirmed that miR-29c inhibited lung cancer metastasis in vivo, including bone and liver metastasis. Taken together, our results demonstrate that miR-29c serves as a tumor metastasis suppressor, which suppresses lung cancer cell adhesion to ECM and metastasis by directly inhibiting integrin β1 and MMP2 expression and by further reducing MMP2 enzyme activity. The results show that miR-29c may be a novel therapeutic candidate target to slow lung cancer metastasis.     

Suppression of hepatic stellate cell activation by microRNA-29b

MicroRNAs (miRNAs) participate in the regulation of cellular functions including proliferation, apoptosis, and migration. It has been previously shown that the miR-29 family is involved in regulating type I collagen expression by interacting with the 3′UTR of its mRNA. Here, we investigated the roles of miR-29b in the activation of mouse primary-cultured hepatic stellate cells (HSCs), a principal collagen-producing cell in the liver. Expression of miR-29b was found to be down-regulated during HSC activation in primary culture. Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs and additionally blunted the increased expression of α-SMA, DDR2, FN1, ITGB1, and PDGFR-β, which are key genes involved in the activation of HSCs. Further, overexpression of miR-29b led HSCs to remain in a quiescent state, as evidenced by their quiescent star-like cell morphology. Although phosphorylation of FAK, ERK, and Akt, and the mRNA expression of c-jun was unaffected, miR-29b overexpression suppressed the expression of c-fos mRNA. These results suggested that miR-29b is involved in the activation of HSCs and could be a candidate molecule for suppressing their activation and consequent liver fibrosis.     

MicroRNA-326 attenuates hepatic stellate cell activation and liver fibrosis by inhibiting TLR4 signaling

Hepatic fibrosis is a chronic inflammatory and reversible repair reaction of the liver under the continuous action of virus or various injuries. In this study, we aimed at identifying the role of miR-326 in the hepatic stellate cell (HSC) activation and liver fibrosis and its potential mechanism. In this study, the liver fibrosis mouse model was developed by injecting CCl₄. Liver tissue morphology was observed and the expression level of α-smooth muscle actin, collagen1α1 and miR-326 was measured. Target gene identification was performed by loss-of-function and gain-of-function. The effect of miR-326 on the expression level of the cytokines associated with the TLR4/MyD88/nuclear factor-κB (NF-κB) pathway was assessed in vitro and in vivo. We show that miR-326 was downregulated in CCl₄-induced fibrotic mice and activated HSCs. The target gene of miR-326 is TLR4. Moreover, miR-326 inhibited the activation of HSCs in vitro through TLR4/MyD88/NF-κB signaling. miR-326 attenuated hepatic fibrosis and inflammation of CCl₄-induced mice in vivo. Our results demonstrate for the first time that miR-326 inhibits HSC activation through TLR4/MyD88/NF-κB signaling. Furthermore, miR-326 plays critical roles in attenuating liver fibrosis and inflammation, suggesting the therapeutic potential of miRNAs.     

Myeloperoxidase and elastase are only expressed by neutrophils in normal and in inflammed liver

Myeloperoxidase (MPO) is involved in acute and chronic inflammatory diseases. The source of MPO in acute liver diseases is still a matter of debate. Therefore, we analysed MPO-gene expression on sections from normal and acutely damaged [carbon tetrachloride-(CCl₄) or whole liver γ-Irradiation] rat liver by immunohistochemistry, real time PCR and Western blot analysis of total RNA and protein. Also total RNA and protein from isolated Kupffer cells, hepatic stellate cells, Hepatocytes, endothelial cells and neutrophil granulocytes (NG) was analysed by real time PCR and Western blot, respectively. Sections of acutely injured human liver were prepared for MPO and CD68 immunofluorescence double staining. In normal rat liver MPO was detected immunohistochemically and by immunofluorescence double staining only in single NG. No MPO was detected in isolated parenchymal and non-parenchymal cell populations of the normal rat liver. In acutely damaged rat liver mRNA of MPO increased 2.8-fold at 24 h after administration of CCl₄ and 3.3-fold at 3 h after γ-Irradiation and MPO was detected by immunofluorescence double staining only in elastase (NE) positive NGs but not in macrophages (ED1 or CD68 positive cells). Our results demonstrate that, increased expression of MPO in damaged rat and human liver is due to recruited elastase positive NGs.     

EPC‐derived exosomes promote osteoclastogenesis through LncRNA‐MALAT1

Bone repair involves bone resorption through osteoclastogenesis and the stimulation of neovascularization and osteogenesis by endothelial progenitor cells (EPCs). However, the role of EPCs in osteoclastogenesis is unclear. In this study, we assess the effects of EPC‐derived exosomes on the migration and osteoclastic differentiation of primary mouse bone marrow‐derived macrophages (BMMs) in vitro using immunofluorescence, western blotting, RT‐PCR and Transwell assays. We also evaluated the effects of EPC‐derived exosomes on the homing and osteoclastic differentiation of transplanted BMMs in a mouse bone fracture model in vivo. We found that EPCs cultured with BMMs secreted exosomes into the medium and, compared with EPCs, exosomes had a higher expression level of LncRNA‐MALAT1. We confirmed that LncRNA‐MALAT1 directly binds to miR‐124 to negatively control miR‐124 activity. Moreover, overexpression of miR‐124 could reverse the migration and osteoclastic differentiation of BMMs induced by EPC‐derived exosomes. A dual‐luciferase reporter assay indicated that the integrin ITGB1 is the target of miR‐124. Mice treated with EPC‐derived exosome‐BMM co‐transplantations exhibited increased neovascularization at the fracture site and enhanced fracture healing compared with those treated with BMMs alone. Overall, our results suggest that EPC‐derived exosomes can promote bone repair by enhancing recruitment and differentiation of osteoclast precursors through LncRNA‐MALAT1.     

MFGE8 protects against CCl4-induced liver injury by reducing apoptosis and promoting proliferation of hepatocytes

Milk fat globule-EGF factor 8 (MFGE8) has been reported to play various roles in acute injury and inflammation response. However, the role of MFGE8 in liver injury is poorly investigated. The present research was designed to clarify the expression and function of MFGE8 in carbon tetrachloride (CCl₄)-induced liver injury. Using serum cytokine arrays, we selected a promising cytokine MFGE8 as the candidate in the process of hepatitis-fibrosis-hepatocellular carcinoma (HCC) progression, based on the elevated expression in both hepatic fibrosis and HCC models. We validated the increased expression of MFGE8 in liver tissues and serum samples of acute and chronic CCl₄-induced mice. Immunohistochemistry staining of mouse liver tissues indicated that elevated MFGE8 expression was mainly derived from the injured hepatocytes. In addition, MFGE8 expression in the supernatant of primary hepatocytes was accumulated with prolongation of culture time, and CCl₄ treatment further increased the expression of MFGE8. Moreover, a strong correlation between serum MFGE8 expression and liver transaminase activities suggested that MFGE8 may be a novel candidate in liver injury. Intriguingly, mice pretreated with MFGE8 were protected from CCl₄-induced liver injury through antiapoptosis role in the early stage and proproliferation role in the late stage. MFGE8 reduced apoptosis by inhibiting the activation of IRE1α/ASK1/JNK pathway and promoted proliferation by phosphorylation of ERK and AKT. Moreover, serum MFGE8 expression was increased in hepatitis patients while decreased in liver cirrhosis patients. All the results suggest MFGE8 as a novel marker and promising therapeutic agent of liver injury.     

miR-140-3p suppresses the proliferation and migration of macrophages

Macrophages benefit myelin debris removal, blood vessel formation, and Schwann cell activation following peripheral nerve injury. Identifying factors that modulate macrophage phenotype may advantage the repair and regeneration of injured peripheral nerves. microRNAs (miRNAs) are important regulators of many physiological and pathological processes, including peripheral nerve regeneration. Herein, we investigated the regulatory roles of miR-140-3p, a miRNA that was differentially expressed in injured rat sciatic nerves, in macrophage RAW264.7 cells. Observations from EdU proliferation assay demonstrated that elevated miR-140-3p decreased the proliferation rates of RAW264.7 cells while suppressed miR-140-3p increased the proliferation rates of RAW264.7 cells. Transwell-based migration assay showed that up-regulated and down-regulated miR-140-3p led to elevated and reduced migration abilities, respectively. However, the abundances of numerous phenotypic markers of M1 and M2 macrophages were not significantly altered by miR-140-3p mimic or inhibitor transfection. Bioinformatic analysis and miR-140-3p-induced gene suppression examination suggested that Smad3 might be the target gene of miR-140-3p. These findings illuminate the inhibitory effects of miR-140-3p on the proliferation and migration of macrophages and contribute to the cognition of the essential roles of miRNAs during peripheral nerve regeneration.     

Protective effect of Idelalisib on carbon tetrachloride-induced liver fibrosis via microRNA-124-3P/phosphatidylinositol-3-hydroxykinase signalling pathway

Liver fibrosis is the repair process of abnormal connective tissue hyperplasia after liver damage caused by different causes. Inhibition of PI3K/Akt signalling pathway can reduce the deposition of extracellular matrix, inhibit the proliferation of hepatic stellate cells (HSCs), and promote its apoptosis to achieve the purpose of therapy. This study aimed to investigate the effect of Idelalisib (PI3K inhibitor) on carbon tetrachloride (CCl₄)-induced liver fibrosis in mice. We used CCl₄-induced liver fibrosis mouse model in vivo and TGF-β1-stimulated HSCs to evaluate the antifibrosis activity of Idelalisib. In vivo, Idelalisib significantly alleviated CCl₄-induced liver damage, collagen deposition, and hydroxyproline accumulation in mice. Immunohistochemistry and Western blot results showed that Idelalisib could significantly inhibit the expressions of COL1 and α-SMA in a concentration-dependent manner. In cell experiments, Idelalisib significantly inhibited the expressions of COL1, SMA, and p-Smad3 in TGF-β-induced HSCs, thereby inhibiting HSC activation. Flow cytometry and Western blot results showed that Idelalisib significantly promoted TGFβ-induced apoptosis of HSCs after 48 h of administration, but had no significant effect after 24 h. Idelalisib promoted the apoptosis of activated HSCs by inhibiting the PI3K/Akt/FOXO3 signalling pathway. To further explore the mechanism by which Idelalisib inhibited PI3K, we predicted the miRNA targeting PI3K through the database and crossed it with the down-regulated miRNA reported in liver fibrosis mice in the past five years. Finally, we identified miR-124-3p and miR-143-3p. We then demonstrated that Idelalisib significantly promoted miR-124-3p and miR-142-3p in vitro and in vivo. Dual-luciferase report analysis showed that Idelalisib significantly inhibited luciferase activity but had no significant effect on the luc-MUT transfection assay. Finally, we demonstrated that Idelalisib reversed the effects of miR-124-3p inhibitor on the PI3K/Akt/FOXO3 asterisk pathway and caspase-3. Idelalisib has potential as a candidate drug for alleviating liver fibrosis.     

Effect of hypoxic treatment on bone marrow cells that are able to migrate to the injured liver

Restricted numbers and poor regenerative properties limit the use of adult stem cells. We tested the effect of hypoxic treatment as a method by which to increase cell migration. Bone marrow cells (BMCs) were cultured under oxygen saturations of 0.1, 3, and 20% for 24 h. After hypoxic treatment, BMCs of apoptotic fraction were decreased. The expression of CXCR4 was noticeably increased in the hypoxia-treated BMCs and their migration in response to SDF-1α was enhanced compared with cells cultured under normoxic condition. Hypoxic BMCs had a higher degree of engraftment to the CCl₄-injured liver than the normoxic cells. Hypoxic treatment of BMCs may have merits in decreasing apoptosis of those cells as well as in enhancing cellular migration to SDF-1α, the chemokine which binds to BMCs expressed CXCR4 and to the injured tissue, such as CCl₄ damaged liver.     

Influence of iNOS and COX on peroxiredoxin gene expression in primary macrophages

Peroxiredoxins (Prxs) are a family of multifunctional antioxidant thiol-dependent peroxidases. This study aimed to examine the regulatory mechanisms of Prx gene expression in murine bone marrow-derived macrophages (BMMs) using standardized serum-free conditions. Stimulation with LPS and IFNγ increased mRNA levels of Prx 1, 2, 4, 5, and 6 in BMMs of both C57BL/6 and BALB/c mice, with Prx 1, 2, 4, and 6 more strongly induced in C57BL/6 BMMs. Further investigations on signaling pathways in C57BL/6 BMMs demonstrated that up-regulation of Prx 5 and 6 by LPS and IFNγ was associated with the activation of multiple protein kinases, most notably JAK2, PI3K, and p38 MAPK. Our experiments also revealed a contribution of inducible NO synthase-derived nitric oxide to the increase in Prx 1, 2, 4, and 6 mRNA expression, whereas NADPH oxidase-derived superoxide was not involved. Furthermore, we could show that LPS- and IFNγ-induced gene expression of Prx 6 was also regulated in an NO-independent manner by cyclooxygenases and prostaglandin E₂. Taken together our results indicate a possible role for Prxs in defense mechanisms of activated macrophages against oxidative stress during inflammation or infection.     

Activation of Mir-29a in Activated Hepatic Stellate Cells Modulates Its Profibrogenic Phenotype through Inhibition of Histone Deacetylases 4

Background

Recent studies have shown that microRNA-29 (miR-29) is significantly decreased in liver fibrosis and that its downregulation influences the activation of hepatic stellate cells (HSCs). In addition, inhibition of the activity of histone deacetylases 4 (HDAC4) has been shown to strongly reduce HSC activation in the context of liver fibrosis.

Objectives

In this study, we examined whether miR-29a was involved in the regulation of HDAC4 and modulation of the profibrogenic phenotype in HSCs.

Methods

We employed miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates to clarify the role of miR-29a in cholestatic liver fibrosis, using the bile duct-ligation (BDL) mouse model. Primary HSCs from both mice were treated with a miR-29a mimic and antisense inhibitor in order to analyze changes in profibrogenic gene expression and HSC activation using real-time quantitative RT-PCR, immunofluorescence staining, western blotting, and cell proliferation and migration assays.

Results

After BDL, overexpression of miR-29a decreased collagen-1α1, HDAC4 and activated HSC markers of glial fibrillary acidic protein expression in miR-29aTg mice compared to wild-type littermates. Overexpression of miR-29a and HDAC4 RNA-interference decreased the expression of fibrotic genes, HDAC4 signaling, and HSC migration and proliferation. In contrast, knockdown of miR-29a with an antisense inhibitor increased HDAC4 function, restored HSC migration, and accelerated HSC proliferation.

Conclusions

Our results indicate that miR-29a ameliorates cholestatic liver fibrosis after BDL, at least partially, by modulating the profibrogenic phenotype of HSCs through inhibition of HDAC4 function.