Retraction Notice to: METTL3-mediated m6A modification of ZBTB4 mRNA is involved in the smoking-induced EMT in cancer of the lung.

[This retracts the article DOI: 10.1016/j.omtn.2020.12.001.].

Therapeutic effect of exosomes derived from hepatocyte-growth-factor-overexpressing adipose mesenchymal stem cells on liver injury.

Adipose mesenchymal stem cell-derived exosomes (ADMSC-Exo) are a new strategy for the treatment of liver injury. However, mesenchymal stem cells (MSCs) exert therapeutic effects mainly by secreting hepatocyte growth factor (HGF). Therefore, we investigated the role of exosomes derived from ADMSC that overexpress HGF (ADMSCHGF-Exo) on liver injury. MATERIAL AND METHODS: ADMSCs were isolated from young BALB/c female mice. Then exosomes derived from ADMSC transfecting negative control (ADMSCNC-Exo) and HGF overexpression (ADMSCHGF-Exo) were isolated and identified by quantitative polymerase chain reaction (qPCR), flow cytometry, western blot, transmission electron microscope and Nanosight particle tracking analysis. These exosomes were injected into male mice via tail vein after inducing liver injury by administering 40% carbon tetrachloride (CCl4)-olive oil twice a week (3 mL/kg, subcutaneously) for 6 weeks. Liver injury and liver collagen fiber accumulation were determined by histopathological analysis. Then, the levels of serum liver function indexes (alanine aminotransferase, aspartate aminotransferase, albumin, total bilirubin), hepatocyte-specific markers (albumin, cytokeratin-18 and hepatocyte nuclear factor 4α), hepatic fibrosis-related proteins (α-smooth muscle actin and collagen I) and Rho GTPase (cell division cycle 42 and ras-related C3 botulinum toxin substrate 1) were determined by Enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, Western blot and qPCR. RESULTS: ADMSCs were identified by high expression of CD105 and CD44 molecules and low expression of CD45 and CD34. ADMSCs-Exo, ADMSCNC-Exo and ADMSCHGF-Exo transfected cells had similar expression of exosome-specific membrane proteins (CD63, CD81 and CD9). Mice with CCl4-induced liver injury exhibited abnormal serum liver function indexes, altered expression of hepatocyte-specific markers, hepatic fibrosis-related proteins and Rho GTPase protein as well as histopathological changes and collagen fiber accumulation in the liver. These changes were reversed by ADMSC-Exo, ADMSCNC-Exo and ADMSCHGF-Exo administration with ADMSCHGF-Exo displaying the most significant impact. CONCLUSIONS: ADMSCHGF-Exo exerted a hepatoprotective effect in mice with experimental liver injury by alleviating hepatic fibrosis and restoring liver function.

Effect of IL-8 on hepatocellular carcinoma-associated metastasis by targeting MMP9 in mice.

Background: Interleukin-8 (IL-8) and matrix metallopeptidase 9 (MMP9) are overexpressed in hepatocellular carcinoma (HCC), and both are related to tumor metastasis, but whether they regulate HCC metastasis is still unclear. Methods: HCC orthotopic implantation and colonization mice models were established in vivo. Model mice were treated with IL-8 and or MMP9 inhibitors, protein kinase C (PKC) inhibitors, or extracellular regulated protein kinases 1/2 (ERK1/2) inhibitors. Liver metastasis and lung metastasis of model mice were confirmed by hematoxylin and eosin staining. The population of circulating tumor cells (CTCs) was detected by flow cytometry. The expression of MMP9 in tumor tissues was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. In vitro, HCC LM6 (HCCLM6) cells were treated with IL-8 combined PKC inhibitor or ERK1/2 inhibitor. The expression of MMP9 was confirmed by qRT-PCR and Western blot, and the activation of the PKC/ERK1/2 signaling pathway was confirmed by Western blot. Results: IL-8 promoted liver metastasis and lung metastasis in orthotopic transplantation model mice, increased the proportion of CTCs and promoted the expression of MMP9 in tumor tissues, but these effects were reversed by PKC inhibitor or ERK1/2 inhibitor. In vivo colonization experiments, IL-8 promoted tumor cell metastasis to the liver and lung, but the MMP9 inhibitor reversed the metastasis-promoting effect of IL-8. In cell experiments, IL-8 promoted the expression of p-PKC and p-ERK1/2 and inhibited the expression of PKC and ERK1/2; the promotion of MMP9 expression by IL-8 was reversed by PKC inhibitor or ERK1/2 inhibitor. Conclusions: IL-8 up-regulated the expression of MMP9 by activating the PKC/ERK1/2 signaling pathway, thereby promoting the metastasis and colonization of HCC.

miR-21 in EVs from pulmonary epithelial cells promotes myofibroblast differentiation via glycolysis in arsenic-induced pulmonary fibrosis.

As an environmental toxicant, arsenic causes damage to various organs and systems of the body and has attracted worldwide attention. It is well-known that exposure to arsenic can induce pulmonary fibrosis, but the molecular mechanisms are elusive. Glycolysis is involved in the process of various diseases, including pulmonary fibrosis. Extracellular vehicles (EVs) are mediators of cell communication through transporting miRNAs. The potential of miRNAs in EVs as liquid biopsy biomarkers for various diseases has been reported, and they have been applied in clinical diagnoses. In the present investigation, we focused on the roles and mechanisms of miR-21 in EVs on arsenic-induced glycolysis and pulmonary fibrosis through experiments with human populations, experimental animals, and cells. The results for arsenicosis populations showed that the serum levels of hydroxyproline, lactate, and EVs-miRNAs were elevated and that EVs-miR-21 levels were positively related to the levels of hydroxyproline and lactate. For mice, chronic exposure to arsenite led to high levels of miR-21, AKT activation, elevated glycolysis, and pulmonary fibrosis; however, these effects were blocked by the depletion of miR-21 in miR-21 knockout (miR-21KO) mice. After MRC-5 cells were co-cultured with arsenite-treated HBE cells, the levels of miR-21, AKT activation, glycolysis, and myofibroblast differentiation were enhanced, effects that were blocked by reducing miR-21 and by inhibiting the EVs in HBE cells. The down-regulation of PTEN in MRC-5 cells and primary lung fibroblasts (PLFs) reversed the blocking effect of inhibiting miR-21 in HBE cells. Thus, miR-21 down-regulates PTEN and promotes glycolysis via activating AKT, which is associated with arsenite-induced myofibroblast differentiation and pulmonary fibrosis. Our results provide a new approach for the construction of clinical diagnosis technology based on analysis of the mechanism of arsenite-induced pulmonary fibrosis.

MicroRNA-191 blocking the translocation of GLUT4 is involved in arsenite-induced hepatic insulin resistance through inhibiting the IRS1/AKT pathway.

Environmental exposure to arsenic can cause a variety of health problems. Epidemiological and experimental studies have established a diabetogenic role for arsenic, but the mechanisms responsible for arsenic-induced impairment of insulin action are unclear. MicroRNAs (miRNAs) are involved in various metabolic disorders, particularly in the development of insulin resistance. The present study investigated whether arsenite, an active form of arsenic, induces hepatic insulin resistance and the mechanisms underlying it. After male C57BL/6J mice were exposed to arsenite (0 or 20 ppm) in drinking water for 12 months, intraperitoneal glucose tolerance tests (IPGTTs) and insulin tolerance tests (ITTs) revealed an arsenite-induced glucose metabolism disorder. Hepatic glycogen levels were lower in arsenite-exposed mice. Further, for livers of mice exposed to arsenite, miR-191 levels were higher, and protein levels of insulin receptor substrate 1 (IRS1), p-IRS1, and phospho-protein kinase B (p-AKT) were lower. Further, glucose transporter 4 (GLUT4) had lower levels on the plasma membrane. For insulin-treated L-02 cells, arsenite decreased glucose consumption and glycogen levels, increased miR-191 levels, and inhibited the IRS1/AKT pathway and the translocation of GLUT4 from the cytoplasm to the plasma membrane. For insulin-treated L-02 cells, the decreases of glucose consumption, glycogen levels, GLUT4 on the plasma membrane, and p-AKT levels induced by arsenite were reversed by SC79 (agonist of AKT) and an miR-191 inhibitor; these effects caused by miR-191 inhibitor were restored by IRS1 siRNA. In insulin-treated L-02 cells, miR-191, via IRS1, was involved in the arsenite-induced decreases of glucose consumption and glycogen levels and in inhibition of the translocation of GLUT4. Thus, miR-191 blocking the translocation of GLUT4 was involved in arsenite-induced hepatic insulin resistance through inhibiting the IRS1/AKT pathway. Our study reveals a mechanism for arsenite-induced hepatic insulin resistance, which provides clues for discovering biomarkers for the development of type 2 diabetes and for prevention and treatment of arsenic poisoning.

miR-21-regulated M2 polarization of macrophage is involved in arsenicosis-induced hepatic fibrosis through the activation of hepatic stellate cells.

Arsenicosis induced by chronic exposure to arsenic is recognized as one of the main damaging effects on public health. Exposure to arsenic can cause hepatic fibrosis, but the molecular mechanisms by which this occurs are complex and elusive. It is not known if miRNAs are involved in arsenic-induced liver fibrosis. We found that in the livers of mice exposed to arsenite, there were elevated levels of microRNA-21 (miR-21), phosphorylated mammalian target of rapamycin (p-mTOR), and arginase 1 (Arg1); low levels of phosphatase and tensin homolog (PTEN); and more extensive liver fibrosis. For cultured cells, arsenite-induced miR-21, p-mTOR, and Arg1; decreased PTEN; and promoted M2 polarization of macrophages derived from THP-1 monocytes (THP-M), which caused secretion of fibrogenic cytokines, including transforming growth factor-ß1. Coculture of arsenite-treated, THP-M with LX-2 cells induced α-SMA and collagen I in the LX-2 cells and resulted in the activation of these cells. Downregulation of miR-21 in THP-M inhibited arsenite-induced M2 polarization and activation of LX-2 cells, but cotransfection with PTEN siRNA or a miR-21 inhibitor reversed this inhibition. Moreover, knockout of miR-21 in mice attenuated liver fibrosis and M2 polarization compared with WT mice exposed to arsenite. Additionally, LN, PCIII, and HA levels were higher in patients with higher hair arsenic levels, and levels of miR-21 were higher than controls and positively correlated with PCIII, LN, and HA levels. Thus, arsenite induces the M2 polarization of macrophages via miR-21 regulation of PTEN, which is involved in the activation of hepatic stellate cells and hepatic fibrosis. The results establish a previously unknown mechanism for arsenicosis-induced fibrosis.

METTL3-mediated m6A modification of ZBTB4 mRNA is involved in the smoking-induced EMT in cancer of the lung.

N6-methyladenosine (m6A) is an epigenetic modification associated with various tumors, but its role in tumorigenesis remains unexplored. Here, as confirmed by methylated RNA immunoprecipitation sequencing (meRIP-seq) and RNA sequencing (RNA-seq) analyses, exposure of human bronchial epithelial (HBE) cells to cigarette smoke extract (CSE) caused an m6A modification in the 3' UTR of ZBTB4, a transcriptional repressor. For these cells, CSE also elevated methyltransferase-like 3 (METTL3) levels, which increased the m6A modification of ZBTB4. RIP-qPCR illustrated that ZBTB4 was the intent gene of YTHDF2 and that levels of ZBTB4 were decreased in an YTHDF2-dependent mechanism. The lower levels of ZBTB4 were associated with upregulation of EZH2, which enhanced H3K27me3 combining with E-cadherin promoter, causing lower E-cadherin levels and induction of the epithelial-mesenchymal transition (EMT). Further, in the lungs of mice, downregulation of METTL3 alleviated the cigarette smoke (CS)-induced EMT. Further, the expression of METTL3 was high in the lung tissues of smokers and inversely correlated with ZBTB4. Overall, our results show that the METTL3-mediated m6A modification of ZBTB4 via EZH2 is involved in the CS-induced EMT and in lung cancer. These results indicate that m6A modifications are a potential therapeutic target of lung damage induced by CS.

Risk of Skin Cancer Associated with Metformin Use: A Meta-Analysis of Randomized Controlled Trials and Observational Studies.

Previous studies demonstrate mixed evidence regarding the association between metformin and skin cancer risk. To synthesize prior evidence and evaluate the association between metformin and skin cancer risk in patients with diabetes/prediabetes, we conducted a meta-analysis. A systematic literature search was performed up to March 23, 2020 to identify randomized controlled trials (RCT) and observational studies of metformin that reported any event of squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and melanoma. In a meta-analysis of 6 trials involving 8,541 patients (Peto method), compared with controls, metformin was not significantly associated with decreased risk of melanoma [OR, 0.82; 95% confidence interval (CI), 0.27-2.43], BCC (OR, 0.75; 95% CI, 0.36-1.57), SCC (OR, 0.98; 95% CI, 0.06-15.60), total nonmelanoma skin cancer (NMSC; OR, 0.69; 95% CI, 0.38-1.24), or total skin cancer (OR, 0.71; 95% CI, 0.42-1.20). This nonsignificant association pattern was consistent with the random-effects meta-analysis of 4 cohort studies with 354,746 patients (melanoma: RR, 0.91; 95% CI, 0.62-1.33; NMSC: RR, 0.65; 95% CI, 0.35-1.18; total skin cancer: RR, 0.83; 95% CI, 0.59-1.16). In conclusion, meta-analyses of both RCT and cohort studies showed no statistically significant association between metformin and skin cancer risks, although suggestive evidence of modestly reduced risks of skin cancer among metformin users was observed. Further studies are needed. PREVENTION RELEVANCE: Meta-analyses of RCT and cohort studies showed no significant association between metformin and skin cancer, although suggestive evidence of modestly reduced skin cancer risks among metformin users was observed. These findings suggest metformin use should not influence current medical decision making for diabetes patients at risk of developing skin cancer.

microRNA-21, via the HIF-1α/VEGF signaling pathway, is involved in arsenite-induced hepatic fibrosis through aberrant cross-talk of hepatocytes and hepatic stellate cells.

Long-term exposure to arsenic, a widely distributed environmental toxicant, may result in damage to various organs, including the liver. Mice exposed chronically to arsenite developed hepatic damage, inflammation, and fibrosis, as well as increased levels of microRNA-21 (miR-21) and hypoxia-inducible factor (HIF)-1α. The levels of miR-21 and HIF-1α were also enhanced in primary hepatocytes and L-02 cells exposed to arsenite. The culture media from these cells induced the activation of hepatic stellate cells (HSCs), as demonstrated by up-regulation of the protein levels of α-smooth muscle actin (α-SMA) and collagen1A2 (COL1A2) and by increased activity in gel contractility assays. For L-02 cells, knockdown of miR-21 blocked the arsenite-induced up-regulation of HIF-1α and vascular endothelial growth factor (VEGF), which prevented the activation of LX-2 cells induced by medium from arsenite-exposed L-02 cells. However, these effects were reversed by down-regulation of von Hippel Lindau protein (pVHL). In arsenite-treated L-02 cells, miR-21 knockdown elevated the levels of ubiquitination and accelerated the degradation of HIF-1α via pVHL. In the livers of miR-21-/- mice exposed chronically to arsenite, there were less hepatic damage, lower fibrosis, lower levels of HIF-1α and VEGF, and higher levels of pVHL than for wild-type mice. In summary, we propose that miR-21, acting via the HIF-1α/VEGF signaling pathway, is involved in arsenite-induced hepatic fibrosis through mediating aberrant cross-talk of hepatocytes and HSCs. The findings provide evidence relating to the pathogenesis of hepatic fibrosis induced by exposure to arsenic.

LncRNA H19-mediated M2 polarization of macrophages promotes myofibroblast differentiation in pulmonary fibrosis induced by arsenic exposure.

Arsenic is a potent toxicant, and long-term exposure to inorganic arsenic causes lung damage. M2 macrophages play an important role in the pathogenesis of pulmonary fibrosis. However, the potential connections between arsenic and M2 macrophages in the development of pulmonary fibrosis are elusive. C57BL/6 mice were fed with drinking water containing 0, 10 and 20 ppm arsenite for 12 months. We have found that, in lung tissues of mice, arsenite, a biologically active form of arsenic, elevated H19, c-Myc, and Arg1; decreased let-7a; and caused pulmonary fibrosis. For THP-1 macrophages (THP-M) and bone-marrow-derived macrophages (BMDMs), 8 µM arsenite increased H19, c-Myc, and Arg1; decreased let-7a; and induced M2 polarization of macrophages, which caused secretion of the fibrogenic cytokine, TGF-ß1. Down-regulation of H19 or up-regulation of let-7a reversed the arsenite-induced M2 polarization of macrophages. Arsenite-treated THP-M and BMDMs co-cultured with MRC-5 cells or primary lung fibroblasts (PLFs) elevated levels of p-SMAD2/3, SMAD4, α-SMA, and collagen I in lung fibroblasts and resulted in the activation of lung fibroblasts. Knockout of H19 or up-regulation of let-7a in macrophages reversed the effects. The results indicated that H19 functioned as an miRNA sponge for let-7a, which was involved in arsenite-induced M2 polarization of macrophages and induced the myofibroblast differentiation phenotype by regulation of c-Myc. In the sera of arseniasis patients, levels of hydroxyproline and H19 were higher, and levels of let-7a were lower than levels in the controls. These observations elucidate a possible mechanism for arsenic exposure-induced pulmonary fibrosis.

MicroRNA-15b in extracellular vesicles from arsenite-treated macrophages promotes the progression of hepatocellular carcinomas by blocking the LATS1-mediated Hippo pathway.

Arsenic, a human carcinogen, causes various human cancers, including those of the skin, lung, and liver. Hepatocellular carcinomas (HCCs), which have high mortality, are common malignancies worldwide. Tumor-associated macrophages (TAMs), which are considered to be similar to M2-polarized macrophages, promote tumor invasion and progression. Small non-coding RNAs (miRNAs) regulate expression of genes involved in progression of various malignancies. Extracellular vesicles (EVs), as mediators of cell communication, pass specific miRNAs directly from TAMs to tumor cells, promoting tumor pathogenesis and metastasis. In HCCs, large tumor suppressor kinase 1 (LATS1), functions as a tumor suppressor. However, the molecular mechanism by which miRNA modulates LATS1 expression in HCCs remains unclear. The results show that exposure to arsenite, increased miR-15b levels and induced M2 polarization of THP-1 cells. Elevated levels of miR-15b were transferred from arsenite-treated-THP-1 (As-THP-1) cells to HCC cells via miR-15b in EVs inhibited activation of the Hippo pathway by targeting LATS1, and was involved in promoting the proliferation, migration, and invasion of HCC cells. In conclusion, miR-15b in EVs from As-THP-1 cells is transferred to HCC cells, in which it targets and downregulates LATS1 expression and promotes the proliferation, migration, and invasion of HCC cells.

Circ0061052 regulation of FoxC1/Snail pathway via miR-515-5p is involved in the epithelial-mesenchymal transition of epithelial cells during cigarette smoke-induced airway remodeling.

Circular RNA (circRNA) has been shown to be widely involved in a variety of lung diseases. Cigarette smoke (CS) may induce epithelial-mesenchymal transition (EMT) of airway remodeling in chronic obstructive pulmonary disease (COPD), however, in which the roles and mechanisms of circRNA have not been elucidated. In this study, we aimed to determine whether circ0061052 is involved in the EMT of human bronchial epithelial (HBE) cells and its potential mechanism for playing a biological role. Cigarette smoke extract (CSE) caused elevated EMT indicators and the increases of circ0061052 in HBE cells. Circ0061052 has a ring structure and is mainly present in the cytoplasm of HBE cells. We analyzed the regulatory relationship between circ0061052 and miR-515-5p using bioinformatics, a luciferase reporter gene, and qRT-PCR. We found that circ0061052 is mainly distributed in the cytoplasm and competitively binds to miR-515-5p, acting as a sponge for miR-515-5p. The luciferase reporter gene showed that miR-515-5p binds to the 3'UTR region of FoxC1 mRNA to inhibit its transcription. For HBE cells, overexpression of miR-515-5p antagonized the CSE-induced EMT. In addition, circ0061052 acts by binding miR-515-5p competitively to regulate the expression of FoxC1/Snail. When circ0061052 siRNA and miR-515-5p inhibitor were co-transfected into HBE cells, the inhibitor reversed the effect of circ0061052 siRNA on reducing EMT. Chronic exposure of mice to CS induced increases of circ0061052 levels, decreases of miR-515-5p levels, and the EMT in lung tissue, which caused dysfunction and airway obstruction. Overall, the results show that, by regulating miR-515-5p through a FoxC1/Snail regulatory axis, circ0061052 is involved in the CS-induced EMT and airway remodeling in COPD.

MircoRNA-143-3p regulating ARL6 is involved in the cadmium-induced inhibition of osteogenic differentiation in human bone marrow mesenchymal stem cells.

Cadmium, which is extensively distributed in the environment, accumulates in organisms through the trophic chain. Although cadmium can cause bone injury, its role in osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs) remains unclear. The present study investigated the effect of cadmium chloride (CdCl2) on osteogenesis of hBMSCs and the underlying mechanism. CdCl2 dose-dependently reduced the viability of hBMSCs. Concentrations of CdCl2 (2.5 and 5.0 µM) increased miR-143-3p levels; decreased levels of adenosine diphosphate-ribosylation factor-like protein 6 (ARL6); inhibited Wnt family member 3A (Wnt3a), ß-catenin, lymphoid enhancer factor (LEF1), and T-cell factor 1 (TCF1); and suppressed osteogenesis of hBMSCs. Inhibition of miR-143-3p or overexpression of ARL6 with lentivirus blocked these CdCl2-induced changes. Luciferase reporter assays confirmed that miR-143-3p binds to the 3'-UTR regions of ARL6 mRNA. Reduced-expression of miR-143-3p enhanced the CdCl2-induced suppression of the osteogenesis of hBMSCs and inhibition of the Wnt/ß-catenin pathway, effects that were reversed by down-regulated expression of ARL6. Thus, miR-143-3p targets ARL6 to down-regulate the Wnt/ß-catenin pathway, which is involved in the suppression of osteogenic differentiation of hBMSCs. The results provide new directions for clinical treatment of bone diseases resulting from cadmium toxicity.

MicroRNA-16, via FGF2 Regulation of the ERK/MAPK Pathway, Is Involved in the Magnesium-Promoted Osteogenic Differentiation of Mesenchymal Stem Cells.

microRNAs (miRNAs) participate in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). However, few reports have discussed the effect of miRNAs on the magnesium chloride (MgCl2)-induced promotion of osteogenic differentiation of BMSCs, a process involved in the healing of bone tissue. As determined in the present investigation, MgCl2 decreased miR-16 levels; increased levels of fibroblast growth factor 2 (FGF2), p-p38, and p-ERK; and promoted the osteogenic differentiation of BMSCs. Enhancement of miR-16 levels by an miR-16 mimic blocked these MgCl2-induced changes. Moreover, luciferase reporter assays confirmed that miR-16 binds to the 3'UTR region of FGF2 mRNA. Down-regulation of FGF2 blocked the MgCl2-induced increases of p-p38 and p-ERK and the promotion of the osteogenic differentiation of BMSCs. Furthermore, over-expression of miR-16 attenuated the MgCl2-induced overproduction of p-p38 and p-ERK1/2 and the high levels of osteogenic differentiation, effects that were reversed by elevated expression of FGF2. In summary, the present findings provide a mechanism by which miR-16 regulates MgCl2-induced promotion of osteogenic differentiation by targeting FGF2-mediated activation of the ERK/MAPK pathway.

Role of B-Cell Lymphoma 2 Ovarian Killer (BOK) in Acute Toxicity of Human Lung Epithelial Cells Caused by Cadmium Chloride.

BACKGROUND B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) is a Bcl-2 family member with sequence homology to pro-apoptotic BAX and BAK, but its physiological and pathological roles remain largely unclear. Exposure of cells to cadmium may cause DNA damage, decrease DNA repair capacity, and increase genomic instability. MATERIAL AND METHODS The present study investigated the effects of BOK on the toxicity of cadmium chloride (CdCl2) to human bronchial epithelial (16HBE) cells. We constructed BOK over-expressing (16HBE-BOK) cells and BOK knockdown (16HBE-shBOK) cells using the BOK-ORF plasmid and BOK-siRNA. qRT-PCR for BOK mRNA expression. We used Trypan blue exclusion assay for cell growth, MTT colorimetric assays for cells inhibition rate, and Comet assays for detecting damaged DNA. RESULTS CdCl2, at various concentrations and exposure times, increased BOK mRNA. 16HBE-BOK cells (BOK over-expressing) proliferated more than 16HBE cells after 72 h; 16HBE-shBOK (BOK knockdown) cells proliferated less. In addition, BOK deficiency enhanced cell death induced by CdCl2. Similarly, CdCl2- and H2O2-induced DNA damage was greater in BOK-deficient cells. CONCLUSIONS These findings support a role for BOK in CdCl2-induced DNA damage and cell death.

NF-κB-regulation of miR-155, via SOCS1/STAT3, is involved in the PM2.5-accelerated cell cycle and proliferation of human bronchial epithelial cells.

Air pollution, especially fine particulate matter (PM2.5, particles <2.5 µm in size), induces adverse health effects on the respiratory system. Uncontrolled proliferation of bronchial epithelial cells, resulting from deregulated cell cycle progression, contributes to pulmonary homeostatic imbalance. Although dysregulation of miRNAs is involved in a variety of pathophysiologic processes, the role of miRNAs in lung injury caused by PM2.5 is unclear. In the present study, we found that different concentrations of PM2.5 caused a biphasic effect on proliferation of human bronchial epithelial (HBE) cells. PM2.5 induced an aberrant cell cycle and proliferation of HBE cells, and up-regulated miR-155 levels with a concentration-dependent manner. High miR-155 expression, mediated by NF-κB activation, produced an accelerated G1/S phase and cell proliferation though the STAT3 pathway, which targeted SOCS1. These findings indicate that NF-κB-mediated miR-155 induces an altered cell cycle through epigenetic modulation of the SOCS1/STAT3 signaling pathway and provide a mechanism for the biphasic effect of different concentrations of PM2.5 in inducing respiratory injury.

Andrographolide antagonizes the cigarette smoke-induced epithelial-mesenchymal transition and pulmonary dysfunction through anti-inflammatory inhibiting HOTAIR.

In cells of the lung surface, cigarette smoke (CS) induces inflammatory and epithelial-mesenchymal transition (EMT), effects that are related to pulmonary dysfunction and Chronic obstructive pulmonary disease (COPD). However, the molecular mechanisms involved remain largely unknown, and potential therapeutic approaches are under development. In the present study, with cell culture and animal studies, we showed that CS exposure causes pulmonary dysfunction and airway remodeling with inflammatory cell infiltration. Consistent with these pulmonary lesions, the inflammatory factors interleukin-6 (IL-6) and interleukin-8 (IL-8) were increased in mice exposed to CS for 4 days. Accordingly, downstream signal transducer and activator of transcription 3 (STAT3) was activated, which up-regulated expression of the lncRNA HOTAIR, and enhancer of zeste homolog 2 (EZH2). In addition, CS exposure led to decreased levels of E-cadherin and to increased N-cadherin, vimentin, and α-SMA, indicating that the EMT was induced in mouse lung tissues. These effects, including increases of IL-6 and HOTAIR, were confirmed in human bronchial epithelial (HBE) cells treated with cigarette smoke extract (CSE). Finally, we established that, in HBE cells, andrographolide reversed the CSE-induced EMT via decreasing IL-6 levels and, in an animal model, prevented CS-induced lung inflammation and small airway remodeling, indicating that it has potential clinical application for CS-induced pulmonary dysfunction and COPD.

Wnt/ß-Catenin Pathway Is Involved in Cadmium-Induced Inhibition of Osteoblast Differentiation of Bone Marrow Mesenchymal Stem Cells.

Cadmium is a common environmental pollutant that causes bone damage. However, the effects of cadmium on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) and its mechanism of action in this process are unclear. Here, we determined the effects of cadmium chloride (CdCl2) on the osteogenic differentiation of BMMSCs and the potential mechanism involved in this process. As determined in the present investigation, CdCl2, in a concentration-dependent manner, affected the viability of BMMSCs and their cytoskeletons. Exposure to 0.1 or 0.2 µM CdCl2 inhibited osteogenic differentiation of BMMSCs, which was reflected in the down-regulation of osteoblast-related genes (ALP, OCN, Runx2, OSX, and OPN); in suppression of the protein expression of alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2); and in decreased ALP activity and capacity for mineralization. Moreover, mRNA microarray was performed to determine the roles of these factors in BMMSCs treated with CdCl2 in comparison to control BMMSCs. As determined with the microarrays, the Wingless-type (Wnt), mothers against decapentaplegic and the C. elegans gene Sam (SMAD), and Janus kinase-Signal Transducers and Activators of Transcription (JAK-STAT) signaling pathways were involved in the effects caused by CdCl2. Moreover, during differentiation, the protein levels of Wnt3a, ß-catenin, lymphoid enhancer factor 1 (LEF1), and T-cell factor 1 (TCF1) were reduced by CdCl2. The current research shows that CdCl2 suppresses the osteogenesis of BMMSCs via inhibiting the Wnt/ß-catenin pathway. The results establish a previously unknown mechanism for bone injury induced by CdCl2.

Regulation of gasdermin D by miR-379-5p is involved in arsenite-induced activation of hepatic stellate cells and in fibrosis via secretion of IL-1ß from human hepatic cells.

Arsenic is an environmental toxicant and human carcinogen. The liver is the main site of arsenic storage and metabolism. Exposure to excessive arsenic causes liver damage and release of pro-inflammatory factors, which in turn lead to liver fibrosis. Gasdermin D (GSDMD), a mediator of pyroptosis, has low expression in hepatic tumor cells. In L-02 cells, arsenite caused increases of GSDMD and cleaved caspase-1 levels and decreases of caspase-1 and miR-379-5p levels. It also promoted the release of IL-1ß in a concentration- and time-dependent manner. Luciferase reporter assays showed that GSDMD was a direct target of miR-379-5p. In L-02 cells, the over-expression of miR-379-5p blocked the arsenite-induced increases of GSDMD levels and the release of IL-1ß, effects that were reversed by up-regulation of GSDMD. LX-2 cells, cultured in the media from arsenite-treated L-02 cells, showed elevated levels of proliferating cell nuclear antigen (PCNA), collagen I, vimentin, and α-smooth muscle actin (α-SMA), which indicated activation of these cells. Activation of LX-2 cells by media from arsenite-treated L-02 cells was inhibited by IL-1ß neutralizing antibody. The media from arsenite-treated L-02 cells transfected with an miR-379-5p mimic inhibited the activation of LX-2 cells, a process that was reversed by up-regulation of GSDMD and by co-treatment with human recombinant IL-1ß. Chronic exposure to arsenite induced, in liver tissue of mice, morphological damage, collagen deposition, and activation of hepatic stellate cells (HSCs). In liver tissue of arsenite-exposed mice, the levels of miR-379-5p were lower, but the levels of GSDMD and cleaved caspase-1 were elevated, and in sera from arsenite-exposed mice, the IL-1ß levels were elevated. These results indicate that, by elevating the secretion of IL-1ß, miR-379-5p regulation of GSDMD is involved in arsenite-induced activation of HSCs and in hepatic fibrosis. This establishes a previously unknown molecular mechanism for arsenite-induced liver damage, inflammation, and fibrosis.

miR-149 Negative Regulation of mafA Is Involved in the Arsenite-Induced Dysfunction of Insulin Synthesis and Secretion in Pancreatic Beta Cells.

Chronic exposure to arsenic, a potent environmental oxidative stressor, is associated with the incidence of diabetes. However, the mechanisms for arsenite-induced reduction of insulin remain largely unclear. After CD1 mice were treated with 20 or 40 ppm arsenite in the drinking water for 12 months, the mice showed reduced fasting insulin levels, a depression in glucose clearance, and lower insulin content in the pancreas. The levels of glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cells isolated from arsenite-exposed mice were low compared with those for control mice. Immunohistochemistry studies showed that arsenite exposure resulted a reduction of insulin content in the pancreas of mice. Exposure of Min6 cells, a pancreatic beta cell line, to low levels of arsenite led to lower GSIS in a dose- and time-dependent fashion. Since microRNAs (miRNAs) are involved in pancreatic ß-cell function and the pathogenesis of diabetes, we hypothesized that arsenite exposure activates miR-149, decreases insulin transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (mafA), and induces an insulin synthesis and secretion disorder. In arsenite-exposed Min6 cells, mafA activity was lowered by the increase of its target miRNA, miR-149. Luciferase assays illustrated an interaction between miR-149 and the mafA 3' untranslated region. In Min6 cells transfected with an miR-149 inhibitor, arsenite did not regulate GSIS and mafA expression. In control cells, however, arsenite decreased GSIS or mafA expression. Our results suggest that low levels of arsenite affect ß-cell function and regulate insulin synthesis and secretion by modulating mafA expression through miR-149.