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.

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.

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.

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.

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.

MALAT1 via microRNA-17 regulation of insulin transcription is involved in the dysfunction of pancreatic ß-cells induced by cigarette smoke extract.

Cigarettes contain various chemicals with the potential to influence metabolic health. Exposure to cigarette smoke causes a dysfunction in pancreatic ß-cells and impairs insulin production. However, the mechanisms for cigarette smoke-induced reduction of insulin remain largely unclear. Data from 558 patients with diabetes showed that, with smoking pack-years, homeostatic model assessment (HOMA)-ß (a method for assessing ß-cell function) decreased and that HOMA of insulin resistance increased. For ß-cells (MIN6), cigarette smoke extract (CSE) increased the levels of thioredoxin-interacting protein (TXNIP) and the long noncoding (lnc)RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), and downregulated the levels of the transcription factor, mafA, and microRNA (miR)-17. MALAT1, one of four lncRNAs predicted to regulate miR-17, was knocked down by small interfering RNA (siRNA). For these cells, an miR-17 mimic inhibited TXNIP and enhanced the production of insulin. Knockdown of MALAT1 induced an increase in miR-17, which suppressed TXNIP and promoted the production of insulin. In the sera of patients with diabetes who smoked, there were higher MALAT1 levels and lower miR-17 levels than in the sera of nonsmokers. Thus, CSE inhibits insulin production by upregulating TXNIP via MALAT1-mediated downregulation of miR-17, which provides an understanding of the processes involved in the reduced ß-cells function caused by cigarette smoke.

NF-κB-regulated miR-155, via repression of QKI, contributes to the acquisition of CSC-like phenotype during the neoplastic transformation of hepatic cells induced by arsenite.

Chronic exposure to arsenite can cause various human tumors. For the initiation and recurrence of human liver cancer, the acquisition of CSC-like properties is essential. In various cancers, microRNAs (miRNAs) act as regulators in induction of CSC-like properties. Liver cancers over-express miR-155, but the mechanism relating miR-155 and arsenite-induced liver cancer is unknown. Here, we show that long-term exposure of L-02 cells to arsenite increases miR-155 levels by activation of NF-κB and leads to the acquisition of CSC-like properties. In spheroids formed from arsenite-transformed L-02 cells, the levels of miR-155 positively relate to the levels of CD90, EpCAM, and OCT4. Inhibition of miR-155, by reduction of SOX2 and OCT4, results in suppression of spheroid formation. Luciferase reporter assays indicate that QKI is a target of miR-155. Inhibition of QKI expression by miR-155 promotes arsenite-induced acquisition of CSC-like properties, whereas QKI over-expression has the opposite effect. Collectively, the findings demonstrate that miR-155, driven by NF-κB, reduces QKI expression and is involved in acquisition of the CSC-like phenotype during neoplastic transformation of hepatic cells induced by arsenite.

Circ100284, via miR-217 regulation of EZH2, is involved in the arsenite-accelerated cell cycle of human keratinocytes in carcinogenesis.

Circular RNAs (circRNAs), a class of noncoding RNAs generated from pre-mRNAs, participate in regulation of genes. The mechanism for regulation, however, is unknown. Here, to determine if, in human keratinocyte (HaCaT) cells, circular RNAs are involved in arsenite-induced acceleration of the cell cycle, a circRNA microarray was performed to analyze the variability of circRNAs in arsenite-treated HaCaT (As-HaCaT) cells and in arsenite-transformed (T-HaCaT) cells in comparison to control HaCaT cells. Among the circRNAs up-regulated in both As-HaCaT cells and T-HaCaT cells, hsa:circRNA_100284 (circ100284) had the greatest increase and was chosen for further research. The presence of circ100284 was confirmed in HaCaT cells. In these cells, arsenite induced increases of EZH2 and cyclin D1 and accelerated the cell cycle. MicroRNA (miR)-217 suppressed the expression of EZH2 was involved in regulation of the cell cycle. Further, in HaCaT cells exposed to arsenite, EZH2 regulated the cell cycle by binding to the promoter of CCND1, which codes for cyclin D1. Moreover, knockdown of circ100284 with siRNA inhibited the cell cycle acceleration induced by arsenite, but this inhibition was reversed by co-transfection with circ100284 siRNA and by a miR-217 inhibitor. Knockdown of circ100284 with siRNA or transfected with miR-217 mimic inhibited the capacity of T-HaCaT cells for colony formation, invasion, and migration, effects that were reversed by co-transfection with a miR-217 inhibitor or by epigenetic expression of EZH2. These results suggest that, in HaCaT cells, arsenite increases circ100284 levels, which act as a sponge for miR-217 and up-regulate the miR-217 target, EZH2, which, in turn, up-regulates cyclin D1and CDK4, and thus accelerates the cell cycle and leads to malignant transformation. Thus, circ100284, via miR-217 regulation of EZH2, is involved in the arsenite-accelerated cell cycle of human keratinocytes in carcinogenesis. This establishes a previously unknown mechanism between arsenite-induced acceleration of the cell cycle and carcinogenesis.

Impaired autophagic flux and p62-mediated EMT are involved in arsenite-induced transformation of L-02 cells.

Autophagy is a catabolic process essential for preserving cellular homeostasis, and the epithelial-to-mesenchymal transition (EMT) is involved during tissue development and cancer progression. In arsenite-treated human hepatic epithelial (L-02) cells, arsenite reduced the autophagic flux, which caused accumulation of p62, an adaptor and receptor of autophagy. Further, in arsenite-transformed L-02 cells, the levels of E-cadherin were attenuated, but the levels of vimentin, which is expressed in mesenchymal cells, and Snail, a transcription regulator of the EMT, were up-regulated. Thus, after chronic exposure of L-02 cells to arsenite, the impaired autophagic flux induced the accumulation of p62, which up-regulated the expression of Snail, a protein involved in arsenite-induced EMT of these cells. Knockdown of p62 by siRNA reversed the arsenite-induced EMT and decreased the capacities of arsenite-transformed L-02 cells for colony formation and invasion and migration. Therefore, in arsenite-induced transformation of L-02 cells, the accumulation of p62, by impairing autophagic flux, mediates the EMT via Snail. These results provide a previously unknown mechanism underlying arsenic toxicity and carcinogenicity.

Epigenetic silencing of miR-218 by the lncRNA CCAT1, acting via BMI1, promotes an altered cell cycle transition in the malignant transformation of HBE cells induced by cigarette smoke extract.

Cigarette smoking is the strongest risk factor for the development of lung cancer, the leading cause of cancer-related deaths. However, the molecular mechanisms leading to lung cancer are largely unknown. A long-noncoding RNA (lncRNA), CCAT1, regarded as cancer-associated, has been investigated extensively. Moreover, the molecular mechanisms of lncRNAs in regulation of microRNAs (miRNAs) induced by cigarette smoke remain unclear. In the present investigation, cigarette smoke extract (CSE) caused an altered cell cycle and increased CCAT1 levels and decreased miR-218 levels in human bronchial epithelial (HBE) cells. Depletion of CCAT1 attenuated the CSE-induced decreases of miR-218 levels, suggesting that miR-218 is negatively regulated by CCAT1 in HBE cells exposed to CSE. The CSE-induced increases of BMI1 levels and blocked by CCAT1 siRNA were attenuated by an miR-218 inhibitor. Moreover, in CSE-transformed HBE cells, the CSE-induced cell cycle changes and elevated neoplastic capacity were reversed by CCAT1 siRNA or BMI1 siRNA. This epigenetic silencing of miR-218 by CCAT1 induces an altered cell cycle transition through BMI1 and provides a new mechanism for CSE-induced lung carcinogenesis.

Mammalian PIWI-piRNA-target complexes reveal features for broad and efficient target silencing.

The PIWI-interacting RNA (piRNA) pathway is an adaptive defense system wherein piRNAs guide PIWI family Argonaute proteins to recognize and silence ever-evolving selfish genetic elements and ensure genome integrity. Driven by this intensive host-pathogen arms race, the piRNA pathway and its targeted transposons have coevolved rapidly in a species-specific manner, but how the piRNA pathway adapts specifically to target silencing in mammals remains elusive. Here, we show that mouse MILI and human HILI piRNA-induced silencing complexes (piRISCs) bind and cleave targets more efficiently than their invertebrate counterparts from the sponge Ephydatia fluviatilis. The inherent functional differences comport with structural features identified by cryo-EM studies of piRISCs. In the absence of target, MILI and HILI piRISCs adopt a wider nucleic-acid-binding channel and display an extended prearranged piRNA seed as compared with EfPiwi piRISC, consistent with their ability to capture targets more efficiently than EfPiwi piRISC. In the presence of target, the seed gate-which enforces seed-target fidelity in microRNA RISC-adopts a relaxed state in mammalian piRISC, revealing how MILI and HILI tolerate seed-target mismatches to broaden the target spectrum. A vertebrate-specific lysine distorts the piRNA seed, shifting the trajectory of the piRNA-target duplex out of the central cleft and toward the PAZ lobe. Functional analyses reveal that this lysine promotes target binding and cleavage. Our study therefore provides a molecular basis for the piRNA targeting mechanism in mice and humans, and suggests that mammalian piRNA machinery can achieve broad target silencing using a limited supply of piRNA species.

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.

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.].

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.

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.

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-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.

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.