| Arsenic compounds, human carcinogens, widely exist in the environment, whose main existence form is sodium arsenite (NaAsO2). Long-term exposure to arsenide causes a variety of diseases, and skin cancer is one of these most serious diseases. Skin cancer is one of the most common malignant tumors, and its incidence is high. What is more, the skin cancer caused by endemic arsenic poisoning has been a major threat to the health of local people. Seeking biomarkers for early diagnosis and targeted therapies of skin cancer have always been hot spots in the field of environmental toxicology. At present, many studies have shown that inflammation and microRNAs are closely associated with human skin cancer, however, their molecular mechanisms of tumorigenesis and tumor progression are still unclear.The change of inflammatory microenvironment is an important factor of tumorigenesis. The secretion of inflammatory cell factor can induce the disorder of intracellular environment, which promotes tumor progression. Epithelial to mesenchymal transition (EMT), involving in a variety of disease process, is a key step in the development of malignant tumor. Long-term exposure to low concentration of arsenite may cause skin tissue injury and inflammation, which promotes skin cancer. EMT, the bridge between tumor and inflammation, is promoted by activating downstream signaling pathways via relevant miRNAs. Signal transducers and activators of transcription 3 (STAT3) pathway is closely related to miR-21 level. MiR-21, one of discovered earliestly carcinogenic miRNAs, is involved in cell growth, apoptosis, migration, invasion, and regulating the EMT process. However, it has not been reported that the roles and its molecular mechanisms of arsenite-induced inflammatory microenvironment change in EMT and malignant transformation of skin cells.Base on above-mentioned reasons, it is used in this investigation that chronic treatment of arsenite has induced the malignant transformation of human keratinocytes (HaCaT). Using a variety of molecular biology methods, we have explored the effects of inflammatory cytokine interleukin-6 (IL-6) on STAT3 activation and miR-21 level to reveal the roles and its molecular mechanisms of inflammatory response and EMT in arsenite-induced malignant transformation of HaCaT cells. The results would provide certain scientific clues for investigating the molecular mechanism of skin injury and cancer induced by environmental chemical, which supplys some scientific basis for seeking biomarkers of early diagnosis and prognosis in skin injury and cancer.Methods1. Effects of arsenite on the EMT in HaCaT cellsTo induce the malignant transformation of HaCaT cells, cells were chronically exposed to 0.0 or 1.0 μM NaAsO2 for 0,10,20, and 30 passages (about 15 weeks). We got the different passages HaCaT cells of control (HaCaT-C) and malignant (HaCaT-As). Western blots were used to detect the levels of E-cadherin and vimentin, which is used to investigat the effects of NaAsO2 on the EMT of HaCaT cells.2. Effects of arsenite on IL-6 levels in HaCaT cellsAfter HaCaT cells were chronically exposed to 0.0 or 1.0 μM NaAsO2 for 0,10, 20, and 30 passages, or acutely exposed to 0.0 or 1.0 μM NaAsO2 for 0,6,12, and 24 h, we tested the levels of IL-6 expression and secretion by real-time polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively, to observe the effects of NaAsO2 on levels of IL-6 mRNA and secretion in HaCaT cells.3. Effects of arsenite on STAT3 activation in HaCaT cellsHaCaT cells were chronically treated by 0.0 or 1.0 μM NaAsO2 for 0,10,20, and 30 passages, or acutely treated by 0.0 or 1.0 μM NaAsO2 for 0,15’,30’,1,3, and 6 h, we detected the the levels of STAT3 and phosphorylation-STAT3 (p-STAT3) by Western blot to observe the effects of NaAsO2 on STAT3 activation in HaCaT cells.4. Effects of arsenite on miR-21 levels in HaCaT cellsAfter HaCaT cells were chronically treated by 0.0 or 1.0 μM NaAsO2 for 0,10, 20, and 30 passages, or acutely treated by 0.0 or 1.0 μM NaAsO2 for 0,6,12, and 24 h, we detected miR-21 levels by quantitative real-time polymerase chain reaction (qRT-PCR) to observe the effects of NaAsO2 on levels of miR-21 in HaCaT cells.5. The roles of IL-6 in arsenite-induced activations of STAT3 and increases of miR-21 levels in HaCaT cellsAfter HaCaT cells were treated by 0.5 μg/mL anti-IL-6 neutralizing antibody or anti-IgG antibody for 3 h, they were exposed to 0.0 or 1.0 μM NaAsO2 for 24 h. The levels of STAT3 and p-STAT3 and miR-21 levels were determinted by Western blots and qRT-PCR. We were going to investigate the roles of IL-6 in arsenite-induced STAT3 activation and improvement of miR-21 levels in HaCaT cells.6. Roles of STAT3 in arsenite-induced upregulation of miR-21 levels in HaCaT cellsHaCaT cells were pre-treated with 25 μM STAT3 siRNA or Con siRNA for 24 h, then exposed to 0.0 or 1.0 μM NaAsO2 for 24 h. Western blots were used to detect levels of STAT3 and its phosphorylation and qRT-PCR was used to measure miR-21 levels. We were going to explore the effects of blocking STAT3 on arsenite-induced upregulation of p-STAT3 and miR-21 levels in HaCaT cells.7. The roles of STAT3 regulating miR-21 in EMT during the malignant transformation of HaCaT cells induced by arseniteAfter arsenite-transformated HaCaT (HaCaT-30T) cells were treated with 25 μM STAT3 siRNA or Con siRNA for 24 h. they were treated with 80 μM miR-21 mimic or miR-NC mimic for 24 h. After HaCaT-30T cells were pre-treated with 25 μM STAT3 siRNA or Con siRNA for 24 h, then they were transfected with 80 μM miR-21 mimic or miR-NC mimic for 24 h. Western blots were used to detect the levels of p-STAT3 and STAT3 and qRT-PCR was used to measure miR-21 levels. The levels of E-cadherin and vimentin were detected by Western blots and immunofluorescence assay. We were going to investigate the roles of STAT3 regulating miR-21 in EMT during arsenite-transformated HaCaT cells.8. Effects of STAT3 regulating miR-21 on the malignant degree and the capacity of invasion and metastas in arsenite-transformated HaCaT cellsAfter HaCaT-30T cells were pretreated with 25 μM STAT3 siRNA or Con siRNA for 24 h, then they were transfected with 80 μM miR-21 mimic or miR-NC mimic for 24 h. Soft agar colony experiments were used to detect the malignant degrees of HaCaT-30T cells. Transwell assay was used to determinte invasion and metastas metastatic properties of HaCaT-30T cells. We were going to investigate the effects of STAT3 regulating miR-21 on the malignant degree and the capacity of invasion and metastas in arsenite-transformated HaCaT cellsResults1. Effects of arsenite on the EMT in HaCaT cellsHaCaT cells chronically treated with 1.0 μM NaAsO2 for 30 passages, During the arsenite-induced transformation of HaCaT cells, there were decreased levels of E-cadherin (one of epithelial cell markers) and increased levels of vimentin (on of interstitial cell markers) with increased treat time and malignant degree. The results indicate that arsenite induces EMT in HaCaT cells.2. Effects of arsenite on levels of IL-6 in HaCaT cellsIn HaCaT cells chronically treated with 1.0 μM NaAsO2 for 30 passages, the mRNA levels and secretion levels of IL-6 were markedly increased with increased treat time and malignant degree. In HaCaT cells treated with 1.0 μM NaAsO2 for 6, 12, and 24 h, IL-6 mRNA and secretion levels were increased in a time-dependent manner. Data indicate that NaAsO2 up-regulateS IL-6 levels, which induces inflammatory response in HaCaT cells.3. Effects of arsenite on STAT3 activation in HaCaT cellsIn HaCaT cells chronically treated with 1.0 μM NaAsO2 for 30 passages, p-STAT3 levels were significantly increased with increased treat time and malignant degree. And in HaCaT cells treated with 1.0 μM NaAsO2for 30 min,1,3, and 6 h, p-STAT3 levels were increased in a time-dependent manner, but STAT3 levels were basically unchanged. The results indicate that NaAsO2 activates STAT3 signaling pathway, which plays an improtant role in arsenite-induced malignant transformation of HaCaT cells.4. Effects of arsenite on levels of miR-21 in HaCaT cellsIn HaCaT cells chronically treated with 1.0 μM NaAsO2 for 30 passages, miR-21 levels were markedly increased with increased treat time and malignant degree. And in HaCaT cells treated with 1.0 μM NaAsO2for 6,12, and 24 h, miR-21 levels were increased in a time-dependent manner. The results show that NaAsO2 induces the up-regulation of miR-21 levels, which plays a key role in arsenite-induced malignant transformation of HaCaT cells.5. The roles of IL-6 in arsenite-induced the activation of STAT3 and the decreases of miR-21 levels in HaCaT cells1.0 μM NaAsO2 caused the increases of p-STAT3 levels and miR-21 levels, however, STAT3 levels were not significantly unchanges. The anti-IL-6 neutralizing antibody blocked 1.0 μM NaAsO2-induced increases of p-STAT3 levels and miR-21 levels in HaCaT cells. The results indicate that anti-IL-6 neutralizing antibody can down-regulate the arsenite-induced STAT3 activation and up-regulation of miR-21 levels, which indicates that IL-6 is involved in arsenite-induced STAT3 activation and improvement of miR-21 levels in HaCaT cells.6. The roles of STAT3 in arsenite-induced increases of miR-21 levels in HaCaT cells1.0 μM NaAsO2 caused the increases of p-STAT3 levels and miR-21 levels, however, STAT3 levels were not significantly unchanges.25 μM STAT3 siRNA blocked the increases of p-STAT3 and miR-21 levels induced by 1.0 μM NaAsO2 in HaCaT cells. Data indicate that the knock-down of STAT3 by siRNA can block NaAsO2-induced the increases of p-STAT3 and miR-21 levels, which suggests that STAT3 plays an important role in arsenite-induced up-regulation of miR-21 levels in HaCaT cells.7. The roles of STAT3 regulating miR-21 in EMT during the malignant transformation of HaCaT cells induced by arseniteThe blockage of STAT3 by siRNA decreased the levels of p-STAT3, STAT3, and miR-21 in HaCaT-30T cells, it induced the increases of protein levels and fluorescence intensities of E-cadherin, however, the changes of vimentin were contrary. MiR-21 mimic, which induced the increases of miR-21 levels in HaCaT-30T cells, blocked the decreases of protein levels and fluorescence intensities of E-cadherin, the changes of vimentin were contrary. The results suggest that the effects of STAT3 knockdown causing EMT reverse (MET) of arsenite-transformated HaCaT cells is blocked by up-regulation of miR-21, which indicats that miR-21 palys an important role in the EMT of arsenite-transformated HaCaT cells.8. Effects of STAT3 regulating miR-21 on malignant content and migration capabilities of arsenite-transformated HaCaT cellsThe cell clone number in soft agar and cell number of invasion and migration in STAT3 siRNA-treated HaCaT-30T cells were significantly lower than those in control HaCaT-30T cells. In HaCaT-30T cells with combined treatment of STAT3 siRNA and miR-21 mimic, the cell clone number in soft agar and cell number of invasion and migration were more than those in HaCaT-30T cells with treatment of STAT3 siRNA alone. The results show that miR-21 up-regulation may block the STAT3 knockdown-induce the decreases of malignant content and migration capabilities in arsenite-transformated HaCaT cells, which suggest that STAT3 activation via increasing miR-21 plays an important role in malignant transformation and migration capabilities in arsenite-transformated HaCaT cells.Conclusions1. The chronic treatment of arsenite induces EMT and malignant transformation of HaCaT cells.2. The inflammatory response of HaCaT cells is caused by the chronic treatment of arsenite, moreover, IL-6, one of inflammatory cytokines, is involved in arsenite-induced the activation of STAT3 activation and increases of miR-21 levels.3. The regulation of miR-21 by STAT3 plays an important role in arsenite-induced EMT and malignant transformation of HaCaT cells. |
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