The Roles Of Reactive Oxygen Species-regulated MIR-21via ERK/NF-κB In The Cell Transformation Induced By Low Levels Of Arsenite

Endemic arsenic poisoning and the arsenic pollutions induced by industry and agriculture damage severely human health. Epidemiological evidences show that the exposure to inorganic arsenic induces many kinds of cancer, such as tumors of skin, lung, and bladder. Although arsenide is confirmed as a group1of carcinogen by the International Agency for Research on Cancer (IARC), the mechanisms underlying arsenide in carcinogenesis are still not well understand. Genetic and epigenetic alterations may contribute to effects of arsenide-induced carcinogenesis. The mechanisms of epigenetic include usually three patterns: the altered mode of DNA methylation, the abnormality of histone modification, and the changes of microRNA (miRNA) levels, etc.Arsenite generates reactive oxygen species (ROS) during its metabolism in cells and body.The relationship between the ROS and the exposure to level of arsenite presents positive correlation relationship. Recent evidences have shown that ROS is involved in arsenic-induced cell injuries. but further investigations will be needed to understand the underlying mechanisms. low levels of ROS are involved in the signal pathways of cell transformation and can affect cytoplasmic and nuclear signal pathways to regulate gene expression and to stimulate cell proliferation, however, high levels of ROS induce the oxidative damages to lead cell death.Because miRNAs are involved in the initiation and development of tumors, which may play as function of oncogenes of tumor suppressors. One of miRNAs, microRNA21(miR-21), a putative oncogene, is overexpressed in a variety of malignancies and is implicated in various malignancy-related processes, including cell proliferation, apoptosis, invasion, and metastasis. The exposure of cells to ionizing radiation generates ROS, which leds to the up-regulation of miR-21. The abnormal expressions of miR-21are associated with hepatocellular carcinogenesis.We have previously reported that long-term exposure of HELF cells to low levels of arsenite induced the malignant transformation and ROS generation. Based on the results and the literature reports, we hypothesize that ROS and miR-21may involve in arsenite-induced malignant transformation of cells and carcinogenesis. It is not reported that the roles of miR-21in cell proliferation and transformation caused by chronic exposure to arsenite. In the present study, arsenite-transformed HELF cells were used to help characterize the possible mechanisms of ROS and miR-21functions in arsenite-inudced carcinogenesis, which will help us understand the mechanisms of arsenide toxicity and carcinogenesis and provide some new clues to find biomarkers and prevention measures of arsenic poisioning.Methods1. The malignant transformation and the changes of growth kinetics induced by low levels of arsenite in HELF cellsThe malignant degree of cells was examined by cell colony after HELF cells were exposed to1.0μM arsenite for10,20, or30passages. The doubling time of cells were detected with cell counting method.2. The effects of low levels of arsenite on miR-21level of HELF cellsAfter HELF cells were exposed to0.0or1.0μM arsenite for10,20, or30passages or exposured to1.0μM arsenite for0,1,3,6,12, or24h,.the level of miR-21was detected with the quantitative real-time polymerase chain reaction (qRT-PCR) after total cellular RNA was isolated.3. The roles of miR-21in arsenite-induced cell proliferation of HELF cellsAfter HELF cells were transfected with150nM anti-miR-nc or anti-miR-21for24h and were exposed to0.0or1.0μM arsenite for24h, the level of miR-21was detected with qRT-PCR and cell proliferation was evaluated by CCK-8.4. Effects of low levels of arsenite on the activations of ERK and NF-κB in HELF cellsThe levels of ERK, NF-κB p65, phosho-ERK, and phosho-NF-KB p65were detected by western blot after HELF cells were exposed to0.0or1.0μM arsenite for10,20, or30passages or exposured to1.0μM arsenite for0,1,3, or6h.5. The roles of ROS in the increases of miR-21level and the activations of ERK and NF-κB induced by low levels of arsenite in HELF cellsHELF cells were cultured in arsenite (0.0or1.0μM) for24h and then exposed to1.5U superoxide dismutase (SOD) for1h or transfected with10nM catalase for1h and then cultured in arsenite (0.0or1.0μM) or hydrogen peroxide (0.0or10.0μM) for24h. The levels of superoxice anion and the levels of peroxides were examined by dihydroethidium (DHE) assay and2’,7’-dichlorofluorescin (DCFH-DA) assay, respectively. The level of miR-21was detected with qRT-PCR. The levels of ERK, NF-κB p65, phosho-ERK, and phosho-NF-κB p65were detected by western blot.6. The roles of ERK and NF-κB in low levels of arsenite-induced increase of miR-21level in HELF cellsAfter cells were treated with10μM U0126(ERK inhibitor) or Bay11-7082(NF-κB p65inhibitor) for3h or transfected with20nM control siRNA or NF-κB p65siRNA, they were exposed to0.0or1.0μM arsenite for24h. The level of miR-21was detected with qRT-PCR. The levels of ERK, NF-κB p65, IκB a, phosho-ERK, phosho-NF-KB p65, phosho-lκB aPten, Pdcd4, Spryl, and Spry2were detected by western blot. The cellular localization of p-NF-KB p65and the binding of NF-κB p65to miR-21promoter were measured by the immunofluorescence staining and ChIP assay, respectively.7. ERK activation was feedback regulated by miR-21through Spry1After HELF cells were transfected with150nM anti-miR-nc or anti-miR-21for24h, they were incubated with0.0or1.0μM arsenite for1or24h. The levels of ERK, phosho-ERK, and Spryl were detected by western blot.8. The effects of miR-21up-regulation on the neoplastic capacity and cell motility of arsenite-transformated HELF cellsAfter HELF cells and the HELF cells treated by1.0μM arsenite for10passages were transfected with150and100nM miR-21mimic, respectively, for24h, their neoplastic capacity was determined by assay for anchorage-independent growth, and their motility was detected by wound-scratch healing assay. The miR-21level was detected with qRT-PCR and the levels of ERK, NF-κB p65, phosho-ERK, phosho-NF-KB p65, and Spry1were detected by western blot. 9. The effects of miR-21knockdown on the neoplastic capacity and cell motility of arsenite-transformated HELF cellsThe HELF cells treated by1.0μM arsenite for30passages were transfected with or without150nM anti-miR-nc or anti-miR-21for24h. The neoplastic capacity and motility of cells were determined by assays for anchorage-independent growth and by wound-scratch healing assay, respectively. The miR-21levels were detected with qRT-PCR nd the levels of ERK, NF-κB p65, phosho-ERK, phosho-NF-κB p65, and Spry1were detected by western blot.Results1. Low levels of arsenite induced the malignant transformation and the changes of growth kinetics in HELF cellsThe colonies in agar of HELF cells treated by1.0μM arsenite for20and30passages were formed more than control HELF cells and HELF cells treated by1.0μM arsenite for10passages. The doubling time of the transformed cells for10,20, or30passages were significantly shorter than that of passage control cells. Data indicated that the long-term exposure of HELF cells to low levels of arsenite induces cell transformation, moreover, there was greater malignancy and proliferation with increased time of exposure.2. The effects of low level arsenite on miR-21levels in HELF cellsmiR-21levels of HELF cells exposed to1.0μM arsenite for10,20, or30passages were greater than the those of control HELF cells. miR-21was overexpressed after HELF cell exposured to1.0μM arsenite for3,6,12, or24h. These results indicated that the levels of miR-21were increased by actue and chronic exposure of HELF cells to arsenite and there was the relationship of time-effect..3. The roles of miR-21in low levels of arsenite-induced cell proliferation of HELF cells150nM anti-miR-21inhibited significantly1.0μM arsenite-induced increases of miR-21levels. The transfection of HELF with anti-miR-21blocked obviously the increases of cell proliferation induced by1.0μM of arsenite. Date indicated that miR-21played an important role in cell proliferation of HELF cells induced by low levels of arsenite.4. Effects of low levels of arsenite on the activations of ERK and NF-κB in HELF cellsThe levels of phosho-ERK and phosho-NF-κB p65in HELF cells exposed to1.0μM arsenite for10,20, or30passages were higher than those in control HELF cells,. moreover, there was increased expressions of phosho-ERK and phosho-NF-κB p65with increased time of exposure to arsenite. The increases of ERK and NF-κB p65levels in HELF cells were induced by1.0μM of arsenite for1,3, and6h. Date suggested that ERK and NF-κB p65were activted by acute or chronic treatment with1.0μM arsenite.5. The roles of ROS in the increase of miR-21level and the activations of ERK and NF-κB induced by low levels of arsenite in HELF cellsThe exposure of HELF cells to1.0μM arsenite induced the generation of ROS including superoxice anion and peroxides and the increases of miR-21, phosho-ERK and phosho-NF-κB p65levels. SOD or Catalase blocked the effects induced by1.0μM arsenite. These results suggested that ROS including superoxice anion and peroxides involved in the increases of miR-21level and the activations of ERK and NF-κB induced by low levels of arsenite.intracellular.6. The roles of ERK and NF-κB in low levels of arsenite-induced increase of miR-21level in HELF cellsThe exposure of HELF cells to1.0μM arsenite induced the increases of miR-21, phosho-ERK, phosho-IκB a, phosho-NF-KB p65, and miR-21levels, the improvement of fluorescence intensity in intranuclear phosho-NF-KB p65, and the decreases of Pte、Pdcd4, and Spry1, the target proteins of miR-21. The inhibition of ERK by U0126prevented significantly the changes induced by1.0μM arsenite. The blockage of NF-κB by Bay11-7082, NF-kB inhibitor, or NF-κB p65siRNA prevented arsenite-induced miR-21upregulation and the decreases of Spry1, Pten, and Pdcd4levels. The examination of putative transcription binding sites revealed that there were two NF-κB binding sites in the miR-21promoter.1.0μM arsenit induced the increases of NF-κB p65binding to miRA-21promoter, however, Bay11-7082blocked the interactions. These results suggested that the arsenite-induced increase of miR-21level was regulated by ERK activation of NF-κB.7. ERK activation was feedback regulated by miR-21through Spry11.0μM arsenite induced the increase of phosho-ERK level at a earlier time (1hour) and the decrease of Spry1level in the at a later time (24hours). The knockdown of miR-21blocked significantly the the increase of phosho-ERK level and the decreases of Spry1levels induced by1.0μM arsenite. These results indicated that miR-21regulated feedback ERK activation.8. The effects of miR-21up-regulation on the neoplastic capacity and cell motility of arsenite-transformated HELF cellsAfter HELF cells and the HELF cells treated by1.0μM arsenite for10passages were transfected with100or150nM miR-21mimic, respectively, cells were obtained in which the levels of miR-21were similar. In HELF cells treated by1.0μM arsenite for10passages, Up-regulation of miR-21by transfection of miR-21-mimic induced the increases of formed colonies in agar and stimulate filling up the wound, the increases of phosho-ERK and phosho-NF-κB p65levels, and the decrease of Spry1level. These results indicated that up-regulation of miR-21promoted the neoplastic transformation and cell migration of HELF cells treated by1.0μM arsenite for10passages and improved feedback the activation of ERK/NF-κB pathway via down-regulaton of Spry1level in low levels of arsenite-treated HELF cells.9. The effects of miR-21knockdown on the neoplastic capacity and cell motility of arsenite-transformated HELF cellsDown-regulation of miR-21by transfection of150nM anti-miR-21induced the decreases of formed colonies in agar and stimulate filling up the wound, the declines of phosho-ERK and phosho-NF-κB p65levels, and the improvement of Spry1level. These results indicated that down-regulation of miR-21prevented the neoplastic transformation and migration of HELF cells treated by1.0μM arsenite for30passages and blocked feedback the activation of ERK/NF-κB pathway via up-regulaton of Spry1level in low levels of arsenite-treated HELF cells.Conclusions1. The chronical and acute exposures to low levels of arsenite induce the increases of miR-21and the activations of ERK and NF-κB in HELF cells.2. miR-21is involved in the cell proliferation and malignant transformation induced by low levels of arsenite in HELF cells.3. the regulation of ERK/NF-κB by ROS is associated with low levels of arsenite-induced increases of miR-21level in HELF cells4. ERK activation is feedback regulated by miR-21through Spryl.5. miR-21promotes the neoplastic capacity and cell motility of arsenite-transformated HELF cells.In summary, low levels of arsenite-induced upregulation of miR-21depends on the ERK/NF-κB pathway, through production of ROS, and is involved in a feedback loop between miR-21and ERK/NF-κB mediated by Spry1, which caused cell abnormal proliferation and thereby promoted the neoplastic transformation of HELF cells induced by a low level of arsenite.