The Roles And Mechanisms Of HIF-2α-mediated MiR-191 In The Malignant Transformation Of Cells Induced By Arsenite

Recently, it has been the research hotspot of arsenite carcinogenic mechanisms that people begin to investigate the role of abnormal micro RNA expression in the malignant transformation of cells caused by arsenite from the aspect of epigenetics. The alterations of mi RNA levels have been implicated in several human tumors, and also associated with tumor initiation, progression, treatment, and prognosis, and they can function as either tumor suppressors or oncogenes. Previous publishes have investigated the function of mi RNA expression in many tumors, however, the roles and molecular mechanisms of cell malignant transformation and carcinogenesis caused by environmental carcinogens remain poorly understood. Mi R-191, which is the highly conservative mi RNA with over-expression in several human tumors, may regulate cell proliferation, differentiation, apoptosis, and migration. Recently, it has been found that mi R-191 levels are regulated by transcription factors, tumor microenvironment(hypoxia), and epigenetics. However, the relationship between Arsenic and mi R-191 has been not still known.Epithelial to mesenchymal transition(EMT), the acquisition of cancer stem cells(CSCs)-like properties, and angiogenesis may affect many kinds of diseases, especially participate in the occurrence, development, and metastasis of malignant tumor. Ourpreviously investigations have found that arsenite could increase the levels of HIF-2αby inhibiting its ubiquitination, which promotes EMT and CSCs-like properties during cells malignant transformation. However, the molecular mechnisms remain largely unknown. According to the former findings that mi RNA can regulate biological process of EMT, CSCs-like properties, and angiogenesis, we speculate that HIF-2α via mi R-191 causes the process of EMT, CSCs-like properties, and angiogenesis, which is involved in the malignant transformation of cells induced by arsenite.Based on our previously results that the malignant transformation of human bronchial epithelial(HBE) cells was induced by arsenite, we will investigate that the effects of HIF-2α-mediatged mi R-191 on EMT, CSCs-like properties, and angiogenesis in arsenite-induced malignant transformation of HBE cells by using many molecular biology methods in order to understand the key molecular mechanisms of arsenite-induced cell malignant transformation. Our results will provide scientific basis for investigating the molecular mechanism of arsenite carcinogenic and looking for new biological targets of arsenite poisoning.Part I The roles of EMT and of CSCs-like properties mediated by mi R-191 in the malignant transformation of HBE cells induced by arseniteObjectiveTo investigate the molecular mechanisms of epithelial-mesenchymal transition(EMT) and acquisition of cancer stem cells(CSCs)-like properties mediated by mi R-191 and their roles in the malignant transformation of HBE cells induced by arseniteMethodsBased on the malignant transformation of HBE cells induced by arsenite.Micro RNA array was used to detecte micro RNA expression profiles of normal HBE cells and arsenite-transformated HBE cells. The levels of mi RNA were detected by quantitative real-time polymerase chain reaction(q RT-PCR) assay. Dual LuciferaseReporter Gene Assays were peformed to determine whether mi R-191 directly binded to3,-untranslated region(3,UTR) of BASP1. EMT index,the levels of BASP1 and its downstream porteins were determined by Western blot. Immunofluorescence microscopy was assessed to confirm the cell localization of some proteins. The index fo CSCs-like properties were analysed by the assay of stem cell suspension into a ball, the flow cytometry screening of side population(SP) cells, and reverse-transcriptase polymerase chain reaction(RT-PCR). The abilities of malignant, migration, and invasion were determinted by the assays of anchorage-independent growth,wound-healing, and Transwell, respectively. Our objective is to investigate the roles of EMT and CSCs-like properties mediated by mi R-191 in the malignant transformation of HBE cells induced by arsenite.Results1. The changes of mi RNA expression profiles in arsenite-transformated HBE cells.By using of mi RNA array, the levels of 51 mi RNA, including the levels of 30 mi RNAs were up-regulated and the levels of 21 mi RNAs were down-regulated, were found the abnormal expression in arsenite-transformated HBE cells. The results of q RT-PCR showed that the levels of mi R-21, mi R-311, and mi R-191 c were increased and the levels mi R-200 c were decreased in transformed cells. Mi R-191 was selected in this study because it was the highest fold change seen from the mi RNA array and q RT-PCR analysis. These results have suggested that mi R-191 may be involved in arsenite-induced malignant transformation of HBE cells.2. The effects of arsenite on mi R-191 levels in HBE cellsHBE cells were exposed to 0.0 or 1.0 μM arsenite for 10, 20, and 30 passages. Our results showed that arsenite increaseed mi R-191 levels, which changes were more significant with longer times of arsenite exposure. After HBE cells were treated by 0.0or 1.0 μM arsenite for 3, 6, 12, and 24 h, data showed that arsenite also increased mi R-191 levels. These results suggest that arsenite regulate the expression of mi R-191.3. The Effects of mi R-191 mediated WT1 via BASP1 on the arsenite-caused activation of Wnt/β-catenin pathway.HBE cells were exposed to 0.0 or 1.0 μM arsenite for10, 20, and 30 passages orfor 3, 6, 12, and 24 h, respectively. Data showed that arsenite reduced the decreases of BASP1 levels and the increases of WT1、Wnt1, and β-catenin levels with longer times of arsenite exposure. The potential binding site of mi R-191 was predicted for the 3,UTR of BASP1 via bio-database. Dual-luciferase analysis showed that the activation of p GL3-BASP1 3’UTR luciferase in HBE cells exposed to arsenite were significantly lower than those in control HBE cells, but the effects were blocked by up-regulation of mi R-191. After the levels of mi R-191 were down-regulated by pre-treatment of 150 n M anti-mi RNA-191 for 24 h, HBE cells were exposed to 0.0 or 1.0 μM arsenite for 24 h.Our data showed that the inhibition of mi R-191 blocked the arsenite-induced decreases of BASP1 levels and increases of of WT1, Wnt1, and β-catenin levels. The up-regulation or down-regulation of BASP1 levels by p EGFP-BASP1 plasmid or si RNA blocked arsenite-caused changes of WT1 levels. Arsenite induced β-catenin into nucleus from cytoplasm in HBE cells under fluorescence microscope. The down-regulation of BASP1 by si RNA blacked arsenite-induced increases of WT1 andβ-catenin levels in HBE cells. These results suggest that mi R-191 regulated WT1 via BASP1 plays important roles in arsenite-caused activation of the Wnt/β-catenin pathway.4. The effects of mi R-191 on EMT and CSCs-like properties in arseinte-transformed HBE cells.After HBE cells were exposed to 0.0 or 1.0 μM arsenite for 30 passages(about 15weeks), then cells were treated by 150 n M anti-mi R-191 or con-si RNA for 24 h. The results showed that knockdown of mi R-191 elevated BASP1 levels, blocked the increases of WT1, Wnt1, and β-catenin levels, decreased the levels of N-cadherin and vimentin, enhanced E-cadherin levels, blocked the formation of spheroids, suppressed the levels of CD133 and CD44 m RNA, and decreased the percentage of SP cells. These results indicate that mi R-191 is involved in EMT and the acquisition of CSCs-like properties of arsenite-transformated HBE cells.5. The effects of mi R-191 on neoplastic capacity, invasion, and migration of arsenite-transformed HBE cells.After HBE cells were exposed to 0.0 or 1.0 μM arsenite for 30 passages(about 15weeks), then cells were treated by 150 n M anti-mi R-191 or con-si RNA. The results showed that down-regulation of mi R-191 decreased the number of colony formation in soft agar, growth area after scratch injury, and the number of invasive cells and metastatic cells in arsenite-transformated HBE cells. These results indicate that mi R-191 is involved in the neoplastic capacity, invasion, and migration of arsenite-transformated HBE cells.ConclusionIn HBE cells, arsenite increases the levels of mi R-191 and WT1, decreases BASP1 levels, and activates the Wnt/β-catenin pathway. The up-regulation of WT1 by mi R-191 inhibiting BASP1, which up-regulated WT levels and activated Wnt/β-catenin pathway,is involved in EMT, CSCs-like properties, and neoplastic transformation of HBE cells and then promotes the metastatic processes.PartⅡ The roles of HIF-2α-regulated mi R-191 in angiogenic and metastatic properties of arsenite-transformaed HBE cellsObjectiveTo investigate the molecular mechanisms of HIF-2α-regulated mi R-191 and the roles in angiogenic and metastatic properties of arsenite-transformaed HBE cells.MethodsBased on the models of arsenite-induced malignant transformation of HBE cells,the effects of arsenite on HIFs, MMP-9, and VEGF levels in HBE cells were determinated by Western blot assay. ELISA and tube formation of human umbilical vein endothelial cell(HUVEC) assays were peformed to detected the levels of VEGF secretion and the angiogenic properties of arsenite-transformaed HBE cells,respectively. Ch IP assay was performed to identify whether HIF-2α binded to the promoter of mi R-191 and Luciferase Reporter Gene assays was uesed to determine whether HIF-2α regulated transcriptly mi R-191 expression. Transwell assay was performed to identify the abilities of migration and invasion of arsenite-transformed HBE cells. We have further investigated the effects of knockdown HIF-2α by si RNAor/and up-regulating mi R191 by mi R-mimic assay on the above indexes. Our objective is to study the effect of HIF-2α regulating mi R-191 on the angiogenic and metastatic properties of arsenite-transformed cells.Results1. The Effect of arsenite on the levels of HIFs, MMP-9, and VEGF in HBE cellsAfter HBE cells were exposed to 0.0 or 1.0 μM arsenite for 10, 20, and 30 passages, our results showed that arsenite induced more increases of HIF-2α,MMP-9,and VEGF levels with longer times of arsenite exposure. After HBE cells were exposed to 1.0 μM arsenite for 3, 6, 12, and 24 h, data showed that arsenite increased the levels of HIF-2α and VEGF. These results indicate that arsenite increased the levels of HIF-2α,MMP-9 and VEGF in HBE cells.2. Arsenite-transformed HBE cells have pro-angiogenic propertiesAfter HBE cells were exposed to 0.0 or 1.0 μM arsenite for 30 passages(about 15weeks), our results showed that arsenite-transformed HBE cells maight secrete more VEGF levels than normal HBE cells. The tube formation of HUVEC was induced by the condition medium from arsenite-transformed HBE cells. These results indicate that arsenite-transformed HBE cells have pro-angiogenic proerties.3. The role of HIF-2α-regulated mi R-191 in arsenite-induced the changes of BASP1 levels and its downstream factors.After HBE cells were pre-treated by 0.0 and 1.0 μM Topotecan(HIF-2α inhibitor)for 3 h or 25 n M HIF-2α-si RNA for 24 h,they were exposed to 0.0 or 1.0 μM arsenite for 24 h. Our data showed that the inhibition or knockdown of HIF-2α blocked arsenite-induced the increases of mi R-191, WT1, and VEGF levels and the decreases of BASP1 levels. After HBE cells were exposed to 0.0 or 1.0 μM arsenite for 24 h, we found that arsenite enchanced the combine with the HIF-2 and the promoter region of mi R-191 by Ch IP assays and Luciferase Reporter Gene assays. After HBE cells were transfected by 25 n M HIF-2α-si RNA and the report gene plasmid for 24 h, then cells were treated by 0.0和1.0 μM arsenite for 24 h. Data showed that arsenite increased the activatives of mi R-191-promoter-Luc, which was blocked by down-regulating HIF-2αlevels. These results suggest that HIF-2α-mediated mi R-191 plays important roles in arsenite-induced the changes of BASP1 and its downstream factors.4. HIF-2α via mi R-191 is involved in angiogenic and metastatic properties of arsenite-transformed HBE cellsAfter HBE cells were exposed to 0.0 or 1.0 μM arsenite for 30 passages(about 15weeks), then arsenite-transformated cells were co-transfected with 25 n M HIF-2α-si RNA or/and 80 n M mi R-191-mimic. Our data showed that down-regulation of HIF-2α decreased mi R-191 levels, which was blocked by up-regulation of mi R-191.The knockdown of HIF-2α by si RNA induced following changes of arsenite-transformated HBE cells: increasing BASP1 levels, decreaseing the levels of WT1, MMP-9, and VEGF, down-regulating the levels of VEGF screation, shortening microtubule length of HUVEC treated by the medium from arsenite-transformated HBE cells, and reducing the number of invasion and migration cells. These changes induced by HIF-2α knockdown were blocked by up-regulation of mi R-191 levels. These results indicate that HIF-2α via mi R-191 plays important roles in angiogenic and metastatic properties of arsenite-transformed HBE cells.ConclusionArsenite increased HIF-2α levels in HBE cells. HIF-2α regulates transcriptly mi R-191 levels by binding to the promoter of mi R-191, which regulates BASP1 and improved the levels of WT1 and VEGF. The mechanism plays important roles in angiogenic and metastatic properties of arsenite-transformed HBE cells.In summary, the novel findings of this study are as follows1. The expression profilings of varied mi RNA have changed during the malignant transformation of HBE cells induced by chronic treatment of arsenite and the up-regulation of mi R-191 is the most obvious.2. The up-regulation of WT1 by mi R-191 inhibiting BASP1, which up-regulated WT1 levels and activated Wnt/β-catenin pathway, is involved in EMT, CSCs-like properties, and neoplastic transformation of HBE cells.3. Arsenite induces the increases of HIF-2α levels and improves the combine withHIF-2α and the promoter of mi R-191, which up-regulates mi R-191 levels.4. HIF-2α regulates transcriptly mi R-191 levels, which regulates BASP1 and improved the levels of WT1 and VEGF. The mechanism plays important roles in angiogenic and metastatic properties of arsenite-transformed HBE cells.