The Epigenetically Silences Of Let-7c Induced The Acquirement Of Tumor Stem Cells Properties Is Involved In Malignant Transformation Of Cells By Arsenite

Arsenic(As)is a widely spread environmental contaminant that can be found in the soil and water and as airborne particles.Epidemiological studies have linked arsenic exposure to the development of lung,bladder,kidney and liver cancer.Exposure to arsenic occurs in the form of either arsenite(As ?)or arsenate(As?).The increased cancer risk to arsenic is attributed to arsenite rather than arsenate,possibly due to the cell's ability to take up arsenite at a faster rate than arsenate.Although arse-nic has been proposed to disturb a variety of cellular processes including cellular redox status and signal transduction,recent evidence suggests that arsenic may promote cellular transformation through chromatin-based mechanisms.Tumor stem cells(TSCs)properties to get involved in a variety of diseases,particularly with the development and progression of cancer related.In recent years,TSCs features access to research and the degree of malignancy and metastasis of tumor cells and its molecular mechanism of the relationship has become a hot spot,but for TSCs characteristics obtained environmental carcinogens in the carcinogenic process of malignant transformation and molecular mechanisms of action and research reports even caused less.Therefore,in-depth study of arsenic-induced cell transformation and epigenetic regulation of specific signaling pathways,especially on the regulation of arsenic-induced malignant transformation of TSCs characteristics obtained in the course of its molecular mechanism of arsenic carcinogenicity for finding early biomarkers and the discovery of new prevention measures have important theoretical and practical value.In the present study,based on the model that arsenite induced cell malignant transformation of human keratinocytes cells,we would use some molecular biology methods to investigate the roles and the mechanisms between the epigenetic regulation of let-7c and Ras/NF-?B influence during arsenite-induced acquirement of TSCs properties and malignant transformation of HaCaT cells to find some molecular mechanisms of arsenite-induced cell mailgnant transformation of HaCaT cells.The results would help us further understanding the molecular mechanisms of arsenic toxicity and carcinogenesis,so as to provide some new clues to find biomarkers and prevention measures of arsenic poisioning.Methods1.Arsenite induces the acquisition of TSCs properties by HaCaT cellsHaCaT cells were exposed to 0.0 or 1.0 ?M NaAsO2 for 0?10?20 and 30 passages(about 15 weeks),respectively.We got the different passages HaCaT cells of control(HaCaT-PC)and malignant levels(HaCaT-As).QRT-PCR was used to detect the mRNA levels of CD34 and K5;Spheroid formation experiment was used to detect the abillity of colony formation.We were going to investigate that the effects of arsenite-induced the acquisition of TSCs properties by HaCaT cells.2.The effects of arsenite-induced activation of Ras/NF-?BHaCaT cells were exposed to 0.0 or 1.0 ?M NaAsO2 for 0?10?20 and 30 passages,respectively.HaCaT cells were exposed to 1.0 ?M NaAsO2 for 0?6?12 and 24 h.Western blot was used to detect the protein levels of k-ras,Rel-A and p-RelA.We were going to investigate the effects of arsenite-induced activation of Ras/NF-?B.3.The effects of the levels of let-7c by arsenite in HaCaT cellsHaCaT cells were exposed to 0.0 or 1.0 ?M NaAsO2 for 0?10?20 and 30 passages(about 15 weeks),respectively.We got the different passages HaCaT cells of control and malignant levels.HaCaT cells were exposed to 1.0 ?M NaAsO2 for 0?6?12 and 24 h.The levels of let-7c were detected with qRT-PCR.We were going to investigate the effects of the level of let-7c by arsenite in HaCaT cells.4.Arsenite-induced the down-regulated levels of let-7c and activation of Ras/NF-?B may be related to methylationThe methprimer software was used to detect the CpG islands in the promoter region of let-7c.After HaCaT cells were treated with 0.0 or 2.5 ?M 5-AZA(an inhibitor of methyltransferases)for 24 h,they were exposed to 0.0 or 1.0 ?M NaAsO2 for 24 h.The levels of let-7c were detected with qRT-PCR.Western blot was used to detect the protein levels of k-ras and p-RelA.HaCaT cells were exposed to 0.0 or 1.0?M NaAsO2 for 30 passages or 24 h,respectively.MSP was used to detect the methylation of let-7c promoter.We were going to investigate the effects of arseuite-induced the levels of let-7c and activation of Ras/NF-?B by methylation.5.The effects of EZH2 and H3KL27me3 are caused by exposure of HaCaT cells to arseniteHaCaT cells were exposed to 0.0 or 1.0 ?M NaAsO2 for 0?10?20 and 30 passages,or 0?6?12 and 24 h,respectively.Western blot was used to detect the protein levels of EZH2 and H3K27me3.We were going to investigate that the effects of EZH2 and H3K27me3 are caused by exposure of HaCaT cells to arsenite.6.The effects of the down-regulated levels of let-7c in HaCaT cells exposed to arsenite by EZH2\H3K27me3.HaCaT cells were transfected by 20 nM of con-siRNA or si-EZH2 for 12 h,respectively,and they were exposed to 0.0 or 1.0 ?M of arsenite for 24 h.Western blot was used to detect the protein levels of EZH2 and H3K27me3;QRT-PCR was used to detect the levels of let-7c.HaCaT cells were exposed to 0.0 or 1.0 ?M NaAsO2 for 24 h,respectively.ChIP was used to experiment if the let-7c promoter region can be combined with EZH2 and H3K27me3.We were going to investigate the effects of down-regulated level of let-7c in HaCaT cells exposed to arsenite by EZH2\H3K27me3.7.The effects of the activation of Ras/NF-KB in HaCaT cells exposed to arsenite by the upregulation of let-7cHaCaT cells were transfected by 50 nM of con-mimic or let-7c-mimic for 6 h,respectively,and they were exposed to 0.0 or 1.0 ?M of arsenite for 24 h.Western blot was used to detect the protein levels of k-ras,RelA and p-RelA.We were going to investigate the effects of the activation of Ras/NF-?B signal pathway in HaCaT cells exposed to arsenite by the upregulation of let-7c.8.The effects of arsenite-induced acquisition of TSCs properties and neoplastic transformation of HaCaT cells by the upregulation of let-7cTransformed HaCaT cells were transfected with 50 nM of con-mimic or let-7c-mimic for 6 h.QRT-PCR was used to detect the mRNA levels of CD34 and K5;Spheroid formation experiment was used to detect the abillity of spheroid formation;Colony formation experiment was used to detect the ability of colonies formation;Nude mice tumorigenic experiment was used to detect the tumorigencity of cells.We were going to investigate the effects of arsenite-induced acquisition of TSCs properties and neoplastic transformation of HaCaT cells by the upregulation of let-7c.Results1.Arsenite induces the acquisition of TSCs properties by HaCaT cells.HaCaT cells were exposed to 1.0 ?M NaAsO2 for 30 passages.During the arsenite-induced transformation of HaCaT cells,there were increased the mRNA levels of K5 and CD34.The spheroid was forming when HaCaT cells were exposed to 1.0 ?M NaAsO2 for 10 passage,these were more and more.Thus,our results indicate that arsenite induces the acquisition of TSCs properties by HaCaT cells.2.The effects of arsenite-induced activation of Ras/NF-?B.HaCaT cells were exposed to 1.0 ?M NaAsO2 for acute or Chronic.The protein levels of k-ras and p-RelA were increased in a time-dependent manner.These datas indicate that Ras/NF-?B is activated by arsenite in HaCaT cells.3.The effects of the levels of let-7c by arsenite in HaCaT cellsHaCaT cells were exposed to 1.0 ?M NaAsO2.After 30 passages,the levels of let-7c in transformed cells were significantly shorter than that of passage control cells.This change was significant in 1.0 arsenite for acute or chronic in a time-dependent mauner.These data indicate that the levels of let-7c are decreased by arsenite in HaCaT cells.4.Arsenite-induced the down-regulated levels of let-7c and activation of Ras/NF-?B may be related to methylationThere were CpG islands in the promoter region of let-7c by the methprimer software.The levels of let-7c were down-regulated and the levels of k-ras and p-RelA were up-regulated in HaCaT cells by exposed to 1.0 ?M NaAsO2 for 24 h.When HaCaT cells were treated with 2.5 ?M 5-AZA and 1.0 ?M NaAsO2 for 24 h,the effects were reversed by 5-AZA.HaCaT cells were exposed to 1.0 NaAsO2.After 30 passages,the methylation of let-7c promoter in transformed cells was not significantly changed than the passage control cells by MSP.Similar results were also observed for cells with longer times of exposure to 0.0 or 1.0 arsenite for 24h.These results suggest that arsenite exposure depletes let-7c through methylation of let-7c;however,such process may not via the DNA methylation.5.The effects of EZH2 and H3K27me3 are caused by exposure of HaCaT cells to arseniteHaCaT cells were exposed to 1.0 ?M NaAsO2 for acute or chronic.The protein levels of EZH2 and H3K27me3 were increased in a time-dependent manner.These datas indicate that during arsenite-induced malignant transformation of HaCaT cells,there are increased expression of EZH2 and H3K27me3.6.The effects of the down-regulated levels of let-7c in HaCaT cells exposed to arsenite by EZH2\H3K27me3.Knock down of EZH2 blocked the expression of H3K27me3,but improved the expression of let-7c induced by 1.0 ?M NaAsO2 in HaCaT cells.Let-7c promoter region can be higher combined with EZH2 and H3K27me3 by arsenite.The results show that NaAsO2 make HaCaT cells let-7c promoter region EZH2/H3K27me3 enrichment,thereby silencing the expression of let-7c.7.The effects of the activation of Ras/NF-?B in HaCaT cells exposed to arsenite by the upregulation of let-7cAfter HaCaT cells were transfected by let-7c-mimic,the levels of k-ras and p-RelA were significantly down-regulated.The data showed overexpression of let-7c attenuated the upregulation of k-ras and p-RelA induced by arsenite.These datas suggest that in arsenite-treated HaCaT cells,let-7c plays an important role in the activation of Ras/NF-?B.8.The effects of arsenite-induced acquisition of TSCs properties and neoplastic transformation of HaCaT cells by the upregulation of let-7cIn transformed HaCaT cells,let-7c over-expression decreased the mRNA levels of CD34 and K5 and partially blocked the formation of spheroids.In agar,transformed HaCaT cells transfected with the let-7c-mimic formed the colonies was more than control transformed HaCaT cells and these cells transfected with the con-mimic.In mice injected with arsenite-transformed HaCaT cells or with such cells transfected by the con-mimic,tumor incidences and tumor volumes were significant.Tumors induced by arsenite-transformed cells and by cells transfected with the con-mimic were composed of undifferentiated,epithelial-like cells.The datas showed overexpression of let-7c inhibited the arsenite-induced acquisition of TSCs properties and neoplastic transformation of HaCaT cells.Thus,let-7c is involved in the arsenite-induced acquisition of TSCs properties and neoplastic transformation of HaCaT cells.Conclusions1.HaCaT cells are transformed by chronic exposure to arsenite,the acquisition of TSCs properties and tumorigenic.2.The decreased level of let-7c induced by arsenite in HaCaT cells.3.H3K27me3 silence of let-7c induced by arsenite in HaCaT cells is mediated by EZH2 and then activates Ras\NF-?B.4.Epigenetically silences of let-7c induced the activation of Ras\NF-?B play important roles in the arsenite-induced acquisition of TSCs properties and neoplastic transformation of HaCaT cells.In a word,the result of present study indicate that arsenite induces silences of let-7c by EZH2\H3K27me3 results in the activation of Ras\NF-?B,which causes the acquisition of TSCs properties,then results in cell malignant transfornation and tumorigenic.Our results suggest that epigenetically silences of let-7c results in the activation of Ras\NF-?B play important roles in the arsenite-induced acquisition of TSCs properties and neoplastic transformation of HaCaT 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