The Effect And Mechanism Of Nickel Chloride Induces Cancer Stem Cell-like Property Of Human Lung Epithelial Cells Through Upregulating The Expression Level Of ALKBH5

BackgroundNickel chloride（NiCl₂） is a common occupational and environmental carcinogen,causing a range of health hazards such as lung cancer.Our previous study indicated that HIF-1αis involved in the regulation of malignant transformation of lung epithelial cells induced by NiCl₂.However,it is unknown whether HIF-1αis also involved in regulating the transformation of lung epithelial cells to lung cancer stem cells（CSCs）by NiCl₂.Numerous studies have shown that m⁶A plays an important role in the occurrence and development of various tumors.The mechanism of m⁶A modification in the regulation of CSC formation has been continuously revealed.However,whether m⁶A modification plays a role in lung cancer induced by the metal carcinogen NiCl₂and the mechanism of NiCl₂-induced CSC formation remains unclear.ObjectiveIn this study,we explore the mechanism of m⁶A modification on NiCl₂in inducing pulmonary epithelial cell to CSC through in vitro models,which is of great significance to elucidate the mechanism of NiCl₂inducing the formation of lung CSCs.MethodsBEAS-2B cells in good growth state were used for treatment,and the following experiments were carried out:（1）To verify the formation of cancer stem cells induced by NiCl₂,BEAS-2B cells were treated with long-term low-dose NiCl₂（0.25 mm-6 months）,and the self-renewal ability and long-term growth ability of cells were detected by free-floating sphere formation assay and plate colony formation assay respectively.q RT-PCR assay was used to detect the expression of marker genes in CSCs after NiCl₂exposure（0.25 m M-0,1,3,6 m）.Western Blot assay was used to detect the expression levels of marker molecular proteins in CSCs treated with NiCl₂for long term（0.25 m M-0,1,3,5,6 m）.（2）In order to investigate the role of m⁶A demethylase ALKBH5 in NiCl₂-induced tumor stem cell formation,q RT-PCR assay was used to detect the gene expression levels of various m⁶A modifiers after short-term exposure to NiCl₂（0.5 m M-0,6,12,24 h）.Western Blot assay was used to detect the protein expression level of ALKBH5 in cells treated with NiCl₂for short term（0.5 m M-0,1,3,6,12,24 h）and long term（0.25 m M-0,1,5,6 m）.Through transfection exogenous ALKBH5 or sh ALKBH5 expression plasmid to increase or decrease in the cell ALKBH5 gene expression level,and then uses m⁶A RNA methylation the quantitative kit detecting m⁶A modified level in the total RNA,respectively by free-floating sphere formation assay and plate colony formation assay to detect cells since the update ability and the ability to grow for a long time,using Western Blot experiments detect cancer stem cells in the cell marker molecular protein expression level;the modification level of m⁶A in different tumor stem cell marker molecules was detected by Me RIP assay.（3）To study the regulatory mechanism of NiCl₂on ALKBH5,using Western Blot test different period by the NiCl₂（0.5 m M-0,1,3,6,12,24 h）and different concentrations（24 h-0.125 0,0.25,0.5,1.0 m M/L）after processing HIF-1αprotein expression level in cells;BEAS-2B cells were evenly planted into four culture plates and treated with different treatments:control group,HIF-1αinhibitor group,nickel chloride exposure group,inhibitor+NiCl₂exposure group.The ALKBH5 m RNA was detected by q RT-PCR assay,and the expression levels of HIF-1α,ALKBH5 and cancer stem cell marker protein were detected by Western Blot assay.Results1 Long-term exposure to NiCl₂ enhanced the self-renewal ability and long-term growth ability of BEAS-2B cells.2 Nickel chloride induced up-regulation of expression levels of marker molecular genes and proteins in CSCs of BEAS-2B cells:after long-term exposure NiCl₂（0.25 m M-1,3,6 m）BEAS-2B cells in the CSCs molecular markers to raise the level of gene expression and CSCs markers protein expression level after the NiCl₂long-term processing（0.25 m M-1,3,5,6 m）were present obvious time-effect relationship,the difference is statistically significant（P<0.05）.3 NiCl₂ exposure can be mediated by the expression level ALKBH5 intracellular RNA m⁶A modification level cut:NiCl₂short-term exposure group（0.5 m M-24 h）and long exposure group（0.25 m M-6 m）RNA in the cell m⁶A modification levels are lower than the control group and NiCl₂short-term treatment can increase cell ALKBH5 gene and protein expression level,the difference is statistically significant（P<0.05）.Knockdown of ALKBH5 reversed the downregulation of RNA m⁶A modification induced by NiCl₂exposure.4 NiCl₂ exposure can regulate the stem cell characteristics of BEAS-2B cells through ALKBH5:Knocking down the expression level of ALKBH5 in BNIT cells can reduce its self-renewal ability and long-term growth ability,and down-regulate the expression level of stem cell marker protein.Knocking down the expression level of ALKBH5 in BEAS-2B cells can down-regulate the expression level of stem cell marker protein induced by nickel chloride.The expression level of ALKBH5 in overexpressed BEAS-2B cells could enhance the long-term growth ability of cells and up-regulate the expression level of stem cell marker protein（P<0.05）.5 NiCl₂ exposure can down-regulate the modification level of various tumor stem cell marker molecules m⁶A:after 24 hours exposure to 0.5 m M/L NiCl₂,the levels of SOX2,NANOG,CD44,CD133 m⁶A in BEAS-2B cells were significantly lower than the control（P<0.05）,while the m⁶A modification level of OCT4 m RNA was not significantly changed.6 NiCl₂ upregulated HIF-1αprotein expression and HIF-1αinhibitors can decrease NiCl₂induced ALKBH5:in different time by NiCl₂（0.5 m M-0,1,3,6,12,24 h）and different concentrations（24 h-0.125 0,0.25,0.5,1.0 m M/L）after processing intracellular HIF-1αprotein expression level;compared with the NiCl₂exposed group,the addition of HIF-1αinhibitor decreased the up-regulated expression of ALKBH5gene and protein in the NiCl₂exposed group.7 HIF-1αinhibitor can down-regulate the expression level of marker protein in CSCs induced by NiCl₂.Compared with NiCl₂exposure group,the addition of HIF-1αinhibitor can reduce the up-regulated expression levels of CSCs marker proteins in NiCl₂exposure group.Conclusions1 NiCl₂ can induce the enhancement of cell self-renewal ability,proliferation and differentiation ability,up-regulate the expression levels of CSC-like markers and promote the transformation of lung epithelial cells to lung cancer stem cells.2 NiCl₂ may regulate the transformation of lung epithelial cells to lung cancer stem cells by up-regulating the expression level of demethylase ALKBH5 and down-regulating the modification level of target gene RNA m⁶A.3 NiCl₂ may regulate the expression level of demethylase ALKBH5 by up regulating HIF-1αexpression level to promote the characteristics of stem cells.