The Effect And Mechanism Of LncRNA-MEG3 In Epithelial Mesenchymal Transformation And Cancer Stem-like Property Of Lung Epithelial Cells Induced By Nickel

Background:nickel chloride（NiCl₂）is widely distributed in both natural and occupational environments,and long-term occupational exposure may lead to malignant tumors such as lung cancer.Our research group previously screened for differential LncRNAs in normal lung epithelial cell line BEAS-2B treated with short-term and long-term nickel chloride,and the results showed that the expression level of LncRNA-MEG3 was decreased,and the down-regulation of LncRNA-MEG3 could enhance HIF-1αprotein translation by down-regulating PHLPP1 transcription.And then promote the malignant transformation of normal lung epithelial cells.However,in the process that nickel compounds promote the transformation of human lung bronchial epithelial cells into malignant cells by inhibiting the expression of LncRNA-MEG3,Whether LncRNA-MEG3 plays a role in Epithelial mesenchymal transformation（EMT）and cancer stem cell（CSC）formation is not fully known.Objective:Human lung bronchial epithelial cell line BEAS-2B was cultured in vitro to investigate the role of LncRNA-MEG3 in inducing epithelial mesenchymal transformation and tumor stem cell like characteristics of human lung bronchial epithelial cells induced by nickel chloride exposure,and to clarify the mechanism of nickel chloride induced epithelial mesenchymal transformation and the formation of lung cancer stem cells.Methods:BEAS-2B cells in good growth state were used for treatment,and the following experiments were performed:Part Ⅰ:The role and mechanism of LncRNA-MEG3 in nickel chloride induced human lung bronchial epithelial cells to acquire cancer stem cell-like characteristics.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 of the cells was detected by suspension pellet forming assay.Western Blot assay was used to detect the expression levels of cancer stem cell marker proteins in cells treated with NiCl₂at different concentrations（24h-0,0.125,0.25,0.5,1.0mM）and different time periods（0.5 mm-0,1,3,6,12,24h）.Meanwhile,the expression level of LncRNA-MEG3 was analyzed by quantitative PCR.The expression level of LncRNA-MEG3 in cells was increased by transfection of exogenous overexpressed LncRNA-MEG3 plasmid.The expression level of LncRNA-MEG3 in cells after overexpression of LncRNA-MEG3 was detected by semi-quantitative PCR assay,and the self-renewal ability of cells was detected by free suspension pellet formation assay.The levels of stem cell marker proteins were detected by Western Blot.In order to investigate the mechanism of LncRNA-MEG3 in the formation of tumor stem cells after exposure to NiCl₂,protein western blotting assay was used to analyze the protein expression levels of Wnt3a,Wnt5a and WIF1 in human lung bronchial epithelial stable cell lines after exposure to nickel chloride,and then q RT-PCR assay was used to analyze the protein expression levels of Wnt3a in cells.The levels of Wnt5a and WIF1 mRNA were analyzed by immunofluorescence assay.Part Ⅱ:The role and mechanism of LncRNA-MEG3 in nickel chloride-induced epithelial interstitial transformation of human lung bronchial epithelial cells.Human bronchial epithelial stable transfer cell lines were exposed to nickel chloride for 24h,and the morphological changes of the cells were observed with phase contrast microscopy.Meanwhile,the expression levels of epithelial interstitium transition related proteins such as E-cadherin and Vimentin were analyzed by western blotting after LncRNA-MEG3 overexpression.And the protein expression levels of p-P42/44,P-p38,P-JNK,TGF-β,p-Smad3,p-Smad2 were changed.Results:1.NiCl₂induced the down-regulation of LncRNA-MEG3 in a time/dose dependent manner.Compared with the control group,the difference was statistically significant（P<0.05）.2.Overexpression of LncRNA-MEG3 could inhibit the cancer stem cell-like characteristics induced by nickel chloride:After overexpression of LncRNA-MEG3,the self-renewal ability of cells was weakened,and the expression level of stem cell marker protein was down-regulated,with statistical significance（P<0.05）.3.Overexpression of LncRNA-MEG3 can inhibit the entry ofβ-catenin protein into the nucleus induced by nickel chloride and up-regulate the expression levels of Wnt3a and Wnt5a mRNA and proteins:After LncRNA-MEG3 was overexpressed,β-catenin protein entered the nucleus decreased,and Wnt3a and Wnt5a mRNA and protein expression levels were down-regulated.Compared with the control group,the difference was statistically significant（P<0.05）.4.Overexpression of LncRNA-MEG3 can inhibit the down-regulation of WIF1 mRNA and protein expression level by nickel chloride:After overexpression of LncRNA-MEG3,the expression level of WIF1 mRNA and protein is up-regulated.Compared with the control group,the difference was statistically significant（P<0.05）.5.NiCl₂induced epithelial mesenchymal transformation of BEAS-2B cells:After exposure to NiCl₂,the morphology of BEAS-2B cells underwent EMT-like changes,and the expression level of epithelial marker E-cadhering decreased,while the expression level of mesenchymal marker Vimentin increased.The results showed that NiCl₂induced EMT in BEAS-2B cells.6.LncRNA-MEG3 regulates EMT formation induced by NiCl₂exposure:Overexpression of MEG3 in BEAS-2B cells reversed EMT-like morphological changes,down-regulation of E-cadherin protein expression and up-regulation of Vimentin protein expression induced by NiCl₂.7.LncRNA-MEG3 reverses NiCl₂-induced EMT by regulating TGF-β/Smad2/3 and MAPKs signaling pathways:The overexpression of LncRNA-MEG3 in BEAS-2B cells reversed the up-regulation of TGF-β1,p-Smad2,p-Smad3,p-P42/44,P-p38 and P-JNK proteins in TGF-β1/Smad2/3 signaling pathway induced by NiCl₂.Conclusion:1.On the one hand,TGF-β/Smad2/3 and MAPKs signaling pathways were activated by nickel chloride exposure,and epithelial interstitial transformation occurred.On the other hand,WIF1 can be down-regulated and Wnt/β-catenin signaling pathway can be activated to promote cell dryness.2.Nickel chloride exposure down-regulates LncRNA-MEG3 expression,and overexpression of LncRNA-MEG3 can activate TGF-β/Smad2/3 and MAPKs signaling pathways on the one hand,and inhibit cell epithelial interstitial transformation.On the other hand,WIF1 was up-regulated,which inhibited the activation of Wnt/β-catenin signaling pathway and inhibited the formation of cancer stem cells.