Effects Of Water-soluble Components Of Atmospheric Particulates From Rare Earth Mining Areas On The Lung Cancer Cell Cycle

To study the effects of Water Soluble Particulate Matters?WSPM?on the proliferation and protein expression profile of human lung adenocarcinoma cell A549in Bayou Obo rare earth mining area,and explore the influence of WSPM on A549cell cycle,find a clear study direction and provide a certain experimental basis for the development of intervention and control methods,this work has been designed.The spring atmospheric particulate matters in the Bayan Obo mining area was collected,the contents of rare earth,heavy metal elements and water-soluble ions in WSPM were analyzed by ICP-AES and IC techniques.Cell proliferation of A549 cells were detected by measuring MTT and CCK8 after exposure to the WSPM.Effects of WSPM on cell cycle and intracellular reactive oxygen?ROS?level of A549 cell were investigated by flow cytometry.The cellular protein expression profiles in A549 cells were built using iTRAQ-based proteomics after WSPM treatment.Hierarchical cluster,Gene Ontology and KEGG Pathway enrichment were applied to analyze the differential expression proteins by bioinformatics.Some of the differentially-expressed proteins were further tested by multiple-reaction monitoring?MRM?and Western blot?WB?.siRNA technique was used to inhibit the expression of several proteins associated.The expression level of cell cycle regulatory factors were also detected by qRT-PCR.Elisa was used to determine the expression of Caspase 3 and other regulatory proteins.At the same time,the effects of LaCl₃,CeCl₃,NdCl₃ and NaF on the cell proliferation,cell cycle and related cell regulatory factors of A549 cell were evaluated.We found that:the main chemical components of WSPM in this mining area included 10 kinds of metal elements and 4 kinds of anions,which can inhibit the proliferation of A549 cells and induce cell arrest in G2/M phase.In this process,the intracellular ROS level was found to be decreased with the increase of exposure dose of WSPM.Compared with the blank control group,the exposure to WSPM10 and WSPM2.5 caused 134 and 116 proteins to be differentially expressed in A549 cells,respectively.33 and 31 differentially-expressed proteins were confirmed further,being consistent with proteomic analysis.The most prominent enrichment in ribosome-associated proteins were shown.These proteins were classified into 6 categories according to their functions,and 7 different proteins associated with ribosome were down-regulated.When inhibiting RPL6,RPL13 or RPL18A gene expression,A549 cells were arrested in G1 phase,affecting the expression of Cyclin D1,p21,RB1,Cyclin A2,Cyclin B1,CDC25A,CDK2,CHEK2and E2F1.La,Ce,Nd and F^-found in WSPM,also inhibited the proliferation of A549 cells.The semi-lethal doses?IC50?of LaCl₃,CeCl₃,NdCl₃ and NaF to A549cells were 1.29,1.14,1.26 and 0.95 mM,respectively.After 24 h exposure to 2 mM NaF,A549 cells were also arrested in G2/M phase while the other three compounds did not have this effect.The four compounds affected cell cycle regulatory factors in A549 cells,mainly focusing on the effect on the expression of CDK2,CDK4,RB1,ATM,TP53 and MDM2 genes.The results are in consistent with the those from WSPM exposure.The expression of caspase 3,6,8,9 were decreased under the exposure to low doses?<100?g/ml?WSPM,while it was increased at high doses?200?g/ml?,and the sub-G1 peak was significantly larger.The expression of NF-?B was not found to be changed during this process?p>0.05?.These results allow us to conclude that WSPM from rare earth mines decreased the amount of proliferation A549 cells and induced cell cycle G2/M phase arrest even apoptosis which may be independent of the NF-?B/MYD88 pathway and be perceived by TLR4 receptor.The disfunction of cell cycle is related to the down-expression of ribosomal protein,but it is not the direct reason for the A549 cell arrest in G2/M phase.Fluorinion,is probably one of the main toxic substances in WSPM,may regulate the A549 cell cycle by affecting the expression of genes such as MDM2,RB1,ATM,TP53,CDK2 and CDK4together with rare earth elements such as Ce,Nd and La.These results has provided evidences for further investigation on the relationship between atmospheric particulate matter and lung cancer in rare earth mine.