1: MicroRNAs Are Involved In BPDE Induced Cellular Response In Human Amnion FL Cells 2: Establishment Of DNA Polymerase Beta Down-regulated And Overexpression Stable Cell Line By Dexamethasone Induction

(±)-anti-benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) is one of the metabolic products of benzo(a)pyrene (BaP), which belongs to the polycyclic aromatic hydrocarbons (PAHs) environmental chemical pollutants. BPDE is considered as the ultimate carcinogen of BaP. BPDE is the most carcinogenetic form and the electrophilic species of it is able to interact with nucleophilic sites on cellular DNA, RNA and protein resulting in bulky-adduct damage. BPDE causing cellular reponse is not a passively accepted process by the cell. BPDE induces bulky-adduct DNA damage, which would activate intracellular nucleotide excision repair (NER) and cell cycle arrest, etc. protective mechanisms, also trigger the low-fidelity translesion replication mechanism that leads to DNA mutations, and stimulate some tumor-promotive stress signaling pathways such as p53, PI-3K/Akt/JNKs, MAPKs/ AP-1, IKKbeta/NF-kappaB was activated after BPDE exposure.In order to a comprehensive understand the early cellular responses of BPDE, our laboratory used genomics and proteomics methods, whole genome and protein levels of FL cells after exposure to BPDE of early cellular responses induced to do a systematic study. Using Affymetrix HG-U133 Set (～33000 genes) whole-genome microarrays, we found 1764 genes expression were significantly altered at 4 h after exposure to BPDE compared with the vehicle exposure. These responsive genes are involved in multiple functions including cell cycle control, signaling molecules, transcription factors, and metabolic enzymes, DNA repair and DNA damage response etc.; and extensive signaling pathways including MAPK, cell cycle, Wnt signaling pathway and TGFbeta pathway. Proteomics results indicated, there were 194 protein spots were significantly altered at 12 h after exposure to BPDE compared with the vehicle exposure. These identified proteins are involved in a variety of cellular process including transcription regulation, cell-cycle control, cell proliferation, signal transduction, cell skeleton, development, metabolism and some proteins with unknown functions.These results reveal the complexity mechanism of cellular response to BPDE. However, the results of cDNA microarray gene expression found in 1764 changed, and only found 194 proteins proteomics change, except for reasons due to technical limitations, there may be exites the mechanism of inhibition many gene expression in the translation levels in cells. Research on this issue, cause us interest.microRNAs are a class of non-coding RNA gene whose products ar～22 nt sequences that play important roles in the regulation of translation and degradation of mRNAs through base pairing to partially complementary sites in the untranslated regions (UTRs) of the message. The relationship between miRNAs and carcinogen exposure were reported. Therefore, we hypothesized that miRNAs also involved in early cell responses of BPDE. In order to confirm this idea, we have examined the BPDE-induced miRNA expression profile by miRNA microarray and quantitative real-time PCR (qRT-PCR) in FL cells. In addition, we have predicted identificated miRNAs target gene and verified the regulation of these specific genes upon BPDE exposure.Analysis of miRNA microarrayWe used miRNA 14.0 version of the LC Sciences company associatedμParaflo (?) microarray to study expression profiles of miRNA of FL cells treated by BPDE for 2h. The results showed that 41 miRNAs and miRNAs* expression changed in BPDE treated compared to control (DMSO treatment) (p<0.1).23 of them were up-regulated and 18 were down-regulated. Moreover, we have focused on 7 miRNAs for further study, includingmiR-509-5p, miR-628-3p, miR-183, miR-100, miR-602, miR-484, miR-99a.Validation of changed miRNAs by qRT-PCRTo verify the 7 miRNAs expression utilized the microarray sample and prepared the fresh sample.The results showed that hsa-miR-509-5p expression significantly up-regulated compared to control (p<0.01), while, other miRNAs expression alteration were not obsevered.Inhibition of miR-509-5p gene up-regulation induced by BPDE in FL cellsTransfected miR-509-5p-specific miRNA inhibitor (100 nM) and its negative control (100 nM) in FL cells, respectively. After 72 h, we treated transfected cells with 0.5μM BPDE, DMSO as control. The results showed miR-509-5p-specific miRNA inhibitor abrogated the BPDE-induced up-regulation of miR-509-5p, suggesting the miR-509-5p is specific regulated by BPDE exposure.miR-509-5p target gene predictionWe predicted the miR-509-5p target gene by Target scan 5.1. The obtained result compared with the previous date of our lab, we found 65 intrested genes. And then, we have selected 11 target genes that are involed in cell basic activity control of miR-509-5p, such as CDC14、DOCK4、EIF5B、IGF1R、MEIS1、NF1、PRKCA、RREB1、RAD23B、TDG、TET1.Validation of miR-509-5p target gene by qRT-PCRTo verify the 11 target gene expression wtih microarray samples, prepared fresh samples, or miR-509-5p know-down samples. The results showed that CDC14B, DOCK4, IGF1R, MEIS1, NF1, PRKCA, RREB1 genes expression were significan down-regulated by BPDE (p<0.01). On the contrary, CDC14B, DOCK4, IGF1R, MEIS1, NF1, PRKCA, RREB1 gene expression were significanly up-regulated in the presence of miR-509-5p-specific miRNA inhibitor (p<0.01).Conclusion:a. miRNAs are involved in the BPDE-induced FL cell early responses.b. CDC14B, DOCK4, IGF1R, MEIS1, NF1, PRKCA and RREB1 gene were regulted by the mir-509-5p in FL cell. These identified target genes are involved in a variety of cellular process including cytoskeletal stability, signal transduction, cell cycle regulation, DNA damage repair, transcriptional regulation and so on. Our results indicated the mir-509-5p plays an important role in the BPDE induced FL cell early responses.This study provides important findings to understand the miRNA-mediated cellular responses induced by BPDE, indicating a new molecular mechanism of BPDE-induced mutagenesis and carcinogenesis. The genome is continuously damaged by a variety of endogenous and exogenous agents. Repair of such damage is a crucial mechanism for maintaining genomic integrity, which is essential for normal developmental and physiological consequences. Among the damage, DNA base modifications, such as deamination, alkylation and oxidation, or apurinic/apyrimidinic (AP) sites due to spontaneous depurination arise most frequently. These small lesions are mainly repaired through base excision repair (BER). Pol P plays a crucial role specifically in in repair of DNA damage and maintaining stability and integrality of genome. However, the recent researches indicate that polymerase P might took part in a wide spectrum of DNA metabolism reactions, including DNA replication, recombination, meiosis and transleisional DNA synthesis and behaves high error rate due to its lack of 3’→5’proofreading activity and low replication fidelity. Down-regulation or mutation of Polβis mutagenic due to deficient in DNA repair, while overexpression of this error-prone Po1βmight perturb the normal function of other accurate polymerases and cause genomic instability as well. Hence, we established the Po1βover-expressed and down-regulated stable cell line with dexamethasone. And we used these constructed cell lines to study the biological function of Pol P and the involvement of Polβin genes mutation and genomic instability.In our present study, we constructed the recombinant dexamethasone (DEX) inducible eukaryotic expression vector, pMAMneo-amp- po1-β-and pMAMneo-amp-pol-(3+. Together with the empty control pMAMneo-amp- we transfected the inducible eukaryotic expression vectors in FL cells. And then we selected cell cluster with MEM medium containing 400μg/mL Geneticin (G418). The expression of Po1βin transgenic cell lines was determined after induced by DEX for 72 hours by qRT-PCR and Western blot. The results showed Po1βexpression was down-regulated in FL-Po1β- cells, and was overecpressed in FL-Po1β+cells. Furthermore, MTT assay demonstrated that the FL-Polβ- cell was sensitive to alkylaing agent MNNG exposure, and FL-Po1β+cells was tolorent to MNNG treatment. Cell cycle analysis showed that pol P expression change does not significantly affect cell cycle distribution.