The Effect Of 3'UTR Alternative Splicing Of ERCC1 On The Expression Of Its Adjacent Overlapping Genes Can Be Associated With The Sensitivity Of Lung Cancer To Cisplatin Treatment

Objective:Lung cancer is the most common incident cancer and the leading cause of cancer death in worldwide,Non-small cell lung cancer?NSCLC?is the most prevalent and heterogeneous subtype of lung cancer,including lung adenocarcinoma?LUAD?and lung squamous cell carcinoma?LUSC?.Despite many advances in treatment,the 5-year overall survival for advanced lung cancer is still dismal.Till now platinum-based postoperative chemotherapy is a standard treatment,and cisplatin is a basic chemotherapy drug of lung carcinoma.But cisplatin-resistance mostly leads to the failure of chemotherapy in the patients.Thus,it is very urgent to find specific and valid biomarkers to predict the adjuvant chemotherapy for NSCLC.DNA repair capacity is a major determinant of cisplatin resistance.Since the excision repair cross-complementation group 1?ERCC1?protein is essential for NER and influence genomic instability,ERCC1 may play a critical role in DNA repair.ERCC1-XPF complex cleave on the 5'side of the DNA lesion.Besides NER,the ERCC1-XPF complex also participates in the repair of DNA interstrand crosslinks and for the completion of homologous recombination.Much of the complexity of biology can be attributed to the regulation of gene expression via changes in the transcriptional and post transcriptional levels.Alternative splicing is a regulated process that occurs in nearly all multi-exon human genes.Different transcriptional regulation and splicing mechanisms lead to the production of multiple products by individual genes.The ERCC1 gene generates a variety of isoforms by alternative splicing,just as previous studies have shown that the protein product of 297aa encoded by ERCC1-202 and ERCC1-208 isoforms have different DNA repair capacity,while the only difference between ERCC1-202 and ERCC1-208 is that ERCC1-202 has a longer 3'UTR.It has been shown that the structural changes of 3'UTR was involved in regulating the function and activation of genes.At genomic level,the transcription termination site of ERCC1 3'UTR is adjacent to a conjunct transcription start site of CD3EAP and PPP1R13L,to form a complex sense–antisense gene arrangement?see Fig1A?.ERCC1,CD3EAP and PPP1R13L work as not only adjacent genes,but also functional related genes,revealing three fates of cells in front of the environmental stress?see Fig1B?.The CD3EAP also called ASE-1?Anti Sense ERCC1?,encodes a nucleolar protein localized to fibrillar centers of the nucleolus and to the nucleolus organizer region of mitotic chromosomes.CD3EAP as a subunit of the RNA polymerase I complex involve in ribosomal RNA transcription,indicating the state of cell proliferation.The gene PPP1R13L also called i ASSP,as an inhibitor of TP53 apoptosis pathway and an inhibitor of the RelA subunit of the transcription factor NF-?B.TP53 and NF-?B plays a pivotal role in the apoptosis and inflammatory response.Therefore,PPP1R13L would influence the availability of relative factors and thus modify the regulation of apoptosis.Such overlapping sense and antisense gene pairs may act predominantly as regulators of genes transcription,which have been implicated in many different cellular processes.Transcription and splicing are tightly coupled,Since the transcription process is strand-specific,it is mysterious how and whether the overlapping genes interfere mutually.Several potential mechanisms could achieve this effect,for example,the overlapping genes cross-talk between their transcription start site,transcription termination site,respective splice sites and shares the modifications of the histones.Nevertheless,the precise function of overlapping relationship is still not elucidated.Here,we hypothesized that the overlap region among ERCC1,CD3EAP and PPP1R13L could play a role in linking upstream and downstream genes and has potential implications of cisplatin resistance in non-small cell lung cancer?NSCLC?.The Cancer Genome Atlas?TCGA?project provides a rich Sequencing source for the investigation of exon and gene expression in cancer.Bioinformatics analysis by the TCGA Data Portal aiming to dissect the effects of transcription and alternative splicing will assist to address this issue.Further,a cisplatin induced DNA damage cell model of NSCLC and other in-vitro experiments were also performed to verify the suggestive evidence,which contribute to provide a clue to find complementary biomarkers related to lung carcinoma.Methods:In this study,the primary cell culture and cisplatin susceptibility test were carried out directly by collecting samples of NSCLC patients.In this study,the clinical samples of 20 patients with NSCLC were collected,and the drug sensitivity of cisplatin was detected.The total RNA of cancer tissue and adjacent tissues was extracted.The total expression level of ERCC1 and the expression of ERCC1-202mRNA were detected by RT-PCR.The association between ERCC1 gene 3'UTR alternative splicing and cisplatin sensitivity was analyzed.By constructing over expression vectors of two ERCC1 variable splice isomers,the effect of variable splicing of 3'UTR on the function of ERCC1 protein was detected.Genome organizations characterization of 19q13.2-3.The genome is a symbol string of construction genes and topology genes,which is ranked according to the generated sequences and spatial topological relations.The genome structure of19q13.2-3 was analyzed by bioinformatics.The data accessed from genome.ucsc.edu was derived from the ENCODE project.UCSC Genome Browser on Human Dec.2013?GRCh38/hg38?Assembly was used to analyze the distribution of H3K27Ac and H3K4Me3,and CpG islands in 19q13.2-3.Heatmap of gene exon expression profiles and correlativity analysis.Exon expression of ERCC1,CD3EAP and PPP1R13L in RNA-seq data of TCGA LUAD and LUSC cohorts were downloaded from TCGA data portal?https://tcga-data.nci.nih.gov/tcga/?.Clinical parameters of LUAD and LUSC cohort were also downloaded from TCGA database.60 cases of LUAD and 51cases of LUSC having matched cancer and adjacent normal tissues were screening.The adjacent normal tissues were defined as a distance that was larger than 2cm from tumor margin.Heatmap of expression of exons in cancer tissue and its adjacent normal control were generated by heatmap package in R version 3.3.3.To analyze the correlation patterns of exons in ERCC1,CD3EAP and PPP1R13L expression,the Spearman's correlation coefficient of each exon pairs was calculated using the corrplot package in R version 3.3.3.Alternativesplicingeventanalysis.TCGASpliceSeq?http://bioinformatics.mdanderson.org/TCGASpliceSeq?is a web based resource that provides a quick,highly visual interface for exploring the alternative splicing patterns of TCGA tumors.The Percent Spliced In?PSI?value,a common,intuitive ration for quantifying splicing event from zero to one?24?,was calculated for seven types of alternative splicing events:Exon Skip?ES?,Mutually Exclusive Exons?ME?,Retained Intron?RI?,Alternate Promoter?AP?,Alternate Terminator?AT?,Alternate Donor site?AD?,and Alternate Acceptor site?AA?.The mean PSI value of splice events in ERCC1,CD3EAP and PPP1R13L on samples from LUAD and LUSC have been loaded into TCGA SpliceSeq,which were filtered by 10%minor splice expression.Cell culture and treatment.A549 and LK2 cells were purchased from the Cell Bank of the Shanghai institute of Biochemistry and Cell Biology,Chinese Academy of Sciences,and cultured in DMEM/F-12?Hyclone,USA?and DMEM?Hyclone,USA?,respectively.The immortalized 16HBE cell line,kindly provided by Prof.Wen Chen?Sun Yat-Sen University,China?was cultured in MEM?Hyclone,USA?.Both cells were supplemented with 10%fetal bovine serum?Hyclone,USA?and maintained at37°C,5%CO₂ in a humidified incubator.Logarithmic growth phase cells were treated with various concentrations of CDDP?cis-Diammineplatinum?II?Dichloride,TCI,Japan?according to the requirement of the following experiments.RNA preparation.A549 and 16HBE cells were treated with 4?g/ml CDDP and only vehicle solutions for indicated time points at 24h,which named group CDDP and Control respectively.Total RNA was extracted using TRIzol?Invitrogen?.RNA integrity was assessed using the Agilent 2100 bioanalyzer?Agilent Technologies,Inc.?.RNA concentrations were determined by the absorbance at 260 nm,and quality control standards were A260/A280?=?1.8–2.1,using NanoDrop 2000?Thermo,America?.Extracted total RNA cryopreservation kept under-80°C and prepared for the following experiment.Affymetrix GeneChip.Quality of the total RNA samples was available.Hybridization to Affymetrix Human Transcriptome Assay 2.0?Affymetrix,Santa Clara,CA,USA?were in accordance with the manufacturer's protocol.All detection sevice were performed on the Affymetrix Microarray-Based Gene Expression Analysis platform by Oebiotech Co.Briefly,the raw data were normalized at the exon level and filtered using Affymetrix Expression Console TM software 1.4 by applying the Just RMA?robust multi-array average?algorithm.The alternative splicing analysis on the selected gene list were performed using the database for annotation,visualization and integrated Affymetrix Transcriptome Analysis Console?TAC?Software version 3.0.1.5.The splice index was the expression of the exon normalized to the expression of the whole gene.Signal Intensity of the Probe selection region?PSR?equations use log2scale data.Score for the Isoform is the sum of the PSR?s?scores.RACE-PCR.Based on the structures of the previously-described mRNAs?depicted in Fig.1A?,primers were designed to clone the termination region sequence of different isoforms?See Table 1?.For 3'-RACE experiments,a first reverse transcription step was performed using 3'Full RACE Core Set Ver.2.0?TAKARA D314,Japan?.Amplification was then performed by two rounds of PCR.The first round was performed with a gene-specific primer from outer and the outer adaptor primer used in3'RACE experiments,while in the second reaction we used a nested primer and the inner adaptor primer for 3'RACE.Gel analysis and sequencing of certain PCR products confirmed gene specific product amplification.Quantitative PCR?qPCR?Assay.1?g of RNA was template reverse transcribed with MMLV reverse transcriptase?TAKARA 047A,Japan?.Amplified by PCR with the primers designed by ourselves,a BLASTN search was performed against GenBank to ensure that all primers were unique to the gene of interest.These sequences of the qPCR primers are listed in Table 1.Quantitative analysis was performed by using the LightCycler 480 II?Roche,US?.The PCR reactions were performed by SYBR Premix Ex TaqTM II?Takara,Japan?.Quantitative real time PCR analysis was carried out using the 2?-Delta Delta C?T??method?2-??Ct?[47].In all qPCR experiments the data were normalized to the expression of the Human GAPDH housekeeping gene.Three independent RNA preparations were tested for each sample,and each reaction was performed in triplicate.Data are representative of three independent experiments.Statistical analysis.Data analysis for Statistical significance and Correlation coefficient were performed using‘‘R''software version 2.13.2.The splicing index differences in lung cancer tissue and its adjacent normal tissues were depicted as the median.The data of PCR were expressed as the Means±SD.Statistical analysis was performed with IBM SPSS?Version 20.0,Inc.,Chicago,IL,USA?and GraphPad Prism?Version 5.0,GraphPad Software;San Diego,CA,USA?.Comparative analysis was made by independent-samples t test to determine the significance between groups.Significance values were set at P-value of<0.05.Results:The sensitivity analysis of lung cancer tissue samples showed that ERCC1-202mRNA was highly expressed in normal tissues,and was low in tumor tissues,and had strong correlation with cisplatin sensitivity.The 3'UTR alterable splicing of the ERCC1 gene plays an important role in the individual differences in the susceptibility to cisplatin induced by DNA repair.Over expression plasmids of two isoforms,were transfected into A549 and HEK293T cells after treatment with cisplatin given,two kinds of expression of intercellular DNA injury there were no statistically significant differences,suggesting that the ERCC1 gene 3'UTR splicing may have no influence on the protein function.Structure Characteristics of 19q13.2-3.Overlapping genes seem to be very close,and as a corollary,this structure may have the property of signal transitive.Direct evidence was that CD3EAP and PPP1R13L share the same first exon transcriptional domain,the transcription termination site of ERCC1 is adjacent to a conjunct transcription start site of CD3EAP and PPP1R13L.Peaks of H3K27 acetylation and H3K4 trimethylation,and CpG island were identified near the first exon of CD3EAP and PPP1R13L,while adjacent the terminal exon of ERCC1,which are often found near active regulatory elements and is conducive to DNA unwinds the double helix.Co-expression genes are often functionally related,and bioinformatics analysis of protein networks reveals that both CD3EAP and ERCC1 bind to TBP,and that PPP1R13L can also associate with transcription initiation complexes via an indirect action.Differentially expressed exons are present in normal tissues and tumor tissues.In order to precisely depict the expression relationship between ERCC1,CD3EAP and PPP1R13L,differential expression analysis of exon resolution in tumor and adjacent normal tissue.Majority of exons were varied expressed between lung cancer tissue and its adjacent normal tissue,the remarkable exons were E11,C2-3 and P11-14.In tumor tissues,the differential expression of exons in a single gene were stronger than that in adjacent normal tissues,which further suggests that the tumor tissue prefer to express a single alternative splicing isoform.According to the order of CD3EAP gene expression from low to high,P1 showed a similar trend to C1,E11 and C3 are similar.Analysis of alternative splice events.Compared with the normal tissue,the PSI of AP-11 decreased and AP-10.2 increased.Alternate Promoter?AP?and Alternate Terminator?AT?of ERCC1 is converge to the gene center,it means that ERCC1 can express a shorter transcript in lung cancer tissues.Transcription related analysis in gene and exon levels.In gene level,CD3EAP was significantly positively related to PPP1R13L in normal tissues and tumor tissues.There were negatively correlated or weakly correlated between CD3EAP and ERCC1 in normal tissues,whereas in tumor tissues CD3EAP was significantly positively related to ERCC1.ERCC1 was negatively correlated with PPP1R13L in normal tissues but there were appearing weakly positive correlations in tumor tissues.In exon level,several exons pairs were found to exist the correlational dependence between the overleaping genes.CD3EAP exon 1?C1?was significantly associated with PPP1R13L exon 1?P1?and CD3EAP exon 3?C3?was significantly associated with exon 11 of the ERCC1 gene?E11?in normal tissues and tumor tissues.We defined the pattern as a collection of exons with similar expression behaviors,it reminded that there are some co-expression patterns of those overlapping genes in the region.DNA damaged Cell model induced by CDDP.The survival rate of 16HBE cells was significantly higher than those in A549 cells.These results suggested that 16HBE cells had a higher tolerance to CDDP than A549.While the average survival rate of LK2cells was in the middle of A549 and 16HBE,and there was significant difference in4?g/ml of CDDP treatment.The cell cycle and apoptotic rate after CDDP treatment were measured using flow cytometry assay.Cell cycle arrest in S phase was observed in the 2?g/ml group.Hypodiploid peak appeared?apoptotic peak?in in the 4?g/ml group but there was no obvious hypodiploid peak appeared in A549 and LK2 cells.With the concentration of cisplatin treatment,apoptosis rate of 16HBE cells was increased gradually,while cell necrosis ratio increased in A549 and LK2.It is suggested that A549 and LK2 have different modes of cell decease from 16HBE.Quantitative change of alternative splicing isoforms.The exons or constructs abundances were described by Human Transcriptome Array.To facilitate the experimental validations,we finally selected several Affymetrix Transcript Isoforms with different architectural feature.Compared with the control,the short transcript containing exons 2 to 10 of ERCC1 gene was up-regulated by 3.28-fold in A549 cells after cisplatin treatment,nevertheless,it down regulated by 2.55-fold in 16HBE cells.The full-length transcript containing exons 1 to 11 of ERCC1 was principal expressed in 16HBE cells which up regulated by5.24-fold.These transcripts containing exons 1 of CD3EAP gene were down regulated in A549 cells,while on the contrary were up-regulated in 16HBE cells.The posterior segments of the PPP1R13L gene containing exons 9 to 14 was up regulated in A549cells?Isoform Score 3.49?.By contrast,the full-length transcript containing exons 1 to14 of PPP1R13L gene was up-regulated in 16HBE cells.These results indicated that the short transcripts of ERCC1?2-10?,CD3EAP?2-3?and PPP1R13L?9-14?are co-expressed in A549 cells.The full-length transcripts of ERCC1,CD3EAP and PPP1R13L genes are co-expressed in 16HBE cells.Relative quantification of the co-expression transcript and 3'RACE identification.The co-expression characteristics of exons or constructs were verified by Real-time PCR,which demonstrated that these clones were indeed differentially expressed.The expression of ERCC1 3'UTR was invariant in A549 cells,down regulation of relative Isoform Score was mainly attributed to the high expression of the short transcript,and the expression of downstream genes CD3EAP and PPP1R13L were increased slightly.The expression of ERCC1 3'UTR was significantly increased in 16-HBE cells,simultaneously the expression of downstream genes CD3EAP and PPP1R13L were enhanced,confirmed the cDNA microarray results.To investigate this further,we used3'rapid amplification of cDNA ends?RACE?-PCR to determine the transcription termination sequence of human ERCC1,CD3EAP and PPP1R13L.The expected bands were obtained in 3'-RACE reaction,revealing the presence of transcription termination sites of ERCC1 3'UTR.Conclusion:Many genes are arranged in complex overlapping and interlaced patterns,the arrangements can potentially contribute to the regulation of gene expression.A region in 19q13.2-3 encompassing the overlapping genes ERCC1,CD3EAP and PPP1R13L was found to be associated with the risk and prognosis of non-small cell lung cancer?NSCLC?.We confirm a hypothesis that there are co-expression patterns among these overlapping genes.The suggestive bioinformatic evidences of TCGA were verified by q-PCR of NSCLC tissue samples.A cisplatin induced DNA damage cell model was performed through microarray chips,q-PCR and 3'RACE to verify and quantify the expression of co-expressed alternative splicing isoforms in NSCLC tissue and cell line as A549 and 16HBE.The expression of CD3EAP exon 1 was shown to be significantly associated with PPP1R13L exon 1 while CD3EAP exon 3 was significantly associated with ERCC1 exon 11 in normal and tumor tissues of NSCLC.Short transcripts of ERCC1,CD3EAP and PPP1R13L are co-expressed in A549 cells and full-length transcripts are co-expressed in 16HBE cells.We described a novel transcriptional regulation pattern based on positional relationships of overlapping genes.The region encompassing the overlapping genes ERCC1,CD3EAP and PPP1R13L could play a role in linking upstream and downstream genes,and the different splicing isoform of ERCC1 can affect the expression of its overlapping genes,which reminded potential implications in cisplatin resistance in NSCLC treatment.We provided evidence here that the different 3'UTR termination sites enable ERCC1 gene not only to produce different mRNAs but also to release distinct sets of alternative splicing interference signal to downstream genes CD3EAP and PPP1R13L,the overlap region between ERCC1,CD3EAP and PPP1R13L could play a role in linking upstream and downstream genes and has potential applications of cisplatin resistance in non-small cell lung cancer?NSCLC?,which reminded a novel transcription regulation pattern.we reveal the elusive,ERCC1 3'UTR could activate the expression of downstream genes CD3EAP and PPP1R13L.Does this co-expression pattern affect the expression of PPP1R13L and further affect apoptosis?The rationale here is that,if cell death processes activated by anticancer chemotherapies and radiotherapies,it is possible to induce apoptosis by reversal cell death mechanisms and enhance the sensitivity of the tumor cells to the chemotherapy.Undoubtedly,more targeted work in this area is required to verify these mechanisms in lung cancer,and more effective cancer therapies that are applicable to a broad range of tumor types will ultimately emerge as a result.In addition to the above research on the 3'UTR alternative splicing of ERCC1 gene and its influence on the expression of downstream overlapping genes,we further explored the reverse overlap gene FOSB of ERCC1 gene at the 5'end.The use of TCGA database,the correlation analysis between the expression of FOSB and ERCC1 gene;comparative non and its influence on the survival rate of patients with lung FOSB expression between small cell lung cancer tissues and adjacent cancer tissues and cancer tissues;also collected clinical specimens of lung cancer tissues,the expression of detecting FOSB gene mRNA and protein.The results showed that the expression level of FOSB gene mRNA in tumor tissues was significantly higher than that in cancer tissue,fosB expression of mRNA gene in predicting the survival rate of patients with LUSC and LUAD has a different meaning,the expression of FOSB represents a longer survival in LUAD,while LUSC was on the contrary,the difference was statistically significant?P<0.05?.There are a variety of alterable splicing forms in the FOSB gene exon.The expression of FOSB mRNA is not consistent with the protein expression.Preliminary analysis shows that the FOSB gene may play an important role in the development of lung cancer and has potential value as a biomarker or therapeutic target for lung cancer.