Research Of Alternative Splicing Induced By Paclitaxel And Function Of Splicing Factor RBM25 In Human Non-Small Cell Lung Cancer

Objective: Lung cancer is one of the malignant tumors with the highest rates of incidence and mortality,providing great threat to human health and life.In recent years,many countries have reported that the incidence and mortality of lung cancer w ere significantly increased.The incidence and mortality of lung cancer was ranked the first place in male and the second in female among all cancers.In addition,non-small cell lung carcinoma(NSCLC)accounts for 83% of lung cancer.Most NSCLC patients with stage III and IV receive chemotherapy in combination with or without ionizing radiation.Paclitaxel,a microtubule inhibitor,is commonly used in advanced NSCLC treatment either in combination with platinum-based agents or as monotherapy.It is well established that Paclitaxel functions by directly binding to polymerized ?-tubulin,thereby resulting in perturbation of microtubules dynamics.Paclitaxel inhibits the dynamic instability of the mitotic spindle,leading to impaired chromosome alignment.Consequently,cells are arrested by the spindle checkpoint at the G2/M phase and eventually go through apoptosis.Multiple cellular pathways are involved in paclitaxelinduced cytotoxicity,including RAS,MYC-controlled pathway,and inhibition of spleen tyrosine kinase.However,further molecular mechanisms about paclitaxel might need to be discovered due to the clinical complexity.Almost ninety-five percentage of genes are alternative spliced in human.Importantly,alternative splicing(AS)of pre-mRNA leads to the production of multiple mature mRNAs and protein isoforms with distinct structural and functional properties.Dysregulation of AS also leads to aberrant protein isoforms,which may contribute to tumor initiation,progression and therapeutic treatment difficulties.Previously,in NSCLC,some pre-mRNA splicing regulators have been demonstrated to be abnormally expressed,including SRSF1,SRSF2,SRPK1 and SRPK2.In addition,Shultz et al employed RNA oligonucleotides to modulate caspase 9 pre-mRNA splicing to promote the production of caspase 9b,resulting in sensitizing non-small cell lung cancer cells to paclitaxel.Moreover,in paclitaxel resistant triple-negative breast cancer cells,aberrant RNA splicing was defined,and the interaction of TRA2 A with the splicing factor hnRNPM can co-regulate alternative splicing.Taken together,these data suggest that as a chemotherapeutic agent,paclitaxel may kill cancer cell by modulating the alternative splicing of cancer-related genes.However,the mechanistic details underlying such splicing regulation upon paclitaxel treatment are still largely unknown.Method: 1.cell viability was tested by MTT assay after cells were treated with gradient concentrations of paclitaxel at 0,5,10,20,50,70,100 nM and then detected 48 h treatment after.The IC50 was calculated using Graphad Prism6.2.RTCA and colony formation assays were performed in A549 and H1299 cells treated for 24 h with different concentrations of paclitaxel.3.Flow cytometry approach was utilized to analyze the cell cycle of cells treated with different dosages of 5,10,20 n M for A549 cells and 20,50,70 nM for H1299 for 12 h respectively.4.Annexin V/PI assay was utilized to examine cell apoptosis.5.High-throughtput mRNA-seq was carried out to detect the gene expression events induced by paclitaxel,and validation by real-time RT-PCR.6.alternative splicing events were validated by RT-PCR.7.Geno ontology analysis of paclitaxel induced gene expression events.8.Functional association networks of paclitaxel induced genes and AS targets were analyzed using the STRING database.9.Western blot were utilized to evaluate the protein level of ECT2-L/S 10.Immunofluorescence was used to observe the cell location of ECT2-L/S after incubated cells with paclitaxel 12 h.Results: 1.Paclitaxel inhibits A549 and H1299 cell growth and migration in a dosedependent manner.2.Treated with distinct concentrations of paclitaxel,A549 and H1299 cells demonstrated a dose-dependent cell cycle arrest at G2/M phase and 10% of apoptosis population was achieved at the maximum concentration of paclitaxel treatment in A549 and H1299 cells respectively.3.high-throughput mRNA sequencing with A549 cells treated with paclitaxel were performed,and we identified 944 genes with significant expression change,Gene ontology analysis showed that DNA replication,mitotic nuclear division,DNA damage,chromosome segregation and G2/M transition of mitotic cell cycle were the most enriched pathways.4.We validated targets induced by paclitaxel with real-time RT-PCR,including CDKN1 A,AURKA,GADD45 A,FOXM1 and hnRNPUL1.5.we obtained 855 paclitaxel-regulated AS events with an obvious change of percent-spliced-in(PSI)values(PSI>0.15),including 552 skipped exon(SE),50 alternative 5? ss exon(A5E),73 alternative 3? ss(A3E),67 retained intron(RI),and 113 mutually exclusive exons(MXE).6.paclitaxel induced splicing events were enriched in groups related to cell response to DNA damage stimulus,preassembly of GPI anchor in ER membrane,DNA-templated transcription,DNA repair and G2/M transition of mitotic cell cycle.7.we demonstrated that exon 4 skipping of ECT2 was significantly induced by paclitaxel,the skipping of exon 8 of ZMIZ2 and exon 6 of FMNL3 were also elevated after paclitaxel treatment.8.A549 and H1299 cells expressing ECT2-S grew evidently slower than control and ECT2-L expressing cells.9.A549 and H1299 cells stably expressing ECT2-S could significantly inhibit cancer cell growth as compared to control cells after treated with paclitaxel at concentration of IC50.10.ECT2-L was predominantly nucleus-localized,whereas ECT2-S was distributed in both cytoplasm and nucleus as examined by immunofluorescence after treated with paclitaxel.11.the relative mRNA levels of ECT2-S were significantly reduced in five out of six tested primary NSCLC specimens compared to the paired normal samples.12.higher expression of ECT2 was related to poor overall survival in patients with lung,breast,ovarian,and gastric cancer.Conclusion: Paclitaxel inhibits cell growth and induced cell cycle arrest at G2/M phase and cell apoptosis.Genes with significant expression change induced by paclitaxel are involved in cell cycle regulation and DNA damage response.Paclitaxel induced splicing events are enriched in many biological functions.Cells expressing ECT2-S sensitive to paclitaxel,which may provide new therapeutic combination to treatment of lung cancer.Objective: RNA alternative splicing(AS),the alternative selection of splice sites within pre-m RNA,leads to the production of multiple m RNAs for a single gene,then alter the composition and function of encoded proteins.The plasticity offered by alternative splicing to remodel the proteome means to provide opportunities for cancer cells to produce proteins that meet the needs of growing and spreading.Almost large areas of tumor biology appear to be affected by alternative splicing changes,including metabolism,apoptosis,cell cycle control,invasion,metastasis and angiogenesis.Because of mutations at the splice sites or splicing-regulatory elements and changes in the expression of splicing regulators,cancer cells exhibit aberrant alternative splicing profiles commonly.Accumulating research reported that recurrent somatic mutations in components of the splicing-associated factors were identified in hematological malignancies,lung and breast cancers,which suggested that alternative splicing might be targets of cancer therapy.The core splicing machinery coupled with associated regulatory proteins in eukaryotes comprises to a large splicing system.Splicing regulators are classified into different groups,such as serine/arginine-rich(SR)-RNA binding protein family,heterogeneous nuclear ribonucleoproteins(hn RNPs),and RNA-recognition motif(RRM)proteins.Members of the RBM proteins contain one or more RRM domains,which spans 80-100 amino acids.It is fairly conserved.The structure formed by the RRM contains four ?-sheets and two ?-helices.As members of the hn RNP particle,the RRM proteins can bind to pre-m RNA,whereas be involved in RNA stability and translation as part of sn RNP.RBM25(RNA-binding motif protein 25)belongs to the RBM protein family which consists of arginine-glutamate(RE)rich domain and the C-terminal proline-tryptophanisoleucine(PWI)domain.RBM25 is localized at splicing-factors-rich nuclear speckles because of RE-rich domain.RBM25 interacts with multiple splicing components such as SRm160/300,U sn RNAs assembled splicing complexes to regulate alternative splicing.RBM25 binds to U1 sn RNP-associated factor Luc7 LA and activates proapoptotic Bcl-x S 5' splice site mediated apoptosis.According to a recent report,RBM25 regulates a large fraction of alternatively spliced human genome,and interacts with components of the early spliceosome SF3 B and regulators of alternative splicing.They identified a mono-methylated at lysine 77 in RBM25,However,the region of RBM25 spanning K77 could bind to SRSF2 with high affinity only when K77 is unmethylated.These findings suggest a pivotal role of RBM25 in regulating alternative splicing and reveal new potential mechanisms.We discovered that depletion of RBM25 inhibits cell proliferations and migration of lung cancer cells.Integrating data from RNA-seq analyses have provided further insights into the effects of RBM25 on regulation of pre-m RNA splicing,including exon skipping,intron retention,selective use of splice sites.Taken together,to exploit the mechanisms of proliferation and migration inhibition,regulation of alternative splicing of RBM25,may provide more potential therapeutic of lung cancer.Methods: 1.H1299 and A549 cells were infected with recombinant lentivirus.Screening with puromycin and maintenance with lower concentration of puromycin.2.Detection of protein levels of RBM25 using Western blot.3.RTCA and colony formation assay were used to evaluate cell proliferation and survival of H1299 and A549 overexpression and knockdown cells.4.RTCA and wound healing assay were applied to assess the changes of cell migration.5.Real-time RT-PCR was utilized to evaluate MRNA levels of RBM25 in lung cancer and paired normal tissue.6.Location of RBM25 were detected by immunofluorescence.7.The morphology of cells were observed by optical microscope 8.High-throughtput m RNA-seq technology to detect the alternatives plicing events induced by RBM25.9.Geno ontology analysis of RBM25 induced AS.10.Functional association networks of RBM25 induced genes analyzed using the STRING databaseResults: 1.H1299 and A549 cells stably knocking down RBM25 were constructed.2.The protein levels of RBM25 protein in knockdown cells were significantly decreased.3.Cell proliferation and survival were affected by RBM25.4.Cell migration decreased after RBM25 knocking down.5.In lung cancer tissues,RBM25 is overexpressed compared to paired normal tissues.6.RBM25 is localized in nucleus in293T cells.7.The morphology of cells stably knocking down RBM25 converts to more tenuous compared to control cells.8.RBM25 regulates most of pre-m RNA splicing,including exon skipping,intron retention,selective usage of splice sites.9.Gene ontology analysis showed that regulation of splicing and m RNA processing were the more enriched pathways.10.Function association networks of RBM25 induced genes involved in nuclear-transcribed m RNA catabolic process,nonsense-mediated decay and m RNA splicing,via spliceosome analyzed by STRING database.Conclusion: We investigated the mechanisms of proliferation and migration inhibition,regulation of alternative splicing of RBM25,may provide more potential therapeutics of lung cancer.