Mechanisms Of The EDB Domain Alternative Splicing Of FN1 Regulating Papillary Thyroid Cancer Proliferation And Invasion

Background:Papillary thyroid cancer(PTC)is the most common pathological type of thyroid carcinoma,and the most common malignancy in the endocrine system,and it’s incidence has been increasing year after year.PTC is clinically and pathologically by overall slow progression and good prognosis,but it is prone to metastasize to the cervical lymph node in the early stage,seriously affects prognosis.Little is known about the molecular mechanisms underlying the biological behavior of PTC.Therefore,it is pertinent to explore the specific mechanisms unique to PTC’ biological behavior,especially those related to invasive metastasis and proliferation.This may offer invaluable theoretical insight and clinical utility for the personalized management of PTC.Fibronectin FN1,an important component of the extracellular matrix,has been reported to play both proand anti-cancer roles in different tumors.The pre-m RNA of FN1 has three alternative splice domains;EDA,EDB,and IIICS.Post-transcription modification by alternative splicing results in various FN1 isoforms include different splicing domains.Splicing factor ZMAT3,a downstream target gene of TP53,affects protein function and regulates alternative splice events via post-transcriptional modification and processing after by binding to RNA.In this study,bioinformatics analysis and in vivo and ex vivo experiments are employed to explore the specific expression of different EDB-FN1 splice isoforms in PTC cells to obtain different biological functions via splice factor ZMAT3 mediation,and found the molecular mechanism of PTC’s biological characteristics of early cervical lymph node metastasis.This mechanism provides a theoretical basis for indepth understanding the biological behavior of PTC and provides an evaluable resource for personalized diagnosis and treatment of PTC.Objective:The project aims to study the invasion and proliferation roles of and mechanism of different EDB-FN1 alternative splice isoforms regulated by splicing factor ZMAT3 in PTC through bioinformatics analysis and a series of experiments,which may provide a theoretical basis for in-depth understanding of the disease’s biological behavior and personalized diagnosis and treatment in PTC.Methods:1.Bioinformatics analysis of TCGA and GEO databases were used to identify the core differential gene FN1 in PTC and normal thyroid,then database analysis,PCR and WB experiments were used to detect and compare the FN1 expression levels in pan-cancer cells;2.PCR experiments were used to detect the expression of FN1 alternative splice domain EDA and EDB in normal thyroid and PTC cells and tissues,and IHC was performed to detect EDB expression in normal thyroid and PTC tissues,and a correlation analysis between EDB expression and cervical lymph node metastasis was performed;3.Lentiviral transfection was performed to construct FN1,EDB,ZMAT3,ITGB1 and splicing factor(SRSF5,SRSF2 and SRSF1)knockdown cell lines.selecting shFN1 monoclonal cells were selected and EDB(+)FN1 and EDB(-)FN1 overexpression cell lines were established based on the selected cells,and ZMAT3 overexpression cell lines were also established,and PCR,q-PCR and WB experiments were performed to verify the expression of all target genes;4.In vitro wound-healing assay,Transwell assay,tube formation assay,CCK8 cell proliferation assay,cell cycle assay,clone formation assay,and an in vivo footpad popliteal lymph node metastasis model,and a subcutaneous tumorigenesis model were constructed to study the roles of different isoforms of EDB-FN1,ZMAT3,and SR splicing factors on the invasion,metastasis and proliferation ability of PTC;5.RNA-seq analysis was performed for control,shFN1,OEEDB(+)FN1,OE-EDB(-)FN1 and CM-shFN1(different EDB-FN1 isoforms conditioned media were co-cultured with shFN1 cells),and mass spectrometry for flag-EDB(+)FN1-and his-EDB(-)FN1-bound proteins was done by tagged antibodies to explore the enriched signaling pathways and regulated downstream genes of different EDB-FN1 isoforms.IP,qPCR,WB and IHC experiments were performed to validate the target genes;6.EDB region downstream intron probes(containing cis-regulatory elements)were constructed,and RNA-pulldown experiments were performed to identify binding proteins;7.While rMATS was used to analyze RNA-seq data of control,shZMAT3 and shSRSF5 for obtaining differential alternative splice events,q-PCR and PCR were performed to validate the regulatory effects to the EDB region of different splicing factors;8.Analysis of the GEO database GSE145555 dataset was done to identify ZMAT3-binding transcripts in eCLIP experiments;9.Chemotherapeutic drug cisplatin was added to PTC cells to induce nuclear damage and activate TP53 expression;PTC cell-conditioned media were co-cultured with vascular and lymphatic cells to explore the roles of secreted FN1;Gene rescue experiments were performed to study the biological function of ZMAT3 by regulating the EDB region;10.ROC and Kaplan-Meier curves for diagnostic efficiency analysis and survival analysis respectively.Results:1.The results from the public data analysis and PCR and WB experiments showed that FN1 is a core differential gene in PTC,specifically expressed in PTC cells compared with pan-cancer cells,and it is positively correlated with lymph node metastasis.2.PCR verified the expression of EDA and EDB domain in normal and PTC cells and tissues,and the results showed that the EDB region was specifically expressed in tumor tissues and cells,not in normal thyroid tissues and cells;and IHC results showed that EDB region expression significantly and positively correlated with lymph node metastasis,and had good efficacy for diagnosing lymph node metastasis by ROC curve analysis(AUC=0.784).3.Establishing respective FN1 and EDB knockdown PTC cell lines,in vitro and in vivo experiments showed that both cell line significantly inhibited cell invasion,metastasis and tube formation ability.Establishing respective EDB(+)FN1 and EDB(-)FN1 overexpression cell lines on bases on shFN1 cells,in vitro and in vivo experiments showed that only the EDB(+)FN1 isoform restored the shFN1 cells invasion and tube formation ability.4.RNA-seq analysis for OE-EDB(+)FN1 and OE-EDB(-)FN1 CM cocultured with shFN1 cells was performed,and tagged antibodies for flagEDB(+)FN1 and his-EDB(-)FN1were used to identify binding protein.IP assays suggested only EDB domain-containing FN1 isoform upregulated ECM-receptor interaction pathway by binding to ITGB1.5.Results from GASE enrichment analysis,qPCR and WB experiments after CM co-culture with endothelial cells showed that EDB domain-contained FN1 isoform promoted lymph node metastasis by upregulating the VEGF signaling pathway,and played a pro-cancer role in PTC.6.In vitro proliferation assays and in vivo subcutaneous tumorigenesis models showed that the EDB-deficient FN1 isoform significantly inhibited cell proliferation,and IHC revealed that k I67 expression was significantly decreased.7.Analysis of RNA-seq data revealed that there was a strong correlation between EDB(-)FN1 and the p53 signaling pathway in PTC cells.When p53 responded to cisplatin stimulation,the trans-activation of the downstream target gene p21 was dependent on FN1 expression and so intracellular FN1 by upregulating p53/p21,inhibited cell proliferation and played an anti-oncogenic role.8.Differential alternative splicing event analysis of RNA-seq,GSE145555 dataset eCLIP experiment data analysis,and RNA-pulldown and mass spectrometry for EDB domain cis-regulatory elements-binding proteins revealed that TP53 downstream target gene,splicing factor ZMAT3 regulated the EDB region by targeting spliceosomes,and knockdown of ZMAT3 promoted EDB domain skipping.9.Functional experiments showed that ZMAT3 promoted cell invasion and metastasis by regulating the alternative splice EDB domain.Conclusion:1.FN1 is a core differential gene and is specifically expressed in PTC cells,and the alternative splice EDB domain is solely expressed in tumors and is closely associated with lymph node metastasis.2.FN1 alternative splice isoforms that possess EDB domains and are secreted extracellularly promote lymph node metastasis via the ECMreceptor interaction and VEGF signaling pathways to play a pro-cancer role in PTC.3.EDB-deficient FN1 isoforms which inhibit cell proliferation ability by upregulating p53/p21,play an anti-oncogenic role in PTC.4.The EDB region in FN1 is regulated by splicing factor ZMAT3 to form different splicing isoforms to play different roles in PTC,which is an important mechanism for biological behavior of lymph node metastasis in early stage of PTC.