| Background: Thyroid cancer is the most common endocrine system malignancy.The incidence rate of thyroid cancer has been increasing year by year.Thyroid cancer is more than 95% of the pathological type of differentiated thyroid carcinoma.Among them,papillary thyroid carcinoma is the most common form.Most papillary thyroid carcinoma patients have a good prognosis after early detection,early standardized operation,standardized TSH inhibition after the operation,and adjuvant treatment with radioactive iodine according to their condition.However,some patients still have early tumor progression or early postoperative recurrence,or even distant metastasis,reducing the disease-free survival rate.Circular RNA is a special cutting form of RNA with a circular structure.In recent years,with the popularization of transcriptome and translation genome sequencing,it has been reported that circ RNA is not completely noncoding RNA.Circ RNA may contain an open reading frame,which can bind ribosomes through an internal ribosome entry site,to translate peptides,which affect cell proliferation,migration,invasion,epithelialmesenchymal transformation,and other biological behaviors.Circ PTPRM is derived from PTPRM(Protein tyrosine phosphate receiver type m,PTPR-μ),formed by circular shearing of exons 9-14.Studies have confirmed that circ PTPRM can affect the proliferation,migration and invasion of liver cancer cells.Through software prediction and evaluation,circ PTPRM has internal ribosomal entry sites and open reading frames,which can encode 187 amino acid peptides.This paper aims to study the role of circ PTPRM translation polypeptide circ PTPRM-187 aa in PTC,find the relationship between circ PTPRM-187 aa and IQGAP1,and try to analyze the changes of the downstream signal pathway to clarify the mechanism of the polypeptide’s influence on the biological behavior of PTC.Methods: Clinical tissue samples were selected,and the expression of circ RNA in PTC tissues and paired adjacent tissues was analyzed by transcriptome high-throughput sequencing data.Circ RNA with high translation potential were evaluated using highthroughput sequencing data.PCR primers and RNase R enzyme were designed to evaluate the circle structure and stability of circ PTPRM.q RT-PCR was used to analyze 100 pairs of PTC tissues and paired adjacent tissues in the thyroid surgery department of the First Affiliated Hospital of China Medical University,and to observe the expression of circ PTPRM.According to the expression of circ PTPRM in PTC and paired adjacent tissues,the relationship between circ PTPRM and clinicopathological data was analyzed.q RT-PCR and fluorescence in situ hybridization were used to determine the expression and subcellular structure of circ PTPRM in normal thyroid cell line Nthy-ori3-1 and four PTC cell lines.Si RNA was used to down-regulate the expression of circ PTPRM,q RTPCR was used to confirm the interference efficiency,CCK-8 and plate cloning experiment was used to detect the effect of down regulating circ PTPRM on the proliferation of PTC cells,scratch experiment and Transwell experiment were used to detect the effect of down regulating circ PTPRM on the migration and invasion of PTC cells,The effect of downregulation of circ PTPRM on EMT related proteins in PTC cells was detected by Western blot.q RT-PCR was used to detect the overexpression efficiency of each exogenous overexpression circ PTPRM configuration,and Western blot was used to detect whether each overexpression configuration can translate peptides.CCK-8 and plate cloning experiments were used to detect the effects of exogenous overexpression circ PTPRM configuration on the proliferation of PTC cells,scratch experiment and Transwell experiment were used to detect the effects of migration and invasion of PTC cells in each group,and Western blot was used to detect the changes of EMT related proteins of PTC cells.Gel electrophoresis and mass spectrometry confirmed whether there was a specific sequence of circ PTPRM translation polypeptide.The cellular localization of FLAG labeled fusion protein was detected by immunofluorescence assay.The proteins interacting with circ PTPRM-187 aa were detected by immunoprecipitation and mass spectrometry,further confirmed by Western blot,and compared with clinicopathological data.The expression of IQGAP1 by CHX treatment and overexpression of circ PTPRM was detected by Western blot;HA labeled ubiquitin was transfected,and the changes of ubiquitination modification of IQGAP1 were detected by immunoprecipitation and Western blot.GSEA analyzed the signal pathways that may be regulated by IQGAP1 and circ PTPRM source gene PTPRM and analyzed the expression relationship between IQGAP1 and key proteins in the corresponding signal pathways.Western blot assay was used to detect the effects of siIQGAP1 and Co-transfection circ PTPRM with si-IQGAP1 on the expression of RAC1,CDC42,and EMT related proteins.Results: 720 circ RNA were differentially expressed in PTC and adjacent tissues,of which301 were up-regulated and 419 were down-regulated.By predicting the ORF and IRES structures of circ RNA,and comprehensively evaluating the junction read and transcriptome differences,seven circ RNA with high translation potential were obtained.Nucleic acid gel electrophoresis confirmed that circ PTPRM did not exist in genomic DNA but was the product of RNA circular shear.q RT-PCR experiments confirmed that circ PTPRM was more tolerant to the digestion of linear RNA digestive enzymes and the natural degradation of temperature and time than m RNA.Tissue q RT-PCR and database data confirmed that circ PTPRM was highly expressed in PTC tissue.The expression of circ PTPRM was positively correlated with the tumor diameter in clinicopathological data.The expression of circ PTPRM was different in PTC cell lines.The relative expression of the TPC1 cell line was the highest,and the expression of BCPAP,K1,and IHH4 was low.Circ PTPRM was localized in the cytoplasm of TPC1.Down regulation of circ PTPRM could weaken the proliferation,migration,and invasion of TPC1 cells,and the expression of E-cadherin was up-regulated,while the expression of N-cadherin and Vimentin were down-regulated.Four exogenous overexpression Vectors,circ PTPRM,circ PTPRMIRESdel,circ PTPRM-ATGmut,and Linear187 aa,could achieve stable overexpression efficiency;Western blot showed that the FLAG labeled fusion polypeptide could be expressed in circ PTPRM group and Linear187 aa group,but not in the circ PTPRMIRESdel group and circ PTPRM-ATGmut group.Both circ PTPRM group and Linear187 aa group could up-regulate the proliferation,migration,and invasion of BCPAP and K1 cell lines,down-regulate the expression of E-cadherin and up regulate the expression of Ncadherin and Vimentin;Circ PTPRM-ATGmut group had no significant effects on cell proliferation,migration,invasion and the expression of EMT related proteins.Gel electrophoresis and mass spectrometry confirmed that the FLAG tag fusion protein in group circ PTPRM originated from circ PTPRM trans junction translation,which was in the cytoplasm.Immunoprecipitation and mass spectrometry confirmed that IQGAP1 interacted with circ PTPRM-187 aa.The high expression of IQGAP1 was correlated with tumor diameter > 2cm and neck lymph node metastasis.Western blot confirmed that the stability of IQGAP1 increased after the interaction between IQGAP1 and circ PTPRM-187 aa.HA labeled ubiquitin immunoprecipitation and Western blot confirmed that circ PTPRM overexpression could down-regulate the ubiquitination modification level of IQGAP1.GSEA analysis found that both IQGAP1 and PTPRM were associated with downstream TGF-β signal pathway was highly enriched,and TCGA data confirmed that the expression of IQGAP1 was related to the expression of RAC1 and CDC42 proteins in Smad independent TGF-β signal pathway.Down regulation of IQGAP1 can inhibit the BCPAP and K1 cell proliferation,migration,and invasion,also inhibit the occurrence of EMT and inhibit the expression of RAC1 and CDC42 proteins in the TGF-β signal pathway;Co-transfection of si-IQGAP1 could restore the enhanced proliferation,migration,invasion and EMT induced by circ PTPRM overexpression,while the upregulation of RAC1 and CDC42 proteins in Smad independent TGF-β signal pathway was also restored.Conclusion: Circ PTPRM is up-regulated in PTC and can translate circ PTPRM-187 aa polypeptide.Circ PTPRM-187 aa binds and inhibits the ubiquitination modification of IQGAP1,regulates the expression of IQGAP1 protein,and up-regulates RAC1 and CDC42 proteins in the Smad independent TGF-β signaling pathway,enhancing the PTC cells proliferation,migration,invasion,and promote the occurrence of EMT. |
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