PDLIM5 Identified By Quantitative Proteomics As A Potential Biomarker For Papillary Thyroid Carcinoma

ObjectivesPapillary thyroid carcinoma is the main type of thyroid cancer.In recent years,the incidence of papillary thyroid carcinoma has continued to rise.Although most of the papillary thyroid carcinoma patients have a good prognosis,there are still some patients with recurrence and distant metastasis.These patients have poor prognosis.Therefore,it is urgent to find new targets for drug therapy.The aim of this study is to identify new potential biomarkers for PTC.MethodsHigh-resolution label-free mass spectrometry was performed on PTC surgical tissues and adjacent normal thyroid tissues from six patients.The differential expression proteins were identified by Label-free quantitative proteomics technique.The differential expression proteins were identified.Gene ontology and protein class analysis of differentially expressed proteins were performed with the PANTHER classification system.Through bioinformatics analysis,the PDZ-LIM protein 5 and PDZ-LIMprotein 1 were selected from the differentially expressed proteins.Besides PDLIM5 and PDLIM1,S100A11 which has been proved to be highly expressed in PTC and can be used as a biomarker for PTC was selected to verify expression level.Western blot and qRT-PCR were used to validate the expression in PTC clinical tissue samples.The Quantitative dot blot analysis(QDB)technique was used to detect the protein level of PDLIM5 and PDLIM1 in 75 clinical tissue samples.According to the results of qRT-PCR,Western blot and QDB,PDLIM5 which shows the most therapeutic target potential was selected for further study.IHC technology was used to analyze the expression of PDLIM5 protein in clinical tissue samples,and the relationship between PDLIM5 and clinicopathological parameters was analyzed.The expression of PDLIM5 protein in the B-CPAP cell line of thyroid papillary carcinoma was knocked down by RNA interference.The effects of the proliferation,migration and invasion ability of PDLIM5 knockdown on the cell proliferation,migration and invasion were detected by real-time cell proliferation detection,plate clone formation experiment,cell scratch and Transwell invasion test.Then we explored the mechanisms underlying the effect of PDLIM5 on PTC.The expression of Ras,Raf-1,p-ERK1/2 and MMP-9 were detected by Western blot.ResultsThrough high-throughput and high resolution mass spectrometric analysis,we screened 2788 proteins in PTC,more than the number of protein screened in any previous mass spectrometric analysis in PTC.49 proteins presented significant differences between PTC tissues and adjacent normal thyroid tissues.Among the differentially expressed proteins,44 proteins were up-regulated whereas 5 proteins were down-regulated in the PTC tissues.Through bioinformatics analysis,the results showed that most of these differentially expressed proteins are cytoskeleton protein and nucleic acid binding protein,whose molecular function is binding.The main pathways are integrin signaling pathway and ubiquitin proteasome pathway.Bioinformatics analysis showed that PDLIM5 and PDLIM1 have potential to become a new biomarker of PTC.The results of Western blot showed that the expression of these three proteins in the cancer tissues was higher than in the normal thyroid tissue.The results of qRT-PCR showed the relative expression of PDLIM5,PDLIM1 and S100A11 mRNA in PTC tissues(3.07+0.41,1.65 + 0.35,3.68+1.05)was higher than in normal thyroid tissue.The results of Western blot and qRT-PCR coincided with the result of mass spectrometry.QDB analysis showed that in the 75 clinical tissue samples,PDLIM5 protein was highly expressed in 54 PTC tissues,and PDLIM1 protein was highly expressed in 56 PTC tissues.The results of ROC curve analysisshowed that the AUC of PDLIM5 is 0.87(P <0.001;95%CI 0.817-0.927),and AUC of PDLIM1 is 0.70(P <0.001,95%CI),PDLIM5 has higher sensitivity and specificity.The analysis of ROC curve and the results of Western blot and qRT-PCR showed that the expression of PDLIM5 protein in PTC carcinoma tissues was more higher and more stable and may became the potential target of the target.IHC results showed that in 62 of 78 PTC patients(79.5%)showed higher PDLIM5 level in cancer tissues than that in normal thyroid tissues.The statistical analysis of the expression of PDLIM5 and the pathological features of the patients showed that the expression of PDLIM5 was positively correlated with the size of the tumor,and was not related to the age,sex,and lymph node metastasis of the patients.PDLIM5 knockdown can inhibited cell proliferation,migration and invasion of B-CPAP cells.The expression of Ras,Raf-1,p-ERK1/2 and MMP-2 was lower in PDLIM5 knockdown cell line..ConclusionThrough label-free quantitative proteomics analysis,we screened the largest number of protein in PTC.We find that PDLIM5 may be a potential target for therapy or a prognosis and treatment biomarker for PTC.We detected for the first time in clinical tissue samples that PDLIM5 levels were increased at both mRNA levels and protein levels.The expression level of PDLIM5 is correlated with the size of tumor,indicatingthat PDLIM5 plays an important role in the development of PTC.PDLIM5 is highly expressed in clinical tissue samples and is associated with the development of PTC.Knocking down PDLIM5 inPTC cell lines can inhibit the malignant biological behavior of cells.Therefore,PDLIM5 has potential value as a therapeutic target for PTC.