Screening And Validation Of Thyroid Carcinoma Biomarker Based On Bioinformatics

ObjectiveThyroid cancer is the most common endocrine malignancy and its incidence hasbeen increasing over the last decades, Papillary Thyroid Carcinoma (PTC) accountsfor nearly80%of human thyroid cancers. Currently many approaches includingserum thyroid-stimulating hormone measurement, diagnostic ultrasound examination,and fine-needle aspiration (FNA) are used for diagnosis and determination of benignversus malignant status. In clinical practice, approximately20%of FNA-derivedcytology reports are classified as "indeterminate" or follicular nodules that do notfulfill either benign or malignant criteria. Therefore, adjunctive diagnostic measuresand molecular-based diagnostic approaches are needed in the preoperative distinctionof these lesions. The thyroid is a radiation sensitivity organ. Radiation is the onlyidentified risk factors of thyroid cancer, but the mechanism of radiation-inducedthyroid cancer is unclear now. A large sample of microarray data was analysis in thisstudy to select genes which related PTC. To investigate the correlation betweenpathological processes associated with thyroid cancer between genes, and thegenes-related radiation in Thyroid Carcinoma. In this study, we want to find sometumor biomarkers, radiation-induced molecular targets and a comparative analysis ofclinical and pathological characteristics in Thyroid Carcinoma.MethodsIn our study,499cases of thyroid cancer tissue microarray were analyzed,455cases were PTC, including58pairs of matched tissue and normal tissue samples.Differentially expressed miRNAs in PTC were screened, and the potential targetgenes based on bioinformatics were screened from the gene microarray data. Thecomparative of clinical and pathological characteristics were analysis between tumorpatients. The radiation-induced miRNAs were screened from the patient betweenhaving history radiation exposure or not. Real-time PCR was used to verify the screening results in202cases of tissue and214cases (tumor:181and normal:33) ofperipheral blood. ROC analyses were used to assess the predictive power of the genequotients.Results1. Differentially expressed miRNAs（1）hsa-miR-21is up-regulate in PTC, and it was significant associated withextrathyroidal extension, tumor size, lymph nodes metastatic and remote metastatic ofPTC patients（P＜0.05）. AUC value of it was0.859, sensitivity was69%, specificitywas93.1%and Youden’s index was0.621.（2）hsa-miR-34a is up-regulate in PTC, and it was significant associated withtumor size and lymph nodes metastatic of PTC patients（P＜0.05）. AUC value of itwas0.944, sensitivity was91.4%, specificity was94.8%and Youden’s index was0.862.（3）hsa-miR-181a is up-regulate in PTC, and it was significant associated withextrathyroidal extension, remote metastatic and history radiation exposure of PTCpatients（P＜0.05）. AUC value of it was0.845, sensitivity was89.7%, specificity was72.4%and Youden’s index was0.621.（4）hsa-miR-221is up-regulate in PTC, and it was significant associated withextrathyroidal extension, tumor size and lymph nodes metastatic of PTC patients（P＜0.05）. AUC value of it was0.961, sensitivity was91.4%, specificity was96.6%andYouden’s index was0.880.（5）hsa-miR-139is down-regulate in PTC, and it was significant associatedwith extrathyroidal extension, tumor size, lymph nodes metastatic, residual tumor andnew tumor event dx indicator of PTC patients（P＜0.05）. AUC value of it was0.900,sensitivity was82.8%, specificity was82.8%and Youden’s index was0.656.（6）hsa-miR-144is down-regulate in PTC, and it was significant associatedwith extrathyroidal extension and lymph nodes metastatic of PTC patients（P＜0.05）.AUC value of it was0.924, sensitivity was94.8%, specificity was81.0%and Youden’sindex was0.758.（7）hsa-miR-152is down-regulate in PTC, and it was significant associatedwith extrathyroidal extension, lymph nodes metastatic and remote metastatic of PTC patients（P＜0.05）. AUC value of it was0.892, sensitivity was86.2%, specificity was91.2%and Youden’s index was0.776.（8）hsa-miR-199a is down-regulate in PTC, and it was significant associatedwith extrathyroidal extension, lymph nodes metastatic and remote metastatic of PTCpatients（P＜0.05）. AUC value of it was0.861, sensitivity was84.5%, specificity was84.5%and Youden’s index was0.690.2. Differentially expressed genes（1）AGRN is up-regulate in PTC, and it was significant associated withextrathyroidal extension and remote metastatic of PTC patient（sP＜0.05）. AUC valueof it was0.911, sensitivity was81.0%, specificity was100.0%and Youden’s indexwas0.81.（2）GALNT7is up-regulate in PTC, and it was significant associated withlaterality extrathyroidal extension and lymph nodes metastatic of PTC patients（P＜0.05）. AUC value of it was0.931, sensitivity was91.4%, specificity was98.3%andYouden’s index was0.897.（3）MGAT4B is up-regulate in PTC, and it was significant associated withextrathyroidal extension, residual tumor, tumor size, lymph nodes metastatic and newtumor event dx indicator of PTC patients（P＜0.05）. AUC value of it was0.955,sensitivity was82.8%, specificity was86.6%and Youden’s index was0.794.（4）RAB34is up-regulate in PTC, and it was significant associated withlaterality, extrathyroidal extension and lymph nodes metastatic of PTC patients（P＜0.05）. AUC value of it was0.96, sensitivity was84.5%, specificity was96.6%andYouden’s index was0.811.（5）RAD23B is up-regulate in PTC, and it was significant associated withlaterality extrathyroidal extension and lymph nodes metastatic of PTC patients（P＜0.05）. AUC value of it was0.902, sensitivity was86.2%, specificity was96.6%andYouden’s index was0.828.（6）TMSB10is up-regulate in PTC, and it was significant associated withextrathyroidal extension, tumor size, lymph nodes metastatic and remote metastatic ofPTC patients（P＜0.05）. AUC value of it was0.823, sensitivity was75.9%,specificity was91.4%and Youden’s index was0.673. （7）ANK3is down-regulate in PTC, and it was significant associated withtumor focality, extrathyroidal extension, tumor size and remote metastatic of PTCpatients（P＜0.05）. AUC value of it was0.91, sensitivity was91.4%, specificity was91.4%and Youden’s index was0.828.（8）NR4A3is down-regulate in PTC, and it was significant associated withgender, vital status, laterality and lymph nodes metastatic of PTC patients（P＜0.05）.AUC value of it was0.753, sensitivity was81.0%, specificity was56.9%andYouden’s index was0.379.（9）CCDC85A is down-regulate in PTC, and it was significant associated withextrathyroidal extension, residual tumor and tumor size of PTC patients（P＜0.05）.AUC value of it was0.899, sensitivity was75.9%, specificity was94.8%andYouden’s index was0.707.（10）AXIN2is down-regulate in PTC, and it was significant associated withlaterality, tumor focality, extrathyroidal extension, tumor size and lymph nodesmetastatic of PTC patients（P＜0.05）. AUC value of it was0.836, sensitivity was70.7%, specificity was91.4%and Youden’s index was0.621.（11）BCL2L11is down-regulate in PTC, and it was significant associated withtumor size of PTC patients（P＜0.05）. AUC value of it was0.956, sensitivity was94.8%, specificity was89.7%and Youden’s index was0.845.（12）MLLT3is down-regulate in PTC, and it was significant associated withtumor focality, extrathyroidal extension, tumor size and lymph nodes metastatic ofPTC patients（P＜0.05）. AUC value of it was0.952, sensitivity was89.7%,specificity was94.8%and Youden’s index was0.845.（13）FBXO30is down-regulate in PTC, and it was significant associated withtumor focality, extrathyroidal extension, residual tumor and tumor size of PTCpatients（P＜0.05）. AUC value of it was0.876, sensitivity was86.2%, specificity was84.5%and Youden’s index was0.707.（14）PKIAis down-regulate in PTC, and it was significant associated with newtumor event dx indicator, tumor focality, extrathyroidal extension, residual tumortumor size and lymph nodes metastatic of PTC patients（P＜0.05）. AUC value of itwas0.862, sensitivity was75.9%, specificity was85.7%and Youden’s index was 0.656.（15）ARHGAP24is down-regulate in PTC, and it was significant associatedwith extrathyroidal extension and lymph nodes metastatic of PTC patients（P＜0.05）.AUC value of it was0.938, sensitivity was87.9%, specificity was94.8%andYouden’s index was0.827.（16）TBC1D4is down-regulate in PTC, and it was significant associated withextrathyroidal extension, tumor size and lymph nodes metastatic of PTC patients（P＜0.05）. AUC value of it was0.966, sensitivity was87.9%, specificity was96.6%andYouden’s index was0.845.（17）BCL2is down-regulate in PTC, and it was significant associated withlaterality, extrathyroidal extension, residual tumor, tumor size and lymph nodesmetastatic of PTC patients（P＜0.05）. AUC value of it was0.942, sensitivity was84.5%, specificity was93.1%and Youden’s index was0.776.（18）TNFRSF11B is down-regulate in PTC, and it was significant associatedwith extrathyroidal extension, residual tumor, tumor size and lymph nodes metastaticof PTC patients（P＜0.05）. AUC value of it was0.921, sensitivity was84.5%,specificity was93.1%and Youden’s index was0.776.（19）TNS1is down-regulate in PTC, and it was significant associated withlaterality, extrathyroidal extension, tumor size and lymph nodes metastatic of PTCpatients（P＜0.05）. AUC value of it was0.916, sensitivity was93.1%, specificity was84.5%and Youden’s index was0.776.（20）MATN2is down-regulate in PTC, and it was significant associated withextrathyroidal extension, residual tumor, tumor size and lymph nodes metastatic ofPTC patients（P＜0.05）. AUC value of it was0.963, sensitivity was89.7%,specificity was94.8%and Youden’s index was0.845.（21）FOS is down-regulate in PTC, and it was significant associated withextrathyroidal extension of PTC patients（P＜0.05）. AUC value of it was0.805,sensitivity was72.4%, specificity was81%and Youden’s index was0.534.3. miRNAand putative target geneshsa-miR-21（target genes：ARHGAP24and MATN2）, hsa-miR-34a （targetgenes：ANK3，FBXO30，MLLT3and PLIA）, hsa-miR-181a（target genes：TBC1D4 and TNS1）, hsa-miR-221（target genes：AXIN2and BCL2L11）, hsa-miR-139（targetgene：GALNT7）and hsa-miR-152（target genes：TMSB10and RAB34） wereidentified。4. Screening radiation-induced miRNAshsa-miR-146b, hsa-miR-181a, hsa-miR-181b, hsa-miR-3065, hsa-miR-342,hsa-miR-34a, hsa-miR-10a, hsa-miR-150, hsa-miR-152, hsa-miR-153, hsa-miR-195,hsa-miR-199a, hsa-miR-199b, hsa-miR-214, hsa-miR-215, hsa-miR-3607,hsa-miR-3652and hsa-miR-365were identified between the patients which hadhistory radiation exposure or not（P＜0.05）.But history radiation exposure was notsignificant associated with clinical and pathological characteristics in PTC（P＞0.05）.5. VerifyingAXIN2by Real-time PCR in tissue and bloodAXIN2was significantly down-regulate in PTC either tissues or blood ofpatients（P＜0.05）. But there are no significant associated between tissues and blood（P=0.06），ROC analyses showed that, the AUC of AXIN2in blood was0.958,sensitivity was90.7%, specificity was93.6%and Youden’s index was0.843.6. Screening the combination biomarkersIn blood of PTC patients, TP53INP1and TP53INP2was significantlyup-regulate.ITGA3was significantly down-regulate（P＜0.05）。The AUC ofTP53INP1in blood was0.783, sensitivity was75.4%, specificity was73.1%andYouden’s index was0.485. The AUC of TP53INP2in blood was0.864, sensitivitywas90.7%, specificity was77.3%and Youden’s index was0.680. The AUC of ITGA3inblood was0.881, sensitivity was96.5%, specificity was77.3%and Youden’s indexwas0.738. The AUC of AXIN2/TP53INP1was0.991sensitivity was97.0%,specificity was95.8%and Youden’s index was0.928. The AUC of TP53INP2/AXIN2was0.969, sensitivity was93.7%, specificity was93.8%and Youden’s index was0.875.Conclusions1. hsa-miR-21, hsa-miR-34a, hsa-miR-181a and hsa-miR-221were up-regulatein PTC.hsa-miR-139, hsa-miR-144, hsa-miR-152and hsa-miR-199a weredown-regulate in PTC. They were significant associated with clinical and pathologicalcharacteristics.2. AGRN, GALNT7, MGAT4B, RAB34, RAD23B and TMSB10were up-regulate in PTC, ANK3, NR4A3, CCDC85A, AXIN2, BCL2L11, MLLT3,FBXO30, PKIA, ARHGAP24, TBC1D4, BCL2, TNFRSF11B, TNS1, MATN2andFOS were down-regulate in PTC. They were significant associated with clinical andpathological characteristics.3. hsa-miR-146b, hsa-miR-181a, hsa-miR-181b, hsa-miR-3065, hsa-miR-342,hsa-miR-34a, hsa-miR-10a, hsa-miR-150, hsa-miR-152, hsa-miR-153, hsa-miR-195,hsa-miR-199a,hsa-miR-199b,hsa-miR-214,hsa-miR-215,hsa-miR-3607,hsa-miR-3652and hsa-miR-365could be the radiation-sensitive miRNAs in Thyroid Carcinoma.4. The potential diagnosis value of hsa-miR-34a, hsa-miR-221, GALNT7andANK3was confirmed.5. AXIN2, TP53INP1, TP53INP2and ITGA3were differentially expressed inblood between the PTC patients and the healthy control. The potential diagnosis valueofAXIN2,AXIN2/TP53INP1and TP53INP2/AXIN2was confirmed in blood.