Transcriptomic And Proteomic Analysis Of Papillary Thyroid Carcinoma And Prediction Of HLA Epitopes

Objective:To provide experimental basis for understanding the biological behavior and the role of immune factors in the occurrence,progression,diagnosis and treatment of thyroid cancer,differential expression genes?DEG?were analyzed by transcriptome sequencing?RNA-seq?,differential expression of proteins?DEP?were analyzed by mass spectrometry?MS?and tumor neoantigens or epitopes were predicted and filtered by combining the HLA I alleles with the result of RNA-seq.Methods:The tumor tissues and corresponding thyroid tissues from 10 patients with pathologically confirmed PTC were obtained for RNA extraction,library construction,and then transcriptome sequencing?RNA-seq?was performed on the Ilumina Hiseq platform.Then genetic variants,fusion genes,gene expressions and differential expression were analyzed by bioinformatics.Total protein was extracted from tumor tissues and adjacent tissues of 17 patients with PTC,then Data Independent Acquisition?DIA?method of mass spectrometry?MS?was used to analyze the differential expression proteins.Immunohistochemical staining of ANXA1,FN1,PROS1,RAB27A,ETHE1,and COX7A2 was performed on 40 cases of PTC patients,and the results were statistically analyzed.HLA I allele was tested by PCR-SSP and analyzed by HLAprofiler and OptiType.The results of single nucleotide variation?SNV?,INDEL and fusion genes obtained from RNA-seq were combined with the corresponding HLA I allele results.The possible antigen epitopes were predicted and filtered using the netMHCpan epitope prediction method by pVACtools software.Results:1.In the VEP predicted consequences of single nucleotide polymorphisms?SNPs,145582?,insertion and deletion of germline mutations?INDEL,18260?,SNVs?32623?and somatic mutation INDEL?9265?,the intron variant and the 3 prime UTR variant were the highest.The proportion of deleterious or probably damaging variants in total genetic variants was predicted by SIFT:1.1%in SNPs and 4.91%in SNVs?p<0.05?;0.5%in SNPs and 1.19%in SNVs as predicted by PolyPhen?p<0.05?.In 10cases,BRAF V600E mutation accounted for 5 cases,KRAS mutation accounted for 1case,and the above two mutations were mutually exclusive in each case.2.The same fusion genes predicted by SOAPfuse and EricScript were 31 in total.Among them,12fusions only existed in cancer tissues,EPS8L2/TALDO1 only existed in 3 cancer tissues,the other 11 fusions were found only once in cancerous tissues.3.When the differential expression criterion was|log₂FoldChange|?1 and Padj?0.05,1464 genes were up-regulated and 2276 genes were down-regulated in cancer tissues for RNA-seq;67 high-expression proteins and 14 low expressions in cancer tissues were identified by MS.There were 26 high-expressed and 8 low-expressed proteins in cancer tissues by combining RNA-seq and MS.The immunohistochemical results showed that the expressions of ANXA1,FN1,PROS1,RAB27A,ETHE1,and COX7A2 in cancer tissue were higher than that of the corresponding thyroid tissue,and the differences were statistically significant?P<0.05?.5.The concordance rate of PCR-SSP method with HLAprofiler and OPtiType was 86.67%?26/30?for 2-digit HLA I allele genotyping,and the concordance rate of the latter two was 96.67%?1/30?for 4-digit HLA I allele genotyping.6.Two nonameric peptide epitopes,KSAELSPFL?KSR1-LGALS9?and RASCQLTVL?FARSA-SYCE2?,were predicted and filtered by genetic fusion data.Three nonameric peptide epitopes were screened by genetic variation data:LAHPGFFYF?P4HA1?,KTYERLFYM?PHLDB3?,LLYSNGYNY?IGKV2?D?-28?.These five epitopes can have high affinity with at least two corresponding HLA I molecules.Conclusions:1.The germline variant of PTC is higher than somatic mutation,and the proportion of potentially deleterious mutations in somatic mutation is relatively higher.The mutation rate of BRAF gene in PTC is higher than that of KRAS,and they are mutually exclusive.2.The reproducibility of fusion gene is low,12 fusins that only existed in cancer tissues may be related to the occurrence and development of PTC,especially EPS8L2/TALDO1 and DUOXA1/DUOX2.3.Combining the results of transcriptome sequencing and mass spectrometry analysis,26 molecules that are highly expressed in cancer tissues may play an important role in the development of PTC.4.The results of immunohistochemistry confirmed that the expressions of ANXA1,FN1,PROS1,RAB27A,ETHE1,and COX7A2 in cancer tissues was higher than that in the corresponding thyroid tissues and were expected as potential tumor markers.5.As for HLA genotyping,HLAprofiler and OptiType were better than PCR-SSP method.6.KSAELSPFL?KSR1-LGALS9?,RASCQLTVL?FARSA-SYCE2?,LAHPGFFYF?P4HA1?,KTYERLFYM?PHLDB3?and LLYSNGYNY?IGKV2?D?-28?have their own HLA class I restrictions,and they can be used for the immunogenic study of PTC.