Preliminary Exploration Of Near-infrared Autofluorescence Mechanism Of Parathyroid Gland Based On TMT Labeling Combined With LC-MS/MS

Objective: In recent years,while thyroid cancer surgery has achieved good results,intraoperative parathyroid gland injury has also increased.At present,Near-infrared autofluorescence(NIRAF)can identify the parathyroid gland during operation and distinguish the normal parathyroid gland,primary hyperthyroidism(PHPT)and secondary hyperthyroidism(SHPT)according to the fluorescence intensity,but the mechanism is not clear.This study first constructed three types of parathyroid differential protein expression profiles based on tandem mass tags(TMT)labeling combined with liquid chromatography tandem mass spectrometry(LC-MS/MS).Based on the conclusion that autofluorescence intensity of normal parathyroid glands are higher than that of PHPT and SHPT,differently expressed proteins(DEPs)which are up-regulated in normal parathyroid glands compared with PHPT and SHPT are used for bioinformatics analysis,screening key genes and biological experiment verification,in order to explore the reasons for the difference in fluorescence intensity between normal parathyroids and PHPT,SHPT parathyroids under near-infrared light.Method:1.4 partially intraoperative misresection normal parathyroid glands confirmed by postoperative pathology,6 parathyroid specimens of PHPT and 6 parathyroid specimens of SHPT patients which are collected from thyroid Department of Yuhuangding Hospital in Yantai.Quantitative analysis is conducted based on TMT marker combined with LC-MS/MS.The DEPs of normal parathyroid glands compared with PHPT and SHPT are screened out under the condition of log2(fold change)absolute value >1.2 and P<0.05.2.The up-regulated DEPs of normal parathyroid glands are imported into DAVID database for GO and KEGG pathway enrichment analysis.Two groups of up-regulated DEPs protein-protein interaction networks(PPI)are obtained from String online database.Cytohubba selected the top 20 key genes in each group.The intersection of two groups of key genes may lead to the difference of autofluorescence generation and intensity.3.In addition,15 normal parathyroid glands,55 parathyroid glands of PHPT and 16 parathyroid glands of SHPT are used to determine the m RNA relative expression levels of the selected key genes with quantitative real-time PCR(qRT-PCR).Among them,the normal parathyroid glands that are mistakenly removed during surgery are used for the experiment and the remaining normal parathyroid tissues are implanted into the patient’s sternocleidomastoid muscle.4.Western blot(WB)is used to verify the protein expression of statistically significant differenced genes screened by qRT-PCR in normal parathyroid glands,PHPT parathyroid glands and SHPT parathyroid glands.Results:1.9007 proteins are identified by TMT labeling combined with LC-MS/MS and8442 proteins are left after removing contaminated proteins.Compared with PHPT,85 DEPs meeting the screening conditions are obtained from normal parathyroid glands.79 DEPs are up-regulated and 6 DEPs are down-regulated.Compared with SHPT,393 DEPs eligible for screening are obtained from normal parathyroid glands.188 DEPs are up-regulated and 205 DEPs are down-regulated.2.In normal parathyroid glands compared with PHPT,79 up-regulated DEPs are involved in 19 biological processes(BP),25 cellular components(CC),14 molecular functions(MF),10 KEGG paths.In normal parathyroid glands compared with SHPT,188 DEPs up-regulated are involved in 42 biological processes(BP),30 cellular components(CC),and 25 molecular functions(MF),8 KEGG paths.3.The PPI network generated by 79 DEPs up-regulated in normal parathyroid glands compared with PHPT contained 74 nodes and 233 interactions;Compared with the 188 DEPs upregulated in the parathyroid of the normal parathyroid,the PPI network consisted of 162 nodes and 676 interactions.4.The top 20 hub genes in each group are calculated by MCC algorithm of Cytohubba plugin.The top 20 hub genes of the two groups obtain COL1A1,COL1A2,COL3A1,COL5A1,DCN and LUM.The 6 genes are regarded as common hub genes.5.The 6 selected common hub genes are collected for qRT-PCR in 15 partially normal parathyroid glands,55 PHPT parathyroid glands and 16 SHPT parathyroid glands samples.It was found that the relative expression level of COL1A2 gene in normal parathyroid glands is higher than that in PHPT and SHPT and had significant differences.WB found that the expression of COL1A2 protein in normal parathyroid glands is increased compared with that in PHPT(P<0.05)and SHPT(P<0.01).Conclusions:1.In this study,8,442 DEPs are identified in normal parathyroid glands,PHPT and SHPT by using TMT labeling combined with LC-MS/MS protein quantitative analysis for the first time.Three types of differential expression protein profiles of parathyroid glands are successfully established.2.Based on the conclusion that the autofluorescence intensity of normal parathyroid glands is higher than that of PHPT and SHPT,79 and 188 DEPs are up-regulated in normal parathyroid glands compared with PHPT and SHPT parathyroid glands respectively.3.Through qRT-PCR and WB experiment verification,it is preliminarily speculated that COL1A2 may cause the difference of parathyroid Autofluorescence and fluorescence intensity between normal parathyroid gland and disease state(including PHPT and SHPT).