| BACKGROUND:Thyroid cancer ranks third in the incidence of malignant tumors in Chinese women,and papillary thyroid cancer is the most common pathological type of thyroid cancer.Factors associated with thyroid cancer recurrence include extra-thyroidal invasion of the primary tumor,tumor metastasis,and aggressive histological subtypes.More than 25% of patients with invasive differentiated thyroid cancer may experience local recurrence.Due to the proximity of the tumor to the recurrent laryngeal nerve as well as occasional involvement of the trachea or oesophagus,surgery for recurrent thyroid cancer is difficult and risky,with far-reaching consequences for patient prognosis.Accurate diagnosis and prediction of recurrence are major challenges for clinicians,and it is therefore essential to explore the molecular mechanisms underlying the development of thyroid cancer.Cuproptosis is a novel form of cell death that has emerged in recent years as a copper-dependent cell death different from apoptosis,necrosis,pyroptosis and iron death.It has been found that abnormal accumulation of cuproptosis and zinc ions often occurs in thyroid cancer tissues,so we believe that exploring the mechanism of cuproptosis-associated death in thyroid cancer may play an important role in understanding the molecular mechanisms of thyroid cancer.Alternative splicing(AS)refers to the process of pre-m RNA to mature m RNA,the same gene produces several different mature m RNAs by different splicing methods,and finally produces different proteins,which is an important guarantee for protein diversity and even functional diversity.PRMT5 is a type II protein arginine methyltransferase that catalyses the transfer of methyl groups from S-adenosylmethionine(SAM)to arginine residues on histones and other proteins.Dysregulation of this methylation is critical for the development of certain cancers.The results of the bioinformatics analyses suggest that PRMT5 may be involved in the molecular mechanisms associated with cuproptosis.Therefore,the aim of this study was first to investigate the role of cuproptosis-related genes in the development of thyroid cancer and additionally to probe the mechanism of PRMT5 regulation of alternative splicing.Methods:Based on TCGA and GEO databases,the differential expression of relevant genes in pan-and thyroid cancers was investigated.Apply KEGG and GO enrichment analyses to investigate the possible biological functions of the differential genes.Clarify the relationship between differential genes and disease-free survival in thyroid cancer using univariate,multivariate regression analyses.Screen and construct thyroid prognosis-related signature gene models by LASSO regression.To construct a prognostic model combining genes and pathological features by Nomogram.RNA and protein expression levels were detected by Q-PCR and Western-blot,RT-PCR was used to assess the level of alternative splicing,CCK-8 was used to assess cell viability,and immunoprecipitation was used to detect protein interactions.RESULT1.differential expression of cuproptosis-related genes in thyroid cancer,functional analysis,and role in prognostic assessmentOf the 19 CRGs associated with thyroid cancer recurrence,16 genes were differentially expressed in thyroid cancer.KEGG enrichment analyses showed that these 19 CRGs were mainly enriched in the cell death,cell cycle,and ribosomal pathways.K-M survival analyses and subsequent multifactorial logistic regressions showed that the expression of BUB1 and GINS2 was a thyroid cancer disease-free survival(DFS)potential risk factor.In addition,LASSO regression screened the following three CRGs associated with DFS: FDX1,BUB1,and RPL3.A new prognostic prediction model was constructed by Nomogram,and the predicted probabilities of 1-,3-,and 5-year survival were close to the actual situation.The mechanisms of action of FDX1,BUB1,and RPL3 were related to immune infiltration.ATM,TP53,CCND1,CDK4,CDK2,PRMT family proteins were found in the functional analysis of the three-gene model,and these genes may be involved in its regulatory mechanism,with ATM and PRMT family proteins appearing at a higher frequency.2.Regulation of thyroid cancer cell death by ATM pathwayFirstly,the correlation between ATM pathway and 7 CRGs was explored based on the raw letter analysis.The results of pan-cancer analysis showed that ATM was expressed at a lower level in most malignant tumors.In thyroid cancer,ATM expression was also lower than that in paraneoplastic tissues,and the expression level was even lower in patients with higher T stage.Secondly,20 pairs of thyroid cancer and paraneoplastic specimens were collected clinically and the ATM expression level was detected by PCR method,and the results showed that ATM expression was lower in cancer tissues compared to paraneoplastic tissues.ATM expression was lower in the N1 group compared to the N0 group,in which no lymph node metastasis occurred.Finally,the role of ATM pathway in thyroid cancer cell death was verified in I131-treated thyroid cancer cells.I131 induced ATM phosphorylation,while the levels of DNA damage and autophagy were significantly increased,suggesting that ATM pathway may be involved in the regulation of cell death in thyroid cancer.The role of ATM pathway in the regulation of thyroid cancer cell death was investigated by means of biological analysis,clinical sample validation and cellular experiments.The main results were:(1)ATM interacted with seven CRGs and was expressed at low levels in most malignant tumors.Compared with normal thyroid tissues,the expression was lower in thyroid cancer tissues,with a concomitant increase in T stage and decrease in expression.(2)Clinical tissue samples were collected to validate the results of the database,and PCR experiments revealed that ATM expression was lower in cancer tissues compared with paraneoplastic tissues,and ATM expression was lower in the N1 group than in the N0 group.(3)At the same time,we constructed the cell model of I131 radiation therapy,and found that the phosphorylation level of ATM increased with the action of I131,and at the same time,the level of cell autophagy and DNA damage increased,suggesting that ATM may be involved in I131-induced cell death.3.Role of PRMT5 short isoform in thyroid cancerThe role of PRMT5 short isoform in pan-cancer and thyroid cancer was explored based on biosignature analysis.Firstly,PRMT5 is highly expressed in most malignant tumors,including: BLCA,BRCA,COAD,GBM,HNSC,LUAD,LUSC,PAAD,PRAD,READ,SARC,UCEC.while it is lowly expressed in thyroid carcinoma,THCA.secondly,among 33 malignant tumors,29 PRMT5 short isoforms have been identified so far.isoforms,of which 17 are translatable transcripts,and PRMT5 long isoform and PRMT5 short isoform include 4 and 2 of these isoforms,respectively,in this study.PRMT5 long isoform is more highly expressed in patients with thyroid cancer,compared with PRMT5 short isoform.PRMT5 short isoform-associated differential genes were mainly enriched in cholesterol metabolism,cytokines,and tyrosine metabolism signalling pathways.Finally,the PRMT5 isoform combined with the clinicopathological features of the nomogram can effectively evaluate the recurrence of thyroid cancer patients.4.Mechanisms of PRMT5 short isoform regulation of alternative splicingFirstly,PRMT5 short isoform specifically regulates the expression level of 139 genes,which are mainly enriched in apoptosis,reactive oxygen response activity,and transcription factor activity,etc.Secondly,PRMT5 short isoform regulates the expression level of 139 genes,which are mainly enriched in apoptosis,reactive oxygen response activity,and transcription factor activity.Secondly,PRMT5 short isoform specifically regulates the selective splicing of 270 bins,and the genes undergoing alternative splicing are mainly enriched in RNA splicing,and the major splicing factors include: hn RNPA1,hn RNPH3,hn RNPA2/B1,and SFPQ.In addition,a series of noncoding RNAs such as LINC1234,SNHG17,and SNHG12 were also regulated by it.Subsequently,cell models of PRMT5 knock down and PRMT5 L/S rescue were constructed in HEK293 and Hela cells,and the arrival of splicing factors and related genes was further verified,suggesting that PRMT5 S is indeed involved in the regulatory mechanism of alternative splicing.Finally,the molecular mechanism of PRMT5 S regulation was explored by immunoprecipitation and mass spectrometry assays.The results showed that in the nucleus,PRMT5 short isoform preferred to bind to hn RNPK,while in the cytoplasm,PRMT5 long isoform preferred to bind to DDX17.In addition,PRMT5 short isoform binds tightly to Histone H1.2 both in the cytoplasm and in the nucleus.Meanwhile,both PRMT5 short isoform and PRMT5 long isoform can maintain the methylation of H4R3.CONCLUSION1.Sixteen cuproptosis-related genes are differentially expressed in thyroid cancer and paracancerous tissues,mainly enriched in cell cycle and cell death pathways.The threegene(BUB1,RPL3,FDX1)prediction model can effectively assess the prognostic recurrence of thyroid cancer patients.The molecular mechanism may be related to immune infiltration,ATM pathway and PRMT5.2.Based on the experimental validation of bioinformatics analysis and clinical specimens,ATM is low expressed in thyroid cancer,and its expression level is even lower in patients with higher T stage.In addition,the ATM signalling pathway is involved in the regulation of I131-induced cell death in thyroid cancer.3.Bioinformatics analysis showed that PRMT5 short isoform was less expressed than PRMT5 long isoform in thyroid cancer patients.Its related differential genes are mainly enriched in cholesterol metabolism and tyrosine metabolism,etc.Nomogram of PRMT5 isoform combined with clinicopathological features can effectively evaluate the recurrence of thyroid cancer patients.4.A series of experiments have confirmed that PRMT5 short isoform not only regulates the expression level of apoptosis and reactive oxygen species-related genes,but also specifically regulates the alternative splicing factors and other genes,and its molecular mechanism may be related to the interaction of histone H1.2,hn RNPK and DDX17. |
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