USP22 Downregulates ERa-mediated Transcription In A Deubiquitinase-independent Way And Its Role In Papillary Thyroid Cancer

Objective: Papillary thyroid cancer(PTC)is the fastest growing tumor of endocrine malignancies.The ratio of male to female is about 1:3 and the reason for the high incidence in women remains unclear.Most PTC have good prognosis,however,relapse and metastasis are still intractable problems in treatment.The expression level of estrogen receptor α(ERα)is related to sex and sex hormone levels.Estrogen can promote the migration and invasion of PTC cells.Ubiquitin specific peptidase(USP22)is an important member of the USP subfamily of deubiquitination enzymes.Although studies have shown that USP22 enhanced ERα-induced transactivation and promoted the occurrence and development of breast cancer,its mechanism in PTC remains unclear.This study aims to elucidate the specific molecular mechanism of USP22 on the occurrence and development of PTC by regulating ERα transcription from epigenetic aspect,and further understand the causes of the high incidence of PTC in female patients and seek new therapeutic targets.Methods: 1.The expression of USP22 in papillary thyroid carcinoma and para-carcinoma normal tissues was detected by Western blot;2.The correlation between USP22 and ERα was analyzed in STRING database,and the interaction between USP22 and ERα was verified by protein immunoprecipitation(Co-IP)and immunofluorescence(confocal);3.The effect of USP22 on ERα-mediated gene transcription and its specific action region were verified by luciferase reporter assay;4.In order to further explore the new mechanism of how USP22 regulates ERα activity,the crucial epigenetic co-regulators of USP22 were identified by mass spectroscopy;5 RNA-sequence was used to investigate the genomic of USP22 in regulating ERα-mediated gene transcription;6.Chromatin immunoprecipitation(Ch IP assay)was used to investigate the recruitment changes of USP22,ERα and co-regulators in the estrogen response element(ERE)of ERα target genes after knocking down USP22,as well as the changes of histone modification levels;7.To explore the effect of USP22 on biological function of PTC through ERα,transwell test,scratch test and plate clone test were used to detect the the proliferation,migration and invasion of PTC cells.Results: 1.The lower expression of USP22 in PTC was negatively correlated with TNM stage;2.STRING database analysis suggested that there was a correlation between USP22 and ER α in PTC.Co-IP and confocal confirmed that USP22 interacted with ERαand co-located with ERα in PTC cells(BCPAP and TPC1);3.Both USP22 and USP22 variants [USP22(HH/AA)],with deubiquitination site mutation,down-regulated ERα-mediated transcriptional activity.USP22 C-terminal motif(USP22-C)also down-regulated ERα-mediated transcriptional activity,with USP22 N-terminal(USP22-N)had almost no effect on ERα induced transactional activity.Moreover,USP22 mainly down-regulated the activity of ERαAF2,the C-terminal ligand-dependent activation function(AF)domain 4.Knockdown of USP22 promoted the protein and m RNA expression levels of CTSD and VEGF,the downstream oncogenic target genes of ERα.While overexpression of USP22 had the opposite effect.5.It was found that USP22 can interact with histone deacetylase 2(HDAC2)and metastesis-associated protein 2(MTA2)to form complex by mass spectrum analysis.Co-IP confirmed that ERα could interact with HDAC2 and MTA2,and knockdown of USP22 did not affect the interaction between ERα and Hd AC2 and MTA2.6.RNA-seq was used to further analyze the effect of USP22 in the regulation of ERα-mediated transcription at the genome-wide.7.Knockdown of USP22 reduced the recruitment of USP22 and complexes in ERE region,but did not affect the recruitment of ERα.8.With E2 stimulated,lower expression of USP22 enhanced the clonogenesis of TPC1 and BCPAP,and promoted the migration and invasion of both cells.Conclusion: 1.USP22 expression was lower in PTC compared with normal thyroid tissue and negatively correlated with TNM stage.2.USP22 did not down-regulate ERα-mediated transcription depending on its deubiquitinase activity,but formed a complex with HDAC2 and MTA2 to inhibit ERα activity.3.Knockdown of USP22 reduced the recruitment of USP22,HDAC2 and MTA2 in ERE region.4.Knockdown of USP22 could promote the proliferation,migration and invasion of PTC cells.