Significance And Molecular Mechanism Of Aberrant Expression Of PAX8 In Copy-number High Molecular Sub-type Group Of Endometrial Cancer

Objective:The purpose of this study was to verify the aberrant expression of PAX8 in endometrial cancer and analyze the relationship between its expression and molecular sub-type of endometrial cancer.The methylation of PAX8 in different cell lines was detected,then the relationship between the expression and methylation of PAX8 and the TP53 mutation high copy array(CNH)typed of endometrial carcinoma was analyzed.si RNA interference technique was used to knockdown the expression of PAX8 in endometrial cancer cells to explore the effect of PAX8 inhibition on downstream signaling pathways and specific molecular mechanism.The effect of PAX8 on the biological behavior of endometrial cancer cells was analyzed by in vitro functional assay.Methods:1.The expression and clinical prognosis of PAX8 in endometrial cancer were analyzed by bioinformatic means;2.The expression of PAX8 m RNA and protein in endometrial cancer cell lines and tissues were detected by q PCR,Western Blot and Immunohistochemistry;3.Methylation of PAX8 in different cell lines were analyzed by bisulfate sequence technology;4.Combined with TCGA database and online visualization analysis website,the relationship between PAX8 expression and methylation and TP53 mutation high copy array(CNH)in molecular sub-type of endometrial carcinoma was explored;5.si RNA interference technology was used to detect the effects of PAX8 inhibition on downstream key signaling pathways;6.The key proteins interacting with PAX8 were identified by immunoprecipitation combined with mass spectrometry,and the specific molecular mechanisms were elucidated by molecular biology and cell biology;7.The effect of PAX8 on the growth of endometrial cancer cells was verified by Ed U proliferation assay.Results:1.PAX8 was not only highly expressed in endometrial cancer cells and tissues,but also positively correlated with pathological grade.The overall survival of the high expression group was significantly shorter than that of the low expression group(HR=2.05,p<0.001);2.Methylation sequence of PAX8 promoter region showed that the methylation of HEC-1b and Ishikawa cell lines were significantly lower than that of He La and benign endometrial adenocarcinoma(12Z)control cells;3.The methylation of PAX8 gene promoter region in the TP53 mutant endometrial carcinoma groups was significantly lower than that in the TP53 wild-type endometrial carcinoma,and the methylation of both groups were lower than those of the normal control group,which was not only corresponding to the high of PAX8 expression of the tumor group.It also indicates that the overall expression of PAX8 in TP53 mutant group may be higher than that in wild-type group;4.In TCGA data,the proportion of high-copy array endometrial carcinoma was significantly higher in the samples of PAX8 copy amplification /m RNA up-regulation(altered group)than in the unaltered group;5.RNA-seq data showed that downregulation of PAX8 expression could lead to changes in ribosome,ribosome synthesis,lysosome,cell cycle and RNA transport;6.q PCR showed that the expression of ribosome,ribosome synthesis and cell cycle related genes decreased with the knockdown of PAX8 expression,while the expression of lysosome-related genes increased with the knockdown of PAX8expression;7.Co-IP experiment showed that DDX5 could interact with PAX8,and the two could co-localize in the nucleus;8.Down regulation of PAX8 expression can inhibit the expression of c-MYC,a critical oncogenic factor of the cell cycle of copy number high groups endometrial cancer cells with TP53 mutations,and the expression of c-MYC is positively correlated with PAX8.Inhibition of PAX8 expression can also hinder the proliferation of endometrial cancer cells.Conclusion:1.In the TP53 mutant high copy number group of endometrial cancer,PAX8 gene with hypo methylation in the promoter region showed high activation.2.DDX5 was not only a PAX8 downstream targeted gene,and may act as a PAX8 transcriptional co-activator.3.PAX8 interacts with DDX5 to indirectly regulate the transcriptional activity of c-MYC,accelerating the endometrial cancer cell cycle and ultimately leading to malignant proliferation of endometrial cancer cells.