Study On The Effect And Mechanism Of PDIA6 Promoting The Malignant Biological Behavior Of Endometrial Cancer

Endometrial cancer was one of the three most common malignancies of female genital tract and the sixth most common cancer among women.In recent years,its incidence rate has been increasing globally,which seriously threatened the physical and mental health of women.Risk factors for the development of endometrial cancer include obesity,aging population,racial differences,endogenous or exogenous estrogen exposure,early menarche,late menopause,metabolic syndrome（including diabetes and polycystic ovary syndrome）and genetic susceptibility,which are closely related.Traditionally,endometrial cancer was divided into type Ⅰ and type Ⅱ,namely estrogen-dependent and estrogen-independent.Type Ⅰ,which accounted for about 90%of endometrial cancers,was usually associated with estrogen and had a better prognosis.Type Ⅱ accounted for about 10%of endometrial cancers and was usually a high-grade tumor with a poor prognosis.Since endometrial cancer was a heterogeneous disease,traditional staging had limitations in terms of reproducibility of diagnosis and prediction of prognosis.For accurate diagnosis and precise treatment,genomics-based TCGA typing classifies endometrial cancer into 4 molecular subtypes:POLE hypermutated,microsatellite instability type（MSI）,low-copy（CN-L）and high-copy（CN-H）.Among them,POLE hypermutated type had the best prognosis,followed by MSI and CN-L,and the worst was CN-H.TCGA typing was of great significance in guiding the selection of treatment methods and predicting the prognosis of patients with endometrial cancer,and more refined typing will have more important clinical significance in the diagnosis and treatment of endometrial cancer.Therefore,exploring the mechanisms underlying the development of endometrial cancer is crucial to improve early diagnosis,treatment and prognosis.Protein disulfide isomerase family A member 6（PDIA6）,a member of the PDI family,was located on the short arm of human chromosome 2,region 2,band 5,also known as ERp5,P5 or TXNDC7.The evidence showed that PDIA6 played an important role in the development of various tumors.Moreover,the role of PDIA6 was two-fold in different tumors.For example,PDIA6 was upregulated in non-small cell lung cancer,pancreatic cancer,bladder cancer,gastric cancer,liver cancer,and melanoma,and promotes the development of these tumors,and was closely associated with their poor prognosis.In contrast,PDIA6 played an inhibitory role in the migration and invasion of glioblastoma cells.However,the role and molecular mechanisms of PDIA6 in endometrial cancer had not been elucidated.Therefore,the aim of this project was to analyze the expression of PDIA6 in endometrial cancer,explored the effects and potential mechanisms of PDIA6 on the malignant biological behavior of endometrial cancer,and provided new ideas for the treatment of endometrial cancer,and further improve the prognosis of endometrial cancer patients.The main research of this topic included the following three parts.Part Ⅰ:Expression and clinical significance of PDIA6 in endometrial cancerObjectThis section aimed to investigate the expression of PDIA6 in endometrial cancer tissues and cells,and the relationship between PDIA6 expression and clinicopathological features.MethodsBioinformatics methods were used to analyze the mRNA and protein expression of PDIA6 between normal endometrial tissues and endometrial cancer tissues from the Gene Expression Profile Microarray dataset（GSE17025）in the GEO database,TCGA database and CPTAC database.Quantitative real-time polymerase chain reaction（qRT-PCR）and western blot assay were performed to detect the mRNA and protein expression levels of PDIA6 in normal endometrial tissues and endometrial cancer tissues.Immunohistochemical assay was performed to analyze the expression of PDIA6 in endometrial cancer tissues and its correlation with clinicopathological factors of endometrial cancer patients.Kaplan-Meier（KM）plotter was used to analyze the correlation between the expression of PDIA6 and overall survival（OS）and recurrence free survival（RFS）of endometrial cancer patients.Results1.Compared with normal endometrial tissues,PDIA6 was highly expressed in endometrial cancer tissues,differences were statistically significant.2.Compared with HESC,PDIA6 was overexpressed in endometrial cancer cell lines（Ishikawa,AN3CA,HEC-1A,RL-95）,differences were statistically significant.3.The high expression of PDIA6 was positively correlated with FIGO stage and lymph node metastasis in endometrial cancer,but not with age,menopause,differentiation degree and tumor size,differences were statistically significant.4.In endometrial cancer patients,patients with high PDIA6 expression showed lower RFS than those with low PDIA6 expression.ConclusionPDIA6 is overexpressed in endometrial cancer tissues and endometrial cancer cell lines.The high expression of PDIA6 is closely related to FIGO stage,lymph node metastasis and lower RFS of endometrial cancer,which could be used as an indicator to determine the prognosis of endometrial cancer.Part Ⅱ:The effect and mechanism of PDIA6 on the malignant biological behavior of endometrial cancerObjectWe aimed to investigate the effect and mechanism of PDIA6 on the proliferation,migration and invasion of endometrial cancer cells.MethodsTransfection of endometrial cancer cells with small interfering and lentivirus to upregulate or down-regulate PDIA6 expression.CCK-8 and clone formation assay were used to detect the effect of PDIA6 on the proliferation of endometrial cancer cells in vitro.A xenograft tumor assay was performed to verify the proliferation role of PDIA6 in vivo.The effect of PDIA6 on migration and invasion of endometrial cancer cells were detected by the Transwell assay and wound scratch assay.Transcriptome sequencing analysis showed that knockdown of PDIA6 caused changes in TGF-β signaling pathway.Western blot was used to verify the related proteins expression of TGF-β signaling pathway after knockdown and overexpression of PDIA6.Results1.Knockdown of PDIA6 attenuated the proliferation of EC cells,while overexpression of PDIA6 had the opposite effect.A xenograft tumor assay in nude mice showed that the tumor formation ability of endometrial cancer cells was significantly decreased after PDIA6 knockdown,while the tumor formation ability was significantly enhanced after PDIA6 overexpression,the difference was statistically significant.2.Knockdown of PDIA6 significantly reduced the migration and invasion of endometrial cancer cells,while overexpression of PDIA6 significantly enhanced the migration and invasion of endometrial cancer cells,the difference was statistically significant.3.Western blot showed that the expression of epithelial marker E-cadherin increased,while the expression of mesenchymal marker N-cadherin,and c-MYC decreased after knocking down PDIA6.After overexpression of PDIA6,the expression of epithelial marker E-cadherin decreased,while the expression of mesenchymal marker N-cadherin,and c-MYC increased.4.PDIA6 was significantly associated with TGF-β signaling pathway,and PDIA6 knockdown could affect the expression of TGFBR1,and the phosphorylation of Smad2 and Smad3.Conclusion1.PDIA6 could promote the proliferation,migration,invasion and epithelialmesenchymal transition of endometrial cancer cells.2.PDIA6 could affect the malignant biological behavior of endometrial cancer by activating TGF-β signaling pathway.Part Ⅲ:Study on the upstream regulatory mechanism of PDIA6 in endometrial cancer cellsObjectWe aimed to explore the regulatory mechanism of PDIA6 expression in endometrial cancer cells.MethodsMiRanda:http://www.miranda.org/,miRmap:https://mirmap.ezlab.org/,PicTar:https://pictar.mdc-berlin.de/and Targetscan7.2:https://www.Targetscan.or/ert₇2/multiple databases were intersected to predict miRNAs and their binding sites that might bind to PDIA6,and then intersected with miRNAs upregulated in both TCGA database and GSE25405 dataset to identify miRNAs that might regulate PDIA6 expression.qRT-PCR was performed by the expression of PDIA6 in endometrial cancer cells after transfection with miR-424-5p mimics and inhibitor.The direct binding relationship between miR-424-5p and PDIA6 was verified by Dual luciferase assay.LncRNA that may bind to miR-424-5p was further predicted through the Starbase:https://starbase.sysu.edu.cn/,and TCGA database.Changes in miR-424-5p expression after knockdown or overexpression of TRPM2-AS were detected by qRT-PCR.A direct binding relationship between TRPM2-AS and miR-424-5p was verified by Dual luciferase assay and RIP assay.Then qRT-PCR was used to examine the expression of TRPM2-AS in normal endometrial tissues and endometrial cancer tissues.Endometrial cancer cells were transfected with small interfering and lentivirus to up-regulate or down-regulate TRPM2-AS expression.CCK-8 and clone formation assay were used to detect the effect of TRPM2-AS on the proliferation of endometrial cancer cells in vitro.A xenograft tumor assay was performed to verify the proliferation role of TRPM2-AS in vivo.Transwell and scratch assays were performed to verify the effect of TRPM2-AS on the migration and invasion of endometrial cancer cells.Western blot was used to detect the changes of E-cadherin,N-cadherin and c-MYC expression after knockdown and overexpression of TRPM2-AS.KEGG enrichment analysis of differential genes in the GEO dataset（GSE40687）revealed a correlation between TRPM2-AS and the TGF-β signaling pathway.Finally,rescue experiments demonstrated that PDIA6 could promote the malignant biological behavior of endometrial cancer cells,which was regulated by TRPM2-AS/miR424-5p.Results1.PDIA6 promoted the malignant biological behavior of endometrial cancer cells by the direct targeted regulation of miR-424-5p.2.MiR-424-5p was a direct target of TRPM2-AS and was negatively regulated by TRPM2-AS.3.Compared with normal endometrial tissues,the expression of TRPM2-AS was upregulated in endometrial cancer.Down-regulation of TRPM2-AS could weaken the proliferation of endometrial cancer cells,while overexpression of TRPM2-AS had the opposite effect.A xenograft tumor assay in nude mice showed that the tumor formation ability of endometrial cancer cells was significantly decreased after TRPM2-AS knockdown,while the tumor formation ability was significantly enhanced after TRPM2-AS overexpression,the difference was statistically significant.4.The migration and invasion of the endometrial cancer cells were significantly weakened after downregulated the expression of TRPM2-AS,while the migration and invasion of the endometrial cancer cells were enhanced after the overexpression of TRPM2AS,the difference was statistically significant.5.Western blot showed that the expression of epithelial marker E-cadherin increased,while the expression of mesenchymal markers N-cadherin and c-MYC decreased after TRPM2-AS knockdown.After overexpression of TRPM2-AS,the expression of epithelial marker E-cadherin decreased,while the expression of mesenchymal markers N-cadherin and c-MYC increased.ConclusionThe promoting role of PDIA6 in the progression of endometrial cancer is regulated by the TRPM2-AS/miR-424-5p axis.