UCHL5 Accelerated The Growth Of Endometrial Cancer Via Activating Wnt/?-catenin Signaling Pathway

Objective:Endometrial cancer(EC)is the most common gynecological malignancy with high mortality.Although most patients are diagnosed early and accepted surgery followed by radiation therapy,chemotherapy,or both,the number of estimated deaths caused by EC was almost 36,000 annually,which accounts for 1.8% of all cancer deaths.Nonetheless,10–15% of the cases without adjuvant therapy have a recurrence of cancer.The prognosis for recurrent cases is very poor,with a 5-year survival rate less than 15%because of the limited choices of treatment and resistance to therapy.Thus,the identification of molecular mechanisms responsible for tumorigenesis and progression of these malignant ECs is imperative in developing therapeutic strategies to improve the prospects of survival.The ubiquitin-proteasome system(UPS)is a highly specific and selective route for cellular protein degradation in all eukaryotic cells.Multiple investigations have demonstrated the UPS involved in various tumor-promoting processes including DNA repair,apoptosis,cell cycle progression,and oncogenic signaling pathways cell cycling.Overall,the human genome encoded over 80 known DUBs,around 40 of which have been associated with various types of cancer.Among them,ubiquitin C-terminal hydrolase L5(UCHL5),a cysteine protease from the family of ubiquitin C-terminal hydrolases(UCHs),can remove Ub from the distal part of the poly-Ub chains and may rescue poorly ubiquitinated proteins from proteolysis.High UCHL5 expression has been reported to correlate with poor survival and increased cancer recurrence in esophageal squamous cell carcinoma,hepatocellular carcinoma,and epithelial ovarian cancer.Additionally,b AP15,an inhibitor of the 19 S proteasome DUBs UCHL5 and USP14,results in cell growth inhibition in several human cancers.Thus,there are sufficient reasons to believe that UCHL5 plays an important role in the development of tumors.The WNT/?-catenin pathway controls various cellular processes such as cell proliferation,differentiation,and maintenance of pluripotency,and also controlling different aspects of cancer biology.The function of Wnt/?-catenin pathway on EC growth had been well established.?-catenin maintains epithelial cell integrity,an important barrier for blocking tumor metastasis.Additionally,hypophosphorylated?-catenin enters the nucleus and binds to transcription factors,leading to transcriptional activation of specific target genes,including Cyclin D1,C-MYC,and MMP-7,which promote cell survival,cell cycle progression,and uncontrolled proliferation.However,the link between UCHL5 and the Wnt/?-catenin pathway and the role of UCHL5 in EC has not yet been completely elucidated to date.In this study,we mainly want to solve the following questions: 1)Whether the expression of UCHL5 is significantly difference between EC and normal endometrium;2)How about the effects of UCHL5 on the growth and survival of EC;3)Whether the Wnt/?-catenin pathway can partly explain the effects of UCHL5 on EC.Our study may contribute to unravel the mechanisms underlying the occurrence and progression of EC and provide new therapeutic targets.Methods:1.Screening and validation of differential expression of UCHL5 in endometrial carcinoma in clinical specimens and cell lines:1)The correlation between UCHL5 expression and gene expression related to proliferation and apoptosis of endometrial carcinoma and the relationship between UCHL5 expression and prognosis of endometrial carcinoma patients;2)Western blot detection of UCHL5 expression in endometrial carcinoma specimens(10cases)and normal endometrial tissue(10 cases);3)Q-PCR and Western blot to detect the differential expression of m RNA and proteins UCHL5 endometrial carcinoma cell lines and normal endometrial epithelium cell lines;2.Clarify the target genes and molecular mechanisms of in vitro cell experiments UCHL5 regulate the growth of endometrial cancer cells:1)Construct UCHL5 gene interference lentivirus transfection HEC1 A cell line,construct UCHL5 gene overexpression lentivirus transfection cell line,Q-PCR and Western blot detection of interference and overexpression efficiency;2)CCK8(0/12/24/48/72 h),cycle(48 h)and apoptosis(48 h)were performed on HEC1 A cell lines with gene interference to detect UCHL5??-catenin?Cyclin D1?C-myc?Survivin and Cleaved-Caspase3 expression;3)AN3-CA cell lines with overexpression of UCHL5 genes were subjected to cell proliferation experiments(0/12/24/48/72 h),flow cytometry to detect cell cycle(48 h)and apoptosis(48 h),and western blot to detect UCHL5??-catenin?Cyclin D1?C-myc?Survivin and Cleaved-Caspase3 expression;4)UCHL5 overexpression of AN3-CA cell lines combined with 10?mol/L of wnt/?-catenin inhibitors,CCK8 cell proliferation test(0/12/24/48/72 h),flow cytometry to detect cell cycle(48 h)and apoptosis(48),Western blot detection of UCHL5 ??-catenin?Cyclin D1?C-myc?Survivin and Cleaved-Caspase3 expression;3.Identify the target genes and molecular mechanisms UCHL5 in vivo animal experiments to regulate the growth of endometrial cancer cells:HEC1A cell lines UCHL5 gene interference were used to construct subcutaneous tumors in nude mice,HE staining analysis,Q-PCR detection of the expression of UCHL5,Western blot detection of the expression of UCHL5??-catenin?Cyclin D1?C-myc?Survivin and Cleaved-Caspase3,and immunohistochemical detection of the expression of UCHL5??-catenin?Cyclin D1?C-myc?Survivin and in paraffin sections.1)Experimental groups were as follows :(1)12 mice in the experimental group with subcutaneous injection of UCHL5 HCE-1-A cell suspension;(2)12 mice in the control group with subcutaneous injection of normal HCE-1-A cell suspension.2)After 12 days of injection,the diameter and volume of subcutaneous tumor were measured every 3 days;3)After 33 days of injection,the mice were killed,the tumor was collected,the diameter was measured and weighed;4)6 mouse tumor samples in each group.The expression of UCHL5 ? ?-catenin ?Cyclin D1?C-myc?Survivin and Cleaved-Caspase3 was detected.5)Remaining 6 mouse tumor samples were taken for paraffin embedding,paraffin sections,and HE staining.Results:1.The expression of UCHL5 was significantly increased in EC tissues;2.UCHL5 richly expressed in endometrial cancer cell lines;3.UCHL5 knockdown inhibited the growth of endometrial cancer cells;4.Upregulation of UCHL5 promoted the growth of endometrial cancer cells;5.UCHL5 activated Wnt/?-catenin signaling and effected the expression of its target genes;6.Wnt/?-catenin signaling pathway inhibitor XAV939 eliminated the disadvantages aroused by UCHL5 overexpression in EC cells;7.UCHL5 silence restricted tumor growth via inhibiting Wnt/?-catenin signaling pathway in vivo.Conclusion:UCHL5 upregulation was observed in EC tissues and cell lines and was closely related with tumor growth via WNT/?-catenin pathway.It may contribute to unravel the mechanisms underlying the occurrence and progression of EC and provide new therapeutic targets.