Molecular Mechanism Of USP14 Stabilizing Estrogen Receptor α And Its Role In Endometrial Cancer

Objective: endometrial cancer(EC)is one of the most common gynecological malignancies in women.The epidemiological data in 2019 showed that endometrial cancer may become the second most frequent cancer after breast cancer.The incidence rate of endometrial cancer is increasing in China and developing countries.Therefore,it is very important to explore the potential molecular mechanism of endometrial cancer.At present,it is recognized that the occurrence of EC is related to the long-term estrogen stimulation without sufficient progesterone antagonism.Estrogen and estrogen receptor α(ERα)play an important role in the occurrence and development of endometrial cancer.Ubiquitin specific protease 14(USP14)is one of the proteasome related deubiquitinases.It is the main regulator of proteasome.USP14 can reversibly bind with proteasome to regulate the degradation of some proteins.However,the regulatory effect of USP14 on ER α and its mechanism in EC have not been reported.This study explored the molecular mechanism of USP14 stabilizing ERα in EC and its effect on the biological function of EC.Methods: 1.The correlation between the expression of USP14 and the survival of patients with EC was analyzed by database,and the expression of USP14 in normal endometrium and endometrial carcinoma tissues was detected by Western blot;2.Gradient overexpression of USP14,Western blot The expression level of ERα protein was detected by blot;3.After treatment with USP14 inhibitor iu1 or overexpression of USP14 plasmid,CHX was added to inhibit the synthesis of whole protein,the degradation of ERα protein was detected;4.The interaction between USP14 and ERα protein and ubiquitination of ERα by USP14 were verified by protein immunoprecipitation(Co-IP);5 The proliferation of Ishikawa or HEC-1A cells was detected by plate cloning test,MTS,flow cytometry and apoptosis test;6.Transwell assay and wound healing assay were used to verify the migration of Ishikawa with the effect of USP14.Results: 1.The overexpression of USP14 protein in endometrial carcinoma was associated with poor survival outcome;2.Overexpression or si RNA silencing of USP14 could up regulate or down regulate ERα protein level,but had no significant effect on ERα m RNA,USP14 could stabilize ERα by affecting ERα degradation;3.USP14 can bind to ERα and change the K48 related ubiquitination level of ERα;4.Knockdown of USP14 can inhibit the expression of target genes related to cancer promotion downstream of ERα;5.USP22 silencing increasing the level of HIF-1α ubiquitination under hypoxia.6.Inhibition of USP14 can reduce the clone forming ability of endometrial cancer cell line,and the inhibition of growth is related to the arrest of G0/G1 phase;7.Under the condition of estrogen stimulation,inhibition of USP14(shusp14 or iu1)reduced the migration ability of Ishikawa cells,on the contrary,overexpression of USP14 enhanced the migration ability of Ishikawa cells.Conclusion: 1.USP14 is highly expressed in endometrial carcinoma and is associated with poor prognosis;2.USP14 can inhibit the degradation of ERα protein by deubiquitination of K48 related ERα;3.USP14 can promote the proliferation and migration of EC cells through estrogen/ERα.