Preliminary Study On The Mechanism Of Progesterone Resistance Induced By LASS2 In Nrf2-Regulated Type Ⅰ Endometrial Cancer

Objective:High-dose progesterone therapy is an important conservative treatment for young endometrial cancer patients who intend to preserve fertility ability,but clinical data shows 30% of the these patients are resistant to progesterone therapy.This study aims to explore the molecular mechanism of Nrf2/LASS2 impacts on endometrial biological function and progesterone sensitivity,to find out molecular marker which could predict progesterone sensitivity,serves as an guidance for clinical and the discovery of targeted drugs.Methods:1.LASS2 gene sequence annalysis,and constructing ARE sequence reporting gene plasmid,Luciferaseassay detcet the relationship of Nrf2-LASS2;2.RT-PCR detects how estrogen effect Nrf2/LASS2 on endometrial cancer;pre-gene ananlysis in TCGA database;3.Western Blot and Immunofluorescence assay detect the differential expression of Nrf2-LASS2 in different types of endometrial cells;4.Immunohistochemical analysis detect the expression of Nrf2-LASS2 in different types of endometrial tissues;5.Apoptotic asssay detects the effcet of LASS2 in endometrial cancer cells apoptosis;6.Transifection of Nrf2/LASS2 Plasmid or siRNA into endometrial cancer cells,Western Blot detects the transfection efficiency,and testing the corresponding influence to cell progestin sensitive;7.Transifection of LASS2/sh LASS2 plasmid and adds Metformin,Western Blot detects the protein expression of Nrf2-LASS2,CCK-8 assay analysis cell proliferation,cloning experiment was used to testing the effect of metformin and sexual hormone on cell cloning ability.Results:1.LASS2 gene analysis found there three ARE sequence,and construct three corresponding gene reporting plsmid,Luciferase assay shows Nrf2 positively regulates LASS2 through binding ARE sequence;2.Exogenous estrogen significantly improve m RNA level of Nrf2-LASS2;TCGA database shows the expression of Nrf2 positive related to patients’ s bad progonosis;and GEPIA analysis displays that the expression of Nrf2 positively correlates with LASS2’s expression;3.Western Blot and Immunofluorescence assay demonstrate the expression of Nrf2/LASS2 protein was highly in endometrial cancer cells compared with benigh endometrial cells;4.Immunohistochemical analysis results consistent to cells that Nrf2/LASS2 highly expressed in endometrial abnormal hyperplastic/cancer tissue compared to benigh(Proliferative/secretory phase)tissues;5.Knockdown LASS2 gene would promote endometrial cancer cell apoptosis;6.the expresssion level of Nrf2/LASS2 negative related with progestin sensitivety;7.Metformin suppress the exppression of Nrf2-LASS2 which help decreases cell preliferation,and incresing progestin sensitive;Sexual hormone and Metformin would influence cells cloning ability,especially metformin could significantly suppress cell cloning.Conclusions:1.Nrf2 regulates LASS2 plays an important role in the occurrence and development of typeⅠendometrial cancer,and positively correlated with the adverse prognosis of the patients.2.Nrf2 regulates LASS2 in endometrial cancer cells which correlated with progesterone sensitivity.3.Metformin inhibits the expression of Nrf2-LASS2 which enhancing the sensitivity of progesterone therapy.