| Background:Progesterone is usually the preferred treatment option for young women with early endometrial cancer and/or atypical endometrial hyperplasia(also known as precancerous lesions)who wish to preserve fertility or some elderly women who are not suitable for surgery.Progesterone resistance(i.e.,insensitivity to progesterone therapy)is the main reason for poor conservative treatment in this population.Therefore,how to predict and evaluate the population of patients who are sensitive to progesterone therapy has become the primary issue that needs to be urgently addressed in clinical treatment.Exploring biomarker and related pathways to predict progesterone resistance in endometrial hyperplasia and endometrial cancer from the perspective of molecular mechanism is the current research hotspot in this field,Previous studies have shown that overexpression of nuclear factor E2 related factor 2(Nrf2)induces endometrial precancerous/cancerous progesterone resistance and is associated with the occurrence,development,and poor prognosis of endometrial cancer,Hypoxia inducible factor-1α(HIF-1α),as a downstream target gene of Nrf2,is involved in the occurrence and development of various malignant tumors.Multiple studies have found that HIF-1αis associated with poor prognosis and chemotherapy resistance in multiple tumors,including endometrial cancer.However,it is unclear whether it plays a role in endometrial hyperplasia and progesterone resistance in endometrial cancer.Serum and glucocorticoid regulated kinase 1(SGK1),as an AGC kinase,is activated by phosphoinositide dependent protein kinase 1(PDK1),There are reports that HIF-1αcan inhibit cell apoptosis by upregulating the expression of SGK1,which plays an important role in regulating tumor growth,metastasis,cancer stem cells,cell cycle,and chemotherapy drug resistance,We found through high-throughput gene sequencing that SGK1 is a significantly different gene between the responsive and unresponsive groups to progesterone therapy.Studies have shown that activation of SGK1can exert antioxidant effects by inducing Nrf2 expression and activity,thereby promoting tumor progression.Given the previous experimental basis,we expect to further explore whether SGK1 is associated with endometrial hyperplasia and progesterone resistance in endometrial cancer through immunohistochemistry and other studies.Objective:Detecting the expression of HIF-1α and SGK1 in endometrial sample tissues with response,partial response,and no response before/after progesterone treatment,Further exploring the progesterone resistance mechanism of endometrial cancer at the molecular level provides a theoretical basis for early prediction and evaluation of endometrial hyperplasia and the optimal patient population for endometrial cancer sensitive to progesterone therapy.Methods:1.Collect a total of 44 patients who underwent endometrial curettage or surgical treatment at our gynecological clinic or ward from January 2017 to October 2022 after receiving progesterone treatment,and retained endometrial tissue samples,Among them,there were 6 cases of simple endometrial hyperplasia(SEH),22 cases of atypical endometrial hyperplasia(AEH),and 16 cases of endometrial cancer(EC).According to the morphological changes of endometrial glands after progesterone treatment,the 44endometrial tissue samples included were divided into a complete response group(CR)of 28 cases,a partial response group(PR)of 4 cases,and a non response group(NR)of 12 cases.2.High-throughput sequencing gene detection was used to screen the significantly different genes between CR and NR endometrial tissue samples before and after progesterone treatment.3.Immunohistochemical methods were used to detect the positive expression of Nrf2,HIF-1α,and SGK1 in CR,PR,and NR before and after progesterone treatment respectively,And compare the differences in the expression of Nrf2,HIF-1α,and SGK1 in different response groups before and after progesterone treatment,and analyze the correlation of the expression of three molecular markers in different response groups to progesterone treatment.4.The RL95-2 endometrial cancer cell line was cultured in vitro,and different doses of progesterone were used to intervene in the cancer cells step by step.The proliferation of the RL95-2 endometrial cancer cell line was detected at 24,48,and 72 hours using the CCK-8 method.5.q RT-PCR and Western Bolt were used to detect HIF-1α,SGK1 m RNA and their encoded proteins expression levels after treatment of RL95-2 endometrial cancer cell lines with different doses of progestin(A1 0μmol/L,B1 0.1μmol/L,C1 1μmol/L,D1 10μmol/L,E1 20μmol/L,F1 40μmol/L).6.Collecting clinical and pathological information of endometrial cancer patients,including age,BMI,pathological type,depth of infiltration,tumor diameter,and degree of differentiation,to analyze the relationship between the expression of HIF-1αand SGK1 in EC tissues and clinical pathological characteristic indicators.7.Statistical data was analyzed using SPSS 23.0,measurement data was represented by`x±s,and counting data was expressed as a percentage(%).Differences were analyzed using Kruskal Wallis rank sum test,and Wilcoxon test was used to determine the differences in positive expression of Nrf2,HIF-1α,and SGK1 in CR,PR,and NR before and after progesterone treatment;Spearman rank correlation analysis was used to analyze the correlation.Graphpad Prism 8.0 software was used to analyze q RT PCR and Western blot results.Fisher’s exact probability method andc~2were used to analyze the relationship between positive expression of HIF-1αand SGK1 and clinical pathological indicators of EC.The difference is statistically significant as P<0.05.Results:1.The positive expression rates of Nrf2,HIF-1α,and SGK1 in the CR,PR,and NR groups before/after progesterone therapy for endometrial hyperplasia and endometrial cancer showed an increasing trend.In pairwise comparison,the positive expression differences of Nrf2,HIF-1α,and SGK1 in CR vs NR were statistically significant(P<0.05),while there was no statistically significant difference in positive expression between CR vs PR and PR vs NR groups(P>0.05).2.The differences of Nrf2,HIF-1α,and SGK1 in CR before progestin therapy vs CR after progestin therapy,PR before progestin therapy vs PR after progestin therapy,and NR before progestin therapy vs NR after progestin therapy were not statistically significant(P>0.05).3.The expression of Nrf2 and HIF-1αwas positively correlated(P<0.05),Nrf2 was positively correlated with SGK1 expression(P<0.05),and HIF-1αwas positively correlated with SGK1 expression(P<0.05)in different response groups before and after progesterone treatment.4.The analysis results of CCK-8 method show that progesterone has a dose-dependent inhibitory effect on the proliferation of endometrial cancer cells,and high-dose progesterone can significantly inhibit the proliferation of endometrial cancer cells.5.The results of q RT-PCR and Western Bolt assay showed that the expression levels of HIF-1αand SGK1 m RNA and their encoded proteins decreased significantly with increasing progestin doses,and the differences of A1vs B1,A1vs C1,A1vs D1,A1vs E1,and A1vs F1 were statistically significant(P<0.05).6.HIF-1αexpression was associated with the depth of infiltration,tumor diameter and differentiation in EC patients(P<0.05),and SGK1 expression was associated with the type of pathology,depth of infiltration,tumor diameter and differentiation in EC patients(P<0.05).Conclusion:1.The high expression of HIF-1αand SGK1 may be associated with endometrial hyperplasia and progesterone resistance in endometrial cancer.2.The expression of Nrf2,HIF-1α,and SGK1 molecules in different response groups before and after progesterone therapy is positively correlated with each other,indicating that Nrf2,HIF-1α,and SGK1 may have a synergistic effect in inducing endometrial hyperplasia and progesterone resistance in endometrial cancer.3.HIF-1αand SGK1 are associated with disease progression and poor prognosis of EC,and may be potentially effective molecular markers for evaluating the poor prognosis of EC. |
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