Inflammasome NLRP3 Mediated By Estrogen In The Development Of Endometrial Cancer

Tthe incidence of endometrial cancer increased year by year,and more cases were seen in young women.The majority of endometrial cancers in clinic are estrogen dependent.It is generally recognized that the long-term estrogen stimulation without progesterone plays an important role in the development of endometrial cancer.Estrogen regulates the downstream signaling molecules to lead to the occurrence of endometrial cancer through estrogen receptor(ER).Epidemiological studies have shown that inflammation plays an important role in the development of endometrial cancer.Inflammasome activation is the central part of the process of inflammation,which continuous expression can result in chronic inflammatory diseases,and eventually lead to malignant pathological changes.The expression of inflammasomes could be regulated by estrogen in the occurrence of various diseases through the ER.In this study,we examined the expression of various inflammasomes in normal human endometrium and endometrial cancer tissue by immunohistochemistry(IHC),quantitative PCR and'Western blotting,studied the effects of NLRP3 on the biological behavior of endometrial cancer cells,investigated the effects of estrogen on the regulation of NLRP3 Inflammasome,and also explored the downstream signaling pathways of NLRP3 in endometrial cancer.Finally,the effect of NLRP3 on the growth of endometrial carcinoma was confirmed by in vivo experiments.Part I Expression profile of inflammasomes in human endometrial cancer tissueObjective To study the expression profie of different subtypes of inflammasomes and the activating products in normal human endometrium and endometrial cancer tissue.Methods Total 12 cases of endometrial cancer patients and 25 cases of vaginal bleeding which diagnosed by pathology in Wuhan Union Hospital,and 19 cases of endometrial carcinoma preserved in Wuhan Union Hospital tissue sample library(average age 41.3±2.579 year)were included in the resarch by IHC.In addition,total 15 cases of endometrial cancer and adjacent normal tissues were examined by qRT-PCR.The text included NLRP3,NLRC4,NLRP7,AIM2,ASC,caspasel,IL-1? and IL-18.At last,detect the expression of NLRP3 by Western blotting.Results There was no significant difference in the expression of NLRC4 or NLRP7 in normal endometrium and endometrial cancer.The expression of NLRP3 in endometrial carcinoma was higher than that in normal endometrium(P<0.05).IHC showed that the positive rate of AIM2 expression in normal endometrium and endometrial cancer was high,and there was no significant difference between them.However,qRT-PCR showed that the expression of AIM2 in endometrial cancer was higher than that in normal endometrium(P<0.05).The expression of ASC,caspasel and IL-1? in endometrial carcinoma was higher than that in normal endometrium(P<0.05).There was no difference about the expression of IL-18 between the two.The expression of NLRP3 was correlated with clinical stage and pathological grading.The higher the staging and grading,the higher the expression of NLRP3.Conclusion NLRP3 and its components and activators expressed higher in endometrial cancer than normal endometrium.NLRP3 may play an important role in the promotion of endometrial cancer by assembling inflammasome.The expression of NLRP3 was positively correlated with clinical stage and pathological staging.Part ? Study on the regulatory effect of NLRP3 expression on malignant progression of endometrial cancer cells Objective To study the effect of NLRP3 inflammasome on the malignant biological behavior of endometrial cancer cells.Methods Establish stable knockdown or overexpression of NLRP3 endometrial cancer cell lines by transfection of shRNA plasmid or lentiviral overexpression vector,Western blotting and qRT-PCR was used to detect the effect of inhibition or over expression of NLRP3,and detect the expression of its activator.Use CCK8,scratch,clone formation and Transwell assay to evaluate the changes of invasion,metastasis and proliferation of endometrial cancer cells after the inhibition or over expression of NLRP3.Detected the proliferation of Ishikawa cells by adding NLRP3 agonists lipopolysaccharide(LPS).Results The proliferation,migration and invasion of Ishikawa and HEC-1A cells decreased significantly after knocked down NLRP3.At the same time,the expression levels of caspase-1 and IL-1? were decreased,but there was no significant change in IL-18 level.The proliferation,migration and invasion of Ishikawa and HEC-1A cells increased significantly after over expression NLRP3.LPS can stimulate the expression of NLRP3 in Ishikawa cells and promote cell proliferation.Conclusion NLRP3 can promote the malignant biological behavior of endometrial cancer,and down regulation of NLRP3 can inhibit the proliferation of endometrial cancer cells.The change of NLRP3 expression was accompanied by the change of caspase1 and IL-1?Regulating the expression and function of NLRP3 inflammasome may be an effective strategy for the prevention and treatment of endometrial cancer.Part III Molecular mechanism of estrogen mediated NLRP3 regulates endometrial cancer through IL-1?/PI3K/AKT signaling pathwayObjective 1.Study the regulation of estrogen receptor(ER)on NLRP3 inflammasome.2.Investigate the signal pathway of NLRP3 in the endometrial cancer.Methods Observe the activation of NLRP3 inflammasome after estrogen stimulates Ishikawa cells.Detect the activation of inflammasome after inhibition of ERa and ER?.Observe the effect of NLRP3 on estrogen-induced cell proliferation by down regulating NLRP3 and then estrogen stimulation on Ishikawa cells.Ishikawa cells over expressing NLRP3,and then add ER? inhibitor to observe the effect of ER? on the proliferation of NLRP3 cells.Detecte AKT signaling after NLRP3 was up-regulated or down-regulated.Detect signal pathway changes by adding inflammasome specific inhibitor YVAD-cmk.Results The expression of NLRP3 and IL-1? increased after estrogen stimulated Ishikawa cells for 24h.The expression of NLRP3 and IL-1? decreased after treatment with ERP specific inhibitor PHTPP in Ishikawa cells.When NLRP3 was reduced,the proliferation of cells was lower than that of negative control group after adding estrogen,and the difference was significant.PHTPP can partly inhibit the proliferation effect of NLRP3.NLRP3 increased the phosphorylation level of PIK3/AKT and increased the expression of BCL-2 in Ishikawa cells.The phosphorylation level of PIK3/AKT decreased after down regulated of NLRP3.The level of AKT and p-AKT decreased after PHTPP or YVAD-cmk treatment.Conclusion Estrogen can regulate the expression of NLRP3 inflammasome through ER? in endometrial cancer.Estrogen regulates PI3K/AKT pathway through NLRP3 inflammasome in the pathogenesis of endometrial cancer.Part IV Effect of NLRP3 on tumor growth in nude miceObjective To study the effect of NLRP3 on tumor growth in vivo.Methods Twelve female 4 to 6 week-old BALB/c nude mice were randomly divided into two groups(6 mice per group).The control group were injected with Ishikawa cells which transfected with blank vector,the experimental group were injected with Ishikawa cells which transfected with shNLRP3.Tumor size was measured with alinear digital caliper every 5 days.Tumor volume was estimated using the equation V =(a×b2)×0.5236,where'a' is the larger imension and 'b' is the perpendicular diameter.Mice were euthanized and tumors were collected after 40 days.The expression of the related proteins was detected by immunohistochemistry and Western blotting.Results 5 nude mice in the control group formed tumor,one of them died of cachexia.4 nude mice in the experimental group formed tumor.After silence NLRP3,the tumor growth is slow,the volume and weight of tumors were reduced.The expression of caspase-1,pro-IL-1?,IL-1?,AKT,p-AKT,Ki67 and VEGF were reduced in the experimental group when compared with the control group.Conclusion After silencing NLRP3,endometrial cancer tumorigenic ability decreased.