Expression Of NR1H3 In Endometrial Carcinoma Tissues And Its Effect On The Proliferation Of Ishikawa Cells In Vitro

Liver X receptors(LXRs)are ligand-activated nuclear transcription factors with two subtypes,NR1H3(LXRa)and NR1H2(LXRβ).NR1H3 and NR1H2 are expressed in many tissues and cells,such as liver,intestine,adipocytes and macrophages.Slightly different NR1H3 is highly expressed in the breast,colon,pancreas,esophagus and liver,and NR1H2 is ubiquitously expressed in various tissues and cells.Recent studies have shown that LXRs also play an important role in a variety of tumors.LXRs agonists also play a role in inhibiting tumor proliferation in a variety of cancer cells,including prostate cancer,breast cancer,liver cancer,lung cancer,cervical cancer,and skin cancer cells,etc.Studies have shown that the metabolic syndrome is closely related to a variety of cancers,including colon cancer,pancreatic cancer,breast cancer,and endometrial cancer.Endometrial cancer is one of the most common malignant tumors in the female reproductive tract.It has been found that metabolic syndrome is an important risk factor for endometrial cancer.Since NR1H3 and NR1H2 play important regulatory roles in the metabolic syndrome,we hypothesized that NR1H3 and NR1H2 may also play a significant role in endometrial cancer.Although studies have been conducted to explore the role and mechanism of NR1H3 and NR1H2 in tumors.However,studies on the roles and mechanisms of NR1H3 and NR1H2 in endometrial cancer remain limited.In this study,we aimed to reveal differences in the expression of NR1H3 and NR1H2 between endometrial cancer tissues and normal endometrial tissues,and found that NR1H3 subtypes are abnormally expressed in endometrial cancer tissues.In addition,we also studied the effects of NR1H3 on the proliferation and cycle of endometrial cancer Ishikawa cells and the possible molecular mechanisms involved.Part I Expression of NR1H3 in endometrial carcinoma tissues and preliminary analysis of the relationship between the expression difference of NR1H3 and clinicopathological parameters of endometrial adenocarcinoma1.Expression of NR1H3 in endometrial carcinoma tissuesObjective:To study the expression difference of NR1H3 and NR1H2 in endometrial cancer tissues and normal endometrial tissues.Methods:In this chapter,90 cases of endometrial tissue were collected,including 30 cases of normal endometrial tissue,20 cases of endometrial polyp tissue,and 40 cases of endometrial adenocarcinoma of different stages.The expression of NR1H3 and NR1H2 protein in different tissues was detected by immunohistochemistry.The expression of NR1H3 protein in endometrial cancer tissue microarray was detected by immunohistochemistry.Results:1.The positive expression rate of NR1H3 protein in endometrial adenocarcinoma was significantly higher than that in normal endometrial tissue and endometrial polyp(P<0.01).There was no significant difference in the positive expression rate of NR1H2 protein in normal endometrial tissues,endometrial polyps and endometrial adenocarcinoma tissues P>0.05).2.The results of tissue microarray showed that the positive expression rate of NR1H3 protein in endometrial adenocarcinoma was significantly higher than that in normal endometrial tissue(P<0.05).3.NR1H3 protein was positively expressed in endometrial carcinoma tissues and mainly located in cytoplasm.Conclusions:NR1H3 protein is highly expressed in endometrial adenocarcinoma.2.Preliminary analysis of the relationship between the expression difference of NR1H3 and clinicopathological parameters of endometrial adenocarcinomaObjective:By analyzing the collected detailed clinicopathological parameters of patients with endometrial adenocarcinoma,including age,clinicopathological stage,degree of differentiation,depth of myometrial invasion and lymph node metastasis,the possible role of NR1H3 in the occurrence and development of endometrial cancer was preliminarily explored and its relationship with prognosis was evaluated.Methods:The expression of NR1H3 protein and its relationship with clinicopathological features of endometrial adenocarcinoma were examined by χ2.Used GraphPad Prism6.0 software to make survival curve,used Bresloff survival analysis to compare the relationship between different expression types and prognosis.Results:1.Reserched for 40 patients with endometrial adenocarcinoma,compared to their NR1H3 protein data of positive expression group and negative expression group through their age,surgical stage,depth of myometrial invasion and lymph node metastasis,found the data without statistically significant(P>0.05).However,in the degree of tumor tissue differentiation,there was a statistically significant difference between G1 and G3,and G2 and G3(P<0.05).2.Compared with NR1H3 protein expression negative group,the overall survival rate of the NR1H3 protein expression positive group patients with endometrial cancer was significantly higher than that of NR1H3 protein expression negative group(P<0.05).Conclusions:The positive expression of NR1H3 protein may indicate that patients with endometrial adenocarcinoma may have a higher degree of tissue differentiation and better prognosis.The NR1H3 protein may plays a role in inhibiting tumor progression to some extent.Part II Effect of NR1H3 on biological behavior of endometrial cancer cell Ishikawa and preliminary analysis of its molecular regulation mechanism1.Effect of NR1H3 on biological behavior of endometrial cancer Ishikawa cellObjective:To study the effect of NR1H3 agonist T0901317 on the proliferation and cycle of endometrial cancer Ishikawa cell line,and to explore the effect of NR1H3 on the biological behavior of endometrial cancer.Methods:Endometrial cancer Ishikawa cells were treated with different concentrations of T0901317 reagent and Ishikawa cells were treated with different time gradients.The expression of NR1H3 protein in Ishikawa cells was detected by immunofluorescence assay.The effect of T0901317 reagent on the proliferation of endometrial carcinoma Ishikawa cells was studied by MTT assay.The effect of T0901317 reagent on Ishikawa cell cycle of endometrial carcinoma was studied by flow cytometry.Results:1.NR1H3 protein was positively expressed in endometrial carcinoma Ishikawa cells and mainly localized in cytoplasm2.The cells were treated with T0901317 for 48 hours and with the increase of T0901317 concentration,the inhibition rate of cell proliferation increased(P<0.05).The inhibition rate of T0901317 treated with 10μM increased with the treatment time P<0.05).2.Flow cytometry showed that the cell cycle showed that the proliferation index of cells treated with T0901317 was 0.37±0.02,which was significantly lower than that treated with DMSO(proliferation index was 0.6±0.03)P<0.01).Conclusions:NR1H3 protein was highly expressed in the Ishikawa cell line,and NR1H3 agonist T0901317 could effectively inhibit the proliferation of endometrial cancer cells and G1/S transformation.2.Preliminary analysis on the molecular regulatory mechanism of NR1H3 on Ishikawa cell biologyObjective:To explore the possible molecular mechanisms involved in the function of NR1H3 agonist T0901317 in the treatment of endometrial cancer Ishikawa cells,and to provide a new theoretical basis for the clinically targeted LXRs treatment of endometrial cancer.Methods:Endometrial cancer Ishikawa cells were treated with different concentration gradients of T0901317 reagent and Ishikawa cells were treated with different time gradients.The effects of T0901317 reagent on the expression of NR1H3,CCND1 and CCNE mRNA and protein in endometrial carcinoma Ishikawa cells were studied by real-time PCR and western blot.Resu ts:1.The mRNA and protein expression levels of NR1H3 gene increased with the increase of T0901317 concentration(P<0.05),and increased with the prolongation of T0901317 P<0.05).2.The mRNA and protein expression levels of CCND1 gene decreased with the increase of T0901317 concentration(P<0.05),and decreased with the prolongation of T0901317(P<0.05).3.The mRNA and protein expression levels of CCNE gene decreased with the increase of T0901317 concentration P<0.05),and decreased with the prolongation of T0901317 P<0.05).Conclusions:NR1H3 agonist T0901317 can effectively promote the expression of NR1H3 gene in Ishikawa cells and inhibit the expression of CCND1 and CCNE genes.The T0901317 may inhibit the proliferation of endometrial cancer cells through the cell cycle pathway.