| Objective:Endometrial carcinoma(EC)is one of the most common malignancies of the female reproductive system.Currently,it is believed that there are mainly two types of endometrial carcinoma.Type ? is estrogen dependent,the most typical histological type is endometrioid adenocarcinoma,accounting for 80-90%of all endometrial carcinoma.Type ? is non-estrogen dependent,such as uterine papillary serous and clear cell carcinoma.The etiology of endometrial carcnoma is not clear,it is relevant with cytokines,oncogenes or tumor suppressor genes,as well as inside or outside environment and other factors.About 10%of endometrial carcinoma is related to genetic,but estrogen is recognized as a high risk factor.With the improvement of modern treatment,the survival rates of patients with endometrial carcinoma have larger increased,but a lot of patients are not sensitive to the traditional treatment.In recent years,the morbidity is rising in the world and the trend is much younger,so it is a serious threat to healthy women.Therefore,it is more urgent to study new therapeutic targets.IL-37(Interleukin-37)is a member of the IL-1 family.It is a new type of negative immune regulator and it has been discovered more than ten years from 2000.The IL-37 gene locates in chromosome 2 of human.According to the different splicing,it can be divided into five different subtypes:IL-37 a-e.IL-37b contains the most complete set of exons,therefore,it maybe the most biologically functional subtype.IL-37 expresses in a variety of tissues and organs of human body and has obvious tissue specificity.Interestingly,there is no mouse-derived IL-37 gene.The studies have shown that IL-37 can inhibit excessive inflammatory responses in the body and tumor occurrence is closely related to inflammation.Therefore,the inhibitory role of IL-37 has also been reported in tumor one after another,such as fibrosarcoma,liver cancer and non-small cell lung cancer and so on.This maybe suggested that IL-37 is a new tumor suppressor gene.In recent years,the role of IL-37 has also been gradually described in gyneological diseases.For example,in cervical cancer,IL-37 inhibits the proliferation and invasion of human cervical cancer cells by inhibiting the phosphorylation of STAT3.Although adenomyosis and endometriosis are benign diseases,they have malignant biological behaviors similar to tumors.It has been reported that the expression of IL-37 is down-regulated in adenomyosis patients.IL-37 suppressed proliferation,adhesion,migration and invasion of human endometrial stromal cells through multiple signaling pathways such as Wnt/?-catenin,MAPK,thus affecting the occurrence and development of endometriosis.However,there are no reports about the expression and roles of IL-37 in endometrial carcinoma so far.Therefore,it is worthy to further explore the expression of IL-37 and the exact molecular mechanism in endometrial carcinoma.In this study,we tested the expression of IL-37 in the endometrial carcinoma tissues and controls,and did further analysis.We explored the effect and molecular mechanism of IL-37 overexpression or knockdown on proliferation,migration and invasion in Ishikawa cells and AN3CA cells.The purpose of this study is to initially investigate the role of IL-37 in development and progression of endometrial carcinoma.We expect to find new therapeutic targets and to explore the feasible treatment for the treatment of endometrial carcinoma.Materials and Methods:1.The expression of IL-37 in endometrial carcinoma tissues or eutopic/ectopic endometrium of adenomyosis patientsForty-six control endometrium and forty endometrial carcinoma tissues were collected.The expression level of IL-37 was detected by immunohistochemistry,and the chi-square test was used for statistical analysis.Twenty-five eutopic or ectopic endometrium of adenomyosis patients and the thirty-eight control endometrium were collected.The expression level of IL-37 was detected by immunohistochemistry,and the chi-square test was used for statistical analysis.2.The effect of progesterone and estradiol on the expression of IL-37 in endometrial cancer cells.Endometrial cancer cells(Ishikawa and HEC-1-A)were treated with different concentrations of progesterone(0.01 ?M,0.1 ?M,1?M,10?M and 20?M).Endometrial cancer cells(Ishikawa and HEC-1-A)were treated with different concentrations of estradiol(0.1nM,1nM,lOnM,100nM,and 10000nM).The protein was collected and the expression of IL-37 was detected by western blot.3.The expression of IL-37 in different endometrial cancer cell lines and the detection of transfection efficiency of IL-37 in endometrial cancer cell lines.The BEL-7402 cell lines were used as the positive control,and the expression of IL-37 were detected by qRT-PCR and western blot in the endometrial cancer cell lines(Ishikawa,HEC-1-A,RL95-2,AN3CA)at mRNA level and protein level.We selected the endometrial cancer cell line(Ishikawa)with lower IL-37 expression,and used overexpression plasmid vectors of IL-37b?1-45,IL-37b?1-20 and IL-37b full length(IL-37b)to transfect into this cell line.Empty plasmid was used as a negative control(MOCK),qRT-PCR and western blot were used to test the overexpression efficiency.Three IL-37 specific siRNAs were synthesized and transfected into the endometrial cancer cell line(AN3CA)with higher IL-37 expression.The irrelevant siRNA was taken as negative control(NC),and the knockdown efficiency was detected by qRT-PCR and western blot.4.The effect and mechanism of IL-37 on proliferation in endometrial cancer cellsIL-37b?1-45,IL-37b?1-20 and IL-37b plasmids were used to transfect into Ishikawa cells.CCK-8 assay and colony formation assay were used to detect the effect of IL-37 overexpression on the ability of proliferation.IL-37 specific siRNAs were used to transfect into AN3CA cells.CCK-8 assay and colony formation assay were used to detect the effect of IL-37 knockdown on the ability of proliferation.Decreased activation of the MAPK or PI3K/AKT signal pathway could inhibit the growth of tumor cells.In order to determine the effect of IL-37 on proliferation-related signal pathway,IL-37b?1-45,IL-37bAl-20 and IL-37b plasmids were used to transfect into Ishikawa cells,the phosphorylation of ERK1/2,JNK1/2,P38,AKT were detected by western blot.5.The effect and mechanism of IL-37 on migration and invasion in endometrial cancer cellsIL-37b?1-45,IL-37b?1-20 and IL-37b plasmids were used to transfect into Ishikawa cells.IL-37 specific siRNAs were used transfect into AN3CA cells.Transwell assay was used to detect the effect of IL-37 overexpression or knockdown on migration and invasion ability in endometrial cancer cells.The migration and invasion of tumor cells are mainly related to the epithelial-mesenchymal transformation(EMT),the movement ability of cells and the degradation of basement membrane.Therefore,we detected the expression of the markers related to EMT,F-actin,and matrix metalloproteinase.Firstly,IL-37b?1-45,IL-37b?1-20,IL-37b plasmids were used to transfect into Ishikawa cells,the expression of the markers related to EMT(glandular epithelial cells markers:E-cadherin;mesenchymal cells markers:N-cadherin,Twist and Vimentin)were detected by qRT-PCR and western blot.Secondly,cytoskeleton is the structure foundation of the cell movement,so we detected the expression of F-actin by immunofluorescence method.PAK(Racl mainly activates p21 activated kinase)is involved in the growth of actin filaments,then the phosphorylation of PAK were detected by western blot after IL-37b?1-45 overexpression in Ishikawa and AN3CA cells.Finally,matrix metalloproteinases-2,9(MMP2 and MMP9)play an important role in the basement membrane degradation.After plasimds were used to transfect into Ishikawa,HEC-1-A and ANCA cells or IL-37 specific siRNAs were used to transfect into AN3CA cells,the expression of MMP2 and MMP9 were detected by qRT-PCR and western blot.In order to determine the exact molecular mechanisms,we did further exploration.Results:1.The expression of IL-37 in endometrial carcinoma tissues and eutopic or ectopic endometrium of adenomyosis patientsImmunohistochemical results showed that the expression of IL-37 was mainly in the cytoplasm of endometrial glandular epithelial cells.The expression of IL-37 in endometrial carcinoma tissues was significantly lower than that in the controls.The expression of IL-37 in secretory phase of control endometrium was higher than that in proliferative phase of control endometrium.In endometrial carcinoma tissues,the expression of IL-37 is not related to the FIGO stage and the degree of differentiation,however,the expression of IL-37 is related to age,estrogen receptor,progesterone receptor and the myometrial invasion.The expression of IL-37 in ectopic endometrium of adenomyosis patients was significantly lower than that in the control group,but the expression of IL-37 has no difference in the eutopic endometrium of adenomyosis patients and the control group.2.The effect of progesterone and estradiol on the expression of IL-37 in endometrial cancer cellsThe expression of IL-37 protein was detected by western blot after treatment with different concentrations of progesterone or estradiol in Ishikawa cells and HEC-1-A cells.It was found that progesterone and estradiol had no significant effect on the expression of IL-37.3.The expression of IL-37 in endometrial cancer cell lines and the detection of transfection efficiencyWe compared the relative expression of IL-37 at mRNA level and protein level in different endometrial cancer cell lines.Finally,Ishikawa cells with lower levels of IL-37 were selected to overexpress,and AN3CA cells with IL-37 higher levels of IL-37 were selected to knockdown.IL-37b?1-45,IL-37b?1-20 and IL-37b plasmids were transfected into the Ishikawa cells,qRT-PCR and western blot results showed that three kinds of plasmids can be highly expressed in the cells.Three IL-37 specific siRNAs were transfected into AN3CA cells,qRT-PCR and western blot results showed that siIL-37-1 and siIL-37-3 could significantly decrease the expression of IL-37.4.The effect of IL-37 and mechansim on proliferation of endometrial cancer cellsIL-37bAl-45,IL-37b?1-20 and IL-37b plasmids were transfected into the Ishikawa cells,the CCK-8 assay and colony formation assay showed that up-regulation of IL-37b expression had no significant effect on proliferation.Similarly,IL-37 specific siRNAs were transfected into AN3CA cells,the CCK-8 assay and colony formation assay showed that down-regulation of IL-37 expression had no significant effect on proliferation.The overexpression plasmid vectors were transfected into the Ishikawa cells.The results showed that IL-37 overexpression had no significant effect on the phosphorylation of ERK1/2,JNK1/2,P38,AKT.5.The effect of IL-37 on migration and invasion in endometrial cancer cellsIL-37b?1-45,IL-37b?1-20 and IL-37b plasmids were transfected into the Ishikawa cells,transwell experiments were used to detect the effect of IL-37 on migration and invasion after 24 hours.The results showed that the migration ability of Ishikawa cells in IL-37b?1-45 or IL-37b?1-20 overexpressed groups were significantly lower than that in MOCK group,the invasion ability of Ishikawa cells in IL-37b?1-45 overexpressed groups were significantly lower than that in MOCK group.Those results suggested that IL-37bA1-45 maybe play an inhibition role in the migration and invasion of endometrial cancer cells.IL-37 specific siRNAs were transfected into AN3CA cells,transwell experiment was used to detect the effect of IL-37 on migration and invasion after 24 hours,the results showed the migration and invasion of AN3CA cells in siIL-37-1 or siIL-37-3 knocdown groups were higher than that in NC group,suggesting that IL-37 down-regulated can promote migration and invasion ability in AN3CA cells.6.IL-37 inhibiting the migration and invasion in endometrial cancer cells by inhibiting Racl/MMP2 or Racl/NF-?B pathway Firstly,IL-37b?1-45,IL-37b?1-20 and IL-37b plasmids were transfected into Ishikawa cells.The qRT-PCR results showed that the expression of E-cadherin,N-cadherin,and Twist had no significance difference at mRNA level and the western blot results showed that the expression of E-cadherin,N-cadherin,and Vimentin also had no significance changes at the protein level.Secondly,IL-37b?1-45 overexpression vector was transfected into AN3CA cells,and it was found that IL-37b?1-45 did not affect the expression of F-actin.Western blot results suggested that IL-37b?1-45 had no significant effect on the phosphorylation of PAK in Ishikawa cells and AN3CA cells.Finally,the expression of MMP2 or MMP9 was detected after up-regulating IL-37 in Ishikawa,HEC-1-A,AN3CA cells or down-regulateing AN3CA cells.qRT-PCR results showed that IL-37 did not influence the expression of MMP2 and MMP9 at mRNA level,and western blot results showed that IL-37 had no effect on the expression of MMP9 protein,but IL-37 inhibited the expression of MMP2 at protein level.The expression of MMPs could be regulated by Racl signaling pathway,and IL-37 has a closed relationship with Racl.Racl activity kit was used to detect the the expression of GTP-Racl after IL-37bA1-45 overexpression in Ishikawa cells,the results showed that IL-37b?1-45 inhibits Racl activation.IL-37b?1-45(amino acid 46-218)and activated Rac1(myc-PRK5-Racl-61L)plasmid vectors were used to co-transfect into Ishikawa cells,and the relationship of them was detected by co-immunprecipitation.The results showed that IL-37b?1-45 directly binds to Racl-61L.In order to determine the accurate mechanism of which IL-37 decreased the migration and invasion of endometrial cancer cells by inhibiting the activation of Racl,then inhibiting the expression of MMP2 and decreasing degradation of the basement membrance.IL-37b?1-45 stable expressed cell line(ISK-LV-IL-37b?1-45)was established in Ishikawa cells,and the Racl-61L plasmid vector was used to transfect into the satble expressed cell line,the inhibitory effect of IL-37 on MMP2 were reversed,that is to say,the expression of MMP2 was up-regulated.Those results suggested that IL-37 inhibited the migration and invasion of endometrial cancer cells by inhibiting the Rac1/MMP2 pathway.Rac1 could regulate the NF-?B(nuclear transcription factor)signal pathway,then regulate the expression of genes.Our results showed that IL-37 inhibited the phosphorylation of P65.The Rac1-61L plasmid vector was used to transfect into ISK-LV-IL-37b?1-45 cell line,this inhibitory effect of IL-37 were reversed,that is to say,the expression of p-P65 were up-regulated,those results showed that the inhibitory effect of IL-37 on the migration and invasion of endometrial cancer cells maybe through the Racl/NF-KB signal pathway.However,the exact mechanism needs further investigation about Rac1/NF-?B affecting endometrial cancer cells migration and invasion.Conclusions:1.The expression of IL-37 is reduced in endometrial carcinoma tissues.2.Progesterone and estradiol have no effect on the expression of IL-37 in endometrial cells.3.IL-37 has no effect on proliferation in endometrial cancer cells.4.IL-37 suppresses the migration and invasion of endometrial cancer cells mediated by Racl/MMP2 or Racl/NF-KB signal pathway.Innovations and Significance:1.The correlation between IL-37 and endometrial carcinoma is demonstrated,and we find the expression of IL-37 is reduced in endometrial carcinoma tissues.2.IL-37 has no effect on the proliferation of endometrial cancer cells,IL-37 suppresses the migration and invasion of endometrial cancer cells,and the mechanism is related to inhibiting the degradation of basement membrane by inactivating Rac1/MMP2 or Racl/NF-KB.3.Our findings reveal that IL-37 could be a new target for the treatment of endometrial carcinoma... |
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