Mechanism Of DUSP5 In Inhibiting The Progrcssion Of Ovarian Cancer By Regulating IL-33 Signaling Pathway

Ovarian cancer?OC?is a major gynecologic malignancy;it is the sixth most prevalent cancer in the world,the fourth most common site of malignancy in females,and the seventh leading cause of cancer death in females.In the clinic,if ovarian cancer?OC?can be detected early,the clinical cure rate will be greatly improved;but it is often asymptomatic in early stages.According to the statistics,approximately 80%patients with OC have a?90%survival rate at early stages,but this decreases to 30%in those with stage? and ? disease.Pelvic examination,ultrasonography,and CA125 detection are routine ovarian cancer screening procedures,but their diagnostic values are limited.Although a variety of targeted drugs have been developed to treat ovarian cancer,the patient overall survival remains suboptimal.Therefore,it is critical to identify the mechanisms of ovarian cancer pathophysiology for use in diagnostic and clinical applications.Interleukin 33?IL-33?was firstly discovered in 2005.Its signaling pathway through the membrane receptor ST2 is thought to activate mitogen-activated protein kinase and nuclear factor?NF?-?B responses,ultimately leading to T helper 2 polarization in vitro reaction.The research on the mechanism of IL-33 in carcinogenesis has recently attracted the attention of many scholars.Several studies have shown that IL-33 has a tumor-promoting effect in cholangiocarcinoma,colon cancer and breast cancer.IL-33 has been identified as a biomarker for the diagnosis and prediction of the prognosis of non-small cell lung cancer.Interestingly,medical studies have shown that IL-33 has an anti-tumor effect on colon cancer,suggesting that IL-33 has a complex role in the pathogenesis of tumors.Studies have shown that IL-33 and ST2 levels are elevated in ovarian cancer,and IL-33 promotes ovarian cancer cell proliferation and inhibits apoptosis by down-regulating p27,Fas,and TRAILR1.It indicates that IL-33 has a significant cancer-promoting effect on ovarian cancer.Therefore,we believe that the inhibition of IL-33 may be a promising novel treatment strategy for ovarian cancer.Dual-specificity phosphatase?DUSPs?are a class of enzyme molecules that both dephosphorylate serine/threonine residues and dephosphorylate tyrosine residues.It has been discovered in the last 10 years that it belongs to a subfamily of the protein tyrosine phosphatase superfamily,which plays an irreplaceable role in the mediated mediation of mitogen-activated protein kinase signaling pathways.It dephosphorylates tyrosine and serine/threonine residues to activate extracellular signal-regulated kinase?ERK?.According to the similarity and structural characteristics of its sequence,it can be divided into atypical and typical.Studies have shown that most of the family members currently known are involved in cell differentiation,proliferation,gene transcription,metabolism,intercellular communication,ion channel,immune response with negative regulators of mitogen-activated protein kinase and the formation and development of tumors.For example,DUSP5 is a direct transcriptional target of the tumor suppressor gene p53 and has a tumor suppressor effect in various types of tumors.The expression of DUSP 5 is down-regulated in prostate cancer and gastric cancer,and the loss of DUSP5 expression is associated with poor prognosis.Defects in mouse epidermal DUSP5 increase H-Ras-driven papilloma formation in a skin cancer carcinogen-inducing model.In gastric cancer,the overexpression of DUSP 5 reduces nuclear p-erk levels and slows cell growth.Low DUSP5 expression is associated with poor prognosis in colorectal cancer.However,the role of DUSP 5 in the progression of ovarian cancer has not been reported.Based on the above information,we consider that since IL-33 is highly expressed in ovarian cancer,and DUSP5 can inhibit the formation of multiple tumors and improve the prognosis,we propose hypotheses:?1?DUSP5 is down-regulated in ovarian cancer;?2?The proliferative,migration,and invasive capabilities of ovarian cancer have been shown to be inhibited by DUSP5;?3?Down-regulation of DUSP5 can enhance the expression andsecretion of IL-33 in ovarian cancer cells;?4?It can reduce the level of IL-33 by up-regulating the expression of DUSP5 and inhibit the expression and secretion of ovarian cancer cells.In short,DUSP5 can affect the development of ovarian cancer by regulating the expression of IL-33 signaling pathway.This study Firstly,detect the expression of DUSP5 in human ovarian cancer tissue,and its effect on the expression and secretion of ovarian cancer cells.Secondly,explore the relationship between the low expression of DUSP5 and the expression of IL-33 and the biological behavior of ovarian cancer cells,and the changes in IL-33 expression and in the biological behavior of ovarian cancer after up-regulation of DUSP5.Part ?:Correlation between expression level and prognosis of DUSP5 in human ovarian cancerObjective:To investigate the expression of DUPS5 in ovarian cancer tissues of human and the expression of DUPS5 in paracancerous tissues.Methods:?1?Collect cancer tissues and paracancerous tissues of patients with ovarian cancer who were treated at the First Affiliated Hospital of Soochow University from 2014 to 2017.?2?Unified real-time polymerase chain reaction?RT-PCR?analysis.?3?Download the expression pattern of DUSP5?NM₀04419.3?from the Gene Expression Omnibus?GEO?dataset?https://www.ncbi.nlm.nih.gov/geo/?.The accession number is GSE49997.Survival analysis was performed to investigate the relationship between DUSP5 level and clinical prognosis.?4?Immunohistochemically staining was performed on 60 human ovarian cancer tissue specimens and 15 paracancerous tissue specimens to further detect the expression level of DUSP5 in human ovarian cancer tissues and paracancerous tissues.To be more objective,this section uses the H-score to quantify the degree of immunohistochemical staining of DUSP5.Results:?1?The expression of DUSP5 in ovarian cancer tissues showed a significant downward trend in comparison to paracancerous tissues.?2?Survival analysis showed that the overall survival of patients with low DUSP5 expression was shortened,HR=0.77?0.69-0.86?,logrankP=4e-06.?3?60 cases of ovarian cancer tissue and 15 paracancerous tissues were compared by H-score method.The results showed that DUSP5 of 15 paracancerous tissues were positive,and the median H value was 79.5.Of the 60 ovarian cancer tissues,42 of the DUSP5 stains were weak or undetectable,and the H score was between 5 and 30.The expression of DUSP5 in OC tissues was significantly down-regulated?P<0.001?.Conclusion:DUSP5 showed a low expression trend in ovarian cancer,and the lower the DUSP5,the shorter the survival time of patients,which suggesting that the low expression of DUSP5 has the potential as a reference indicator for the prognosis of ovarian cancer.Further analysis is required by larger samples.Part ?:Effect of DUSP5 on the biological behavior of OCObjective:To investigate the effects of DUSP5 on the biological behavior of ovarian cancer cells,such as proliferation,migration and invasion,and to analyze the role of DUSP5 in the development and progression of ovarian cancer.Methods:?1?The silencing efficiency of DUSP 5 in human ovarian cancer cell lines SK-OV-3 and Cavo-3 cell lines were examined by quantitative polymerase chain reaction?real-time PCR?and western blot methods.?2?The effect of DUSP5 silencing on the proliferation of SK-OV-3 and Cavo-3 cells was detected by CCK8 method.?3?Using the Wound healing assay and the Invasion assay to study the effects of siRNA silencing DUSP5 on the migration and invasion of SK-OV-3 and Cavo-3 cells.?4?Quantitative polymerase chain reaction?real-time PCR?and western blot were used to verify the overexpression efficiency of DUSP5 in human ovarian cancer cell lines SK-OV-3 and Cavo-3.?5?The effect of overexpression of DUSP 5 on the proliferation of SK-OV-3 and Cavo-3 cells by CCK8 assay.?6?Using Wound healing assay and Invasion assay to study the effects of DUSP5 expression on migration and invasion of SK-OV-3 and Cavo-3 cell lines.Results:?1?After silencing DUSP5,the value-adding ability of SK-OV-3 and Cavo-3 cells was significantly increased?P<0.05?.?2?After silencing DUSP5,the migration and invasion ability of SK-OV-3 and Cavo-3 cells were significantly increased?P<0.05?.?3?After overexpressing DUSP5,the proliferation of SK-OV-3 and Cavo-3 cells was significantly inhibited?P<0.05?,but there was no significant change in cell migration?P>0.05?and invasion?P>0.05?.Conclusion:DUSP5 can inhibit the the biological behavior of OC,but the mechanism of its action is still unclear,and further experimental exploration is needed.Part ?:In OC,the down-regulation of DUSP5 expression affects the expression and secretion of IL-33Objective:To explore the expression levels and secretion changes of IL-33 in SK-OV-3 and Cavo-3 cells after human intervention which resulted in down-regulation of DUSP5 expression.Methods:?1?Transfection of SK-OV-3 and Cavo-3 cells with siRNA,silencing DUSP5 expression;transfection experiments consisting of four treatment types:si-NC,si-DUSP5,oe-NC,oe-DUSP5.?2?The level of IL-33 mRNA in SK-OV-3 and Cavo-3 cells were detected before and after DUSP5 silencing.?3?The protein expression levels of IL-33 in SK-OV-3 and Cavo-3 cells before and after silencing DUSP5 were detected by enzyme-linked immunosorbent assay?ELISA?and Western blotting.?4?Luciferase assay was performed using the wild-type IL-33 promoter?pGL3-2.5k-luc?to investigate whether down-regulation of DUSP5 induced IL-33 transcription.Results:?1?Silencing DUSP5 significantly increased IL-33 transcription;?2?ELISA results showed that IL-33 secretion was significantly increased in SK-OV-3 and Cavo-3 cells after silencing DUSP5.?3?Western blotting results showed that the levels of precursor IL-33 and mature IL-33 were significantly increased in SK-OV-3?P<0.001?and Cavo-3?P<0.001?cells after silencing DUSP5.?4?Luciferase assay showed that IL-33-Luc was activated in SK-OV-3?P<0.01?and Cavo-3?P<0.001?cells after down-regulation of DUSP5,ie IL-33 promoter activity significantly enhanced.Conclusions:The down-regulation of DUSP5 can enhance the expression and secretion of IL-33 in ovarian cancer cells,suggesting that DUSP5 may affect the occurrence and development of ovarian cancer through IL-33 signaling pathway,which needs further experiments to confirm.Part ?:DUSP5 relies on IL-33 signaling to inhibit the development and progression of ovarian cancerObjective:Further explore the relationship between DUSP5 and IL-33 signaling pathways.Methods:?1?Dealing the SK-OV-3 and Cavo-3 cells with IL-33 neutralizing antibody?anti-IL-33?.?2?The effect of IL-33 neutralizing antibody on the proliferation of SK-OV-3 and Cavo-3 cells was detected by CCK8 method.?3?Analyzing the effect of IL-33 neutralizing antibody on migration ability of SK-OV-3 and Cavo-3 cells by silencing DUSP5 by cell scratch assay.?4?Analyzing the effect t of IL-33 neutralizing antibody on invasion ability of SK-OV-3 and Cavo-3 cells by Transwell assay when silencing DUSP5.?5?Recombinant IL-33 was added to wild-type and DUSP5 overexpressing SK-OV-3 and Cavo-3 cells,and then the cell migration and invasion ability were detected by CCK8 method,cell scratch test and Transwell method.Results:?1?CCK8 results showed that anti-IL-33 treated SK-OV-3 and Cavo-3 cells after DUSP5 silencing,inhibited the proliferation of the two groups of cells with increased DUSP5?P<0.05?.?2?Cell scratch test showed that anti-IL-33 treatment of SK-OV-3 and Cavo-3 cells inhibited the migration ability of silencing DUSP5 on the two groups?P<0.05?.?3?Transwell results showed that anti-IL-33 treatment of SK-OV-3 and Cavo-3 cells inhibited the invasive ability of DUSP5 on the two groups?P<0.05?.?4?CCK8 results showed that the addition of recombinant IL-33 protein in DUSP5 overexpressing cells significantly reduced the proliferation of DUSP5 overexpression?P<0.05?.?5?Cell scratch test and Transwell test results showed that the addition of recombinant IL-33 protein significantly promoted the migration ability of the two groups of cells in wild-type and DUSP5 overexpressing SK-OV-3 and Cavo-3 cells.<0.05)and invasive ability?P<0.05?.Conclusions:This part of the experiment suggests that IL-33 has a clear carcinogenic effect,and the function of DUSP5 is largely dependent on the transcriptional repression of IL-33 expression,that is,DUSP5 regulates IL-33 signaling pathway to affect the occurrence and development of ovarian cancer.