| Background and Objective:Ovarian cancer（OC）is a type of malignancy in the ovary,and its mortality rate is higher than endometrial cancer and cervical cancer,ranking the top of reproductive system tumors[1].In 2019,it is reported that 22,530 patients were diagnosed with ovarian cancer in the United States,of which 13,980 patients died[2].Moreover,as life expectancy increases,so does the number of diagnosed cases each year.It cause a serious threat to women’s lives and health.The most common pathological type of OC is the type originating from ovary’s germinal epithelium,namely epithelial ovarian cancer（EOC）,which accounts for about 80-90%[3].Among woman patients over 40 years old,more than 90 percent are epithelial ovarian cancers,and the risk increases with age.In addition,since the anatomical location of ovaries is hidden,so in its early stage,the obvious symptoms in ovarian cancer is absent and its effective screening methods are not available,therefore 70%of patients have metastasis once detected.The pathogenesis and metastasis mechanisms of ovarian cancer are still unclear,and there is no significant breakthrough in treatment.Currently,the main treatment options for epithelial ovarian cancer are tumour cell reduction surgery and platinum-based chemotherapy,immunotherapy and targeted therapy.Although these treatments largely improve the overall survival of patients,75%of patients relapse within two years and the five-year survival rate remains low,at around 30%-40%[3].The high mortality may be due to a number of factors,including the lack of symptoms and high specificity and sensitivity markers in its early stage,distant invasion and metastasis[4].Thus,it is very important to identify new predictive biomarkers for early diagnosis and have a better understanding of the mechanism of ovarian cancer metastasis,which is also an urgent issue for improving the efficacy of ovarian cancer treatment.Cysteine-rich intestinal Protein 1（CRIP1）belongs to the LIM/dizinc finger protein family and has a unique dizinc finger motif.It is called cysteine-rich intestinal Protein because it is structurally rich in cysteine residues and is highly expressed in the intestine when first discovered.It was first recognized as an intracellular zinc transport and absorption protein[5].Recently more and more scholars focus CRIP1 on tumors,they find that the expression of CRIP1 is closely relevant to a number of human malignant tumors and affects its prognosis to some extent.However,the expression and function of CRIP1 seems to be controversial in different tumors.In metastatic colorectal cancer（CRC）,CRIP1 was overexpressed and down-regulation of CRIP1 was found to inhibit cell migration and invasion in the cell lines SW620and HT29[6].Thus,it may function as an oncogene to regulate the migration and invasion of CRC cells,and may be regarded as a new promising biomarker for poor prognosis and the metastasis of colon cancer.Similar results were also found in cervical cancer[7],acute myeloid leukemia[8]and endometrial cancer[9].In contrast,CRIP1 expression led to a favourable outcome and fewer metastases in osteosarcoma[10]and breast cancer[11].However,the current studies of CRIP1 are finite and the underlying mechanisms of CRIP1-mediated tumour metastasis are largely unknown.Moreover,the relationship between ovarian cancer and CRIP1 has not yet been discussed.In this study,our objective was to describe CRIP1 expression patterns in epithelial ovarian cancer and reveal its biological characteristics and possible pathogenesis.First,we used bioinformatics methods to screen out the oncogene CRIP1 in EOC from the TCGA database,showing that ovarian cancer with the high expression of CRIP1 had a poor prognosis.Subsequently,we used tissue samples and cell models to verify its expression.Then,we performed in vitro experiments to explore its function in invasion,migration and EMT,and further uncovered the possible mechanisms.Our results will provide new insights into the biological function and the underlying metastasis mechanisms of CRIP1 in epithelial ovarian cancer.It will also provide a new marker for metastasis or progression of epithelial ovarian cancer and a new therapeutic target for treatment.Methods:1.Using the online Gene Differential Expression Analysis tool,we analysed the differential gene expression of ovarian cancer and normal ovarian tissues from The Cancer Genome Atlas（TCGA）database.Significant survival genes were obtained through the‘Survival’and‘Surv Miner’packages of the R software.After excluding published genes,genes that are both upregulated and meaningful for survival will be obtained,then the overall survival（OS）map was made to identify the target gene.2.Immunohistochemistry（IHC）was used to reveal CRIP1’s expression level in50 epithelial ovarian cancer tissues and 26 para-cancer tissue samples,and its correlation with clinicopathological parameters was analyzed.3.Western blot and q RT-PCR were conduct to identify the CRIP1 protein and m RNA expressions level in human epithelial ovarian cancer cells（A2780 and OVCAR3）and normal ovarian epithelial cells（IOSE80）.CRIP1 expression was down-regulated by three different short interfering RNA（si RNA）in OVCAR3 cell line,and the transfection effect was detected by Western blot and q RT-PCR.4.In order to observe the changes of biological behavior in epithelial ovarian cancer cell line OVCAR3 after si CRIP1,various cell function experiments related to tumorigenesis were performed including the CCK8 assay,Ed U,Annexin V-FITC/PI apoptosis assay,wound-healing and transwell assay.5.The expression of epithelial-mesenchymal-transition（EMT）related markers,such as E-cadherin,N-cadherin and Vimentin,was performed by Western blot to illustrate the relationship between CRIP1 and EMT.6.KEGG pathway enrichment analysis was conducted to reveal the signalling pathways in which CRIP1 is involved in ovarian cancer pathogenesis.Then,we detect the related proteins levels of Wnt/β-catenin pathway（β-catenin,GSK-3β,P-GSK-3β）and its target genes（MMP2 and MMP9）by Western blot.Results:1.Online gene differential expression analysis tool revealed that 2613 genes were significantly upregulated in ovarian cancer tissues,1378 significant genes were obtained through survival analysis,and 358 genes were excluded from Pub Med publication.After taking the intersection of the three genes,51 target genes were finally obtained.51 target genes were ranked by-log10（P-value）,and survival and hazard ratio（HR）analysis was performed on the top five genes.Finally,the target gene CRIP was selected to study.2.The IHC score demonstrated that the CRIP1 protein was expressed at a higher level in tumours than in tumour-adjacent tissues,and was associated with a higher pathological stage,grade and positive lymphatic metastasis,whereas no relation was detected with age,tumour diameter or CA125 level.3.Compared with IOSE80,the m RNA level of CRIP1 was significantly up-regulated in OVCAR3（P<0.01）,whereas CRIP1 expression was relatively unchanged in A2780（P=0.9350）.The western blot also showed the same result at the protein level.After silencing the CRIP1 gene using si RNA in OVCAR3 cells,including si-168,si-229 and si-276,si-229,si-229 showed the best silencing effect in protein and m RNA levels.4.Cell function experiments showed that si-CRIP1 had no significant effect on cell proliferation and apoptosis,but could exert an inhibitory effect on cell migration and invasion in OVCAR3 cells.5.After silencing the CRIP1 gene using si RNA,we observed that the epithelial marker E-cadherin was up-regulated,and the mesenchymal markers N-cadherin and vimentin were down-regulated.6.KEGG pathway enrichment analysis showed that the Wnt/β-catenin signalling pathway was significantly enriched.Western blot results showed that thatβ-catenin,p-GSK-3β,and downstream genes including matrix metalloproteinase（MMP-2 and MMP-9）were markedly downregulated after CRIP1 silencing,while GSK-3βremained unchanged.Conclusion:1.CRIP1 is a tumorigenic gene of epithelial ovarian cancer.High expression of CRIP1 is associated with higher tumor stage,positive lymph node metastasis and poor prognosis.2.CRIP1 is highly expressed in epithelial ovarian cancer tissues and cells,which can promote the invasion and migration of tumor cells.3.CRIP1 may regulate EMT by regulating Wnt/β-catenin signaling pathway,thereby promoting invasion and metastasis of epithelial ovarian cancer.This study is the first to demonstrate that CRIP1 acts as an oncogene and may promote tumour metastasis by regulating the EMT-related Wnt/β-catenin signalling pathway,suggesting that CRIP1 may be an important biomarker for metastasis and progression of epithelial ovarian cancer,and an important molecular target for the treatment. |
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