Comprehensive Study Of WNT5A/ROR2 Signal Pathway In The Development Of Ovarian Cancer

Ovarian cancer (OC) is one of the most common gynecologic malignancies.Clinically its incidence is increasing gradually and proceeding apace, so it's the most lethal gynecologic malignancy worldwide. Due to lack of typical clinical symptoms in early stage and the majority of women are diagnosed after the primary tumor has already metastasized, resulting in a 5-year survival rate that has changed little over the past 20 years. The common clinical manifestation of ovarian cancer is its ability to migrate and seed in the peritoneal cavity with exfoliated tumor cells and the formation of ascites, which is the main cause of death of the OC. At present, the underlying molecular mechnism for its onset remains unclear, there will be of great importance in finding key pathways and specific molecules and exploring the related mechanisms of development of ovarian cancer.There are multitudinous signal pathways in development of cancer. Wnt signaling pathway involved in embryonic development and cell differentiation, which is associated with a variety of known high-risk carcinomas when aberrantly activated.According to Wnt transduction mode, this multifaceted signaling network is traditionally described through the following two distinct mechanisms: canonical Wnt pathway (?-catenin dependent); and non-canonical Wnt pathway (P-catenin independent). The non-canonical Wnt pathway regulates cytoskeleton-mediated processes and establishment of polarity by activating two sub-pathways (Wnt-Ca2+and Wnt-PCP). Recently, experiments had revealed that WNT5A, as a prototypical non-canonical Wnt protein,is up-regulated and contributes to cancer cell migration and invasion in several tumor cell types. It's worth noting that, in different kinds of tumor tissues, WNT5A appears to have dual roles and can act to promote or suppress carcinogenesis due to activation of different ligand-receptor pairings.Receptor tyrosine kinase-like orphan receptor 2 (ROR2) genes, first identified in a neuroblastoma cell line, is located on chromosomes 9. ROR2 is a transmembraneprotein of the receptor tyrosine kinase (RTK) family. ROR2 is 'normally expressed at high levels during embryonic developmental processes, playing a key role in cell movement and polarity. Mutations of the ROR2 receptor, have been shown to be responsible for the Robinow syndrome and Brachydactyly B1, both characterized by skeletal dysplasia syndrome. Furthermore, ROR2, acting as a receptor or co-receptor for WNT5A, has been shown to regulate WNT5A-induced non-canonical (?-catenin independent) pathway, which regulates cell growth, differentiation,and tumor formation. To date, tumor suppressive relationships of ROR2 have been established with colon cancer, hepatocellular carcinoma, and multiple hematologic malignancies;however, there are several researches indicating that ROR2 is up-regulated and promotes tumor growth,migration,and invasion in a multitude of tumor types,such as melanomas, osteosarcomas, and renal cell carcinoma cells. Accordingly similar to WNT5A, ROR2 appears to have dual roles and can act to suppress or promote carcinogenesis in different tissues.It has been documented that the role of WNT5A in ovarian cancer is very complex.It can be acted as an oncogene to promoting the migration and invasion of ovarian cancer cells or as a tumor suppressor gene to suppressing epithelial ovarian cancer by promoting cellular senescence. The expression of ROR2 in ovarian cancer is up-regulated,while its regulatory mechanisms of downstream signaling pathways in ovarian cancer are not elucidated. Our study uses molecular biology techniques to explore the role of WNT5A/ROR2 signaling pathway in the development of ovarian cancer and the specific mechanisms.PATR IROR2 expression in ovarian cancer tissues and the relationship of ROR2 with the expression of WNT5AObjective:To detect ROR2 expression in ovarian tissue samples. To evaluate the relationship of ROR2 expression and clinical pathological features and the expression of WNT5A.To preliminarily explore the effect of ROR2 in occurrence and progression of ovarian cancer.Methods:1 .Immunohistochemical analysis of ROR2 expression in Human ovary tissue microarrays (OV1005a and OV808) including 3 normal ovarian epithelial tissues; 18 benign cystadenomas, 7 borderline cystadenomas, 45 primary malignant epithelial ovarian cystadenomas and 50 metastatic ovarian tumor tissues. The SPSS statistical analysis software was used to analyze the correlation between ROR2 expression and clinical pathological parameters.2.Immunohistochemical was also used to detect the expression of ROR2 and WNT5A in 36 serous ovarian cancers. Spearman to analyze the relationship between ROR2 and WNT5A.Results:1 .The expression of ROR2 in normal ovarian epithelial tissues, benign cystadenomas, borderline cystadenomas, primary malignant epithelial ovarian cystadenomas and metastatic ovarian tumor tissuesImmunohistochemical analysis of ROR2 expression in 3 normal ovarian epithelial tissues, 18 benign cystadenomas, 7 borderline cystadenomas, 45 primary malignant epithelial ovarian cystadenomasand 50 metastatic ovarian tumor tissues. The results showed that ROR2 was expressed in the cytopasm in normal ovarian epithelial tissues and benign cystadenomas. In borderline cystadenomas, primary malignant epithelial ovarian cystadenomas, and metastatic ovarian tumor tissues, ROR2 was expressed both in the nucleus and cytopasm. The positive rate of ROR2 expression in normal and benign tumor tissues, borderline ovarian tumors, malignant epithelial ovarian cancer and metastatic disease tissues was increased gradually,in order,28.57% (6/21),57.14 % (4/7), 60.00 % (27/45) and 96.00 % (48/50). The positive rate of ROR2 was extremely higher in malignant epithelial ovarian cancers and metastatic tumor tissues than that in normal and benign tissues (malignant epithelial ovarian cancers: p=0.0174, metastatic tumor tissues: p < 0.0001), and was apparently highest in themetastatic tumor tissues (malignant epithelial ovarian cancers vs metastatic tumor tissues: p <0.0001).2.The expression of ROR2 in malignant epithelial ovarian cancer and the correlation between ROR2 expression and clinical pathologic characteristicsImmunohistochemical analysis of ROR2 expression in malignant epithelial ovarian cancer, the results showed that the positive rate of ROR2 expression in ovarian cancer grading 1, 2 and 3 was 57.69% (15/26),86.66%(39/45) and 87.50%(21/24)respectively.The ROR2 positive rate was observably higher in grading 2 and 3 (1 vs 2,p = 0.0058; 1 vs 3, p = 0 .0190).The positive rate of ROR2 expression in ovarian cancer stage I/II and III/VI was 50% (5/10), 62.85% (22/35) respectively. According to statistic analysis, there was no significant difference (p>0.05). There was no statistical difference of the correlation between ROR2 expression and patient age (p>0.05).3.The expression of ROR2 and WNT5A in serous ovarian cancer and the correlation between themImmunohistochemical analysis of ROR2 and WNT5A expression in serous ovarian cancers, the results showed that ROR2 and WNT5A were both expressed in the cytopasm and nucleus, and mainly expressed in the cytopasm. In 16 cases of ROR2 strongly positive tissues, the cases of WNT5A strongly positive expression was 12,accounted for 75%. In 12 cases of ROR2 medium positive tissues, the cases of WNT5A medium positive expression was 7, accounted for 58.3%. In 6 cases of ROR2 weakly positive tissues, the cases of WNT5A weakly positive expression was 5,accounted for 83.5%. In 2 cases of ROR2 negative tissues, the cases of WNT5A negative expression was 2, accounted for 100%. Spearman to analyze the relationship between ROR2 and WNT5A, the results showed that there was a correlation between them in serous ovarian cancers(r= 0.440,p<0.05).Conclusions:1.The positive rate of ROR2 was extremely higher in malignant epithelial ovarian cancers and metastatic tumor tissues and was apparently highest in the metastatic tumor tissues, which indicated that ROR2 may be involved in the migration and invasion of ovarian cancer cells and plays a pivotal role as an oncogene in the development of ovarian cancer.2.The positive rate of ROR2 was associated with the ovarian cancer grade but not ovarian cancer stage or patient age.3.ROR2 and WNT5A were both expressed in serous ovarian cancers and there was a correlation between them.PATR ?Study of the effect of targeted silencing ROR2 on the invasion ability of ovarian cancer cellsObjective:To investigate the effect of targeted silencing ROR2 on ovarian cancer cell proliferation, migration and invasion and explore the possible molecular mechanisms in the pathogenesis of ovarian cancer.Methods:1.To detect the expression of ROR2 gene in SKOV3, A2780, OVCAR3 and 3AO ovarian cancer cell lines by Western blot.2.Recombinant WNT5A treated with 3AO and OVCAR3 cell lines with lower expression of ROR2s; iRNA-ROR2 interference treated with A2780 and SKOV3cell lines with higher expression of ROR2 by lipo-2000; Western blot technology and Immunofluorescence staining were used to detect the expression of ROR2 and WNT5A in ovarian cancer cells and analyze the correlation between ROR2 and WNT5A.3.Silencing the expression of ROR2 in SKOV3 and A2780 cell lines by transient transfection of shRNA plasmids interference by lipo-2000; Western blot and Immunofluorescence staining were used to confirm the transfection efficiency; CCK8 assay was used to detect the cell proliferation after the changes of ROR2 expression;Wound healing assay was used to determine the cell migration and Transwell assay was used to detect the cell invasion; Western blot was used to detect some protein related with EMT, and association with WNT5A/ROR2 signaling pathway correlation.Results:1.The expression of ROR2 gene in SKOV3, A2780, 3AO and OVCAR3 ovarian cancer cell linesTo detect the expression of ROR2 gene in SKOV3, A2780, 3AO and OVCAR3ovarian cancer cell lines by Western blot, the results showed that the levels of ROR2 expression were significantly higher in SKOV3 and A2780 cells than those in 3AO and OVCAR3 cells. While the expression of ROR2 gene in SKOV3 and A2780 cell lineswas no significant difference (p>0.05), and so was in 3AO and OVCAR3 celllines(p>0.05).2. The relationship between ROR2 and WNT5A genes in ovarian cancer cell linesRecombinant WNT5A treatment with 3AO and OVCAR3 cell lines with lower expression of ROR2, to detect the expression of ROR2 gene by Western blot, the results showed that the expression of ROR2 gene increased remarkably both in 3AO and OVCAR3 cells after rWNNT5A treatment (3AO cells: p<0.01; OVCAR3 cells:p<0.05) .Western blot analysis showed that silencing of ROR2 by siRNA in A2780 and SKOV3 markedly decreased ROR2 expression(p<0.01), while the expression level of WNT5A protein was almost no change (p>0.05). Immunofluorescence assay also confirmed that the intensity of ROR2 decreased significantly after the transfection with siROR2, but WNT5A had no change.3.The expression of ROR2 gene in SKOV3 and A2780 cell lines of transient transfected shROR2 plasmidsshROR2 plasmids interference target sequences treated with A2780 and SKOV3cell lines with higher expression of ROR2 by lipo-2000, to detect the expression of ROR2 gene by Western blot, the results showed that the expression of ROR2 protein decreased significantly and similarly by each of the three shROR2 plasmids in transient-transfected SKOV3 and A2780 cell lines (p<0.01).Immunofluorescence assay also confirmed that the intensity of ROR2 decreased significantly after the transfection with shROR2.4.The influence of knockdown of ROR2 gene on the proliferation of SKOV3 and A2780 cell linesThe CCK8 assay showed that silencing of ROR2 significantly suppressed the proliferation of the transient-transfected SKOV3 and A2780 cell lines (p <0.01);While after transient transfection 24h, there was no significantly change of proliferation in SKOV3 cell lines (p> 0.05), and after transient transfection 24h and 48h, there was no significantly change of proliferation inA2780 cell lines (p> 0.05).5. The influence of knockdown of ROR2 gene on the migration and invasion of SKOV3 and A2780 cell linesWound healing assay was used to determine the influence of silencing of ROR2 gene on the cell migration of SKOV3 and A2780 cell lines, the results showed that after silencing of ROR2 gene by transient transfection, at 24h and 48h, the migration of the transient-transfected SKOV3 cell line was significantly decreased (p< 0.01).The migration of the transient-transfected A2780 cell line was also significantly decreased (24h: p< 0.05; 48h: p< 0.01). Transwell assay was used to detect the influence of silencing of ROR2 gene on the cell invasion of SKOV3 and A2780 cell lines, the results showed that the invasion of the transient-transfected SKOV3 and A2780 cell lines was significantly decreased (p< 0.01).6.The influence of knockdown of ROR2 gene on the EMT of SKOV3 and A2780 cell linesTo detect correlation factor of EMT by Western blot analysis, we found that ROR2 knockdown in SKOV3 and A2780 cell lines led to the upregulation of epithelial markers (E-cadherin and Keratin) and downregulation of mesenchymal markers(N-cadherin and vimentin) (p< 0.05). Besides, EMT key transcription factors, Snail,Slug and ZEBlwere also significantly decreased (p< 0.05).7.The influence of knockdown of ROR2 gene on the non-canonical Wnt pathway of SKOV3 and A2780 cell linesBased on western blot analysis to detect downstream targets of non-canonical Wnt pathway, we found that knockdown of ROR2 in SKOV3 SKOV3 and A2780 cell lines led to a substantial decrease in ?-catenin-independent targets,including Racl,RhoA, JNK,p-PKC?,and NF-?Bp65 (p<0.05).;Conclusions:1.ROR2 expressed in SKOV3?A2780?3AO and OVCAR3 cell lines,and the levels of ROR2 expression were significantly higher in SKOV3 and A2780 cells than those in 3 AO and OVCAR3 cells.2.The expression of ROR2 and WNT5A was positively correlated, ROR2 gene was affected by the change of WNT5A gene , while WNT5A gene was not affected by the change of ROR2 gene.3.ROR2 expression might play a role as a tumor promoter in ovarian cancer cell proliferation,colony formation,migration,invasion and other malignant behavior,which may primarily be involved in reversing EMT transition by the inactivation of non-canonical Wnt signaling.PATR?Establishment of shROR2stably transfected cell line and ovarian tumor xenograft modelsObjective:To establish shROR2 stably transfected cell line and ovarian tumor xenograft models to investigate the role of ROR2 on cell proliferation, migration and invasion,adhesion, morphologic state and tumor formation of SKOV3 cell line.Methods:1.We successfully established shROR2 stably transfected SKOV3 cell line and shNC stably transfected SKOV3 cell line by G418 screening. To determine the effect of stabilizing screen, the fluorescent expression of the cells were observed and photographed by fluorescence microscope and the expression of ROR2 was detected by Western blot.2.Cell morphological and digestive state alterations were observed and photographed with the Inverted Microscope.3.In stably transfected SKOV3 cells, we detected the cell proliferation, migration and invasion after the changes of ROR2 expression by CCK8 assay, Colony formation assay, Wound healing and Transwell assay respectively.4.We biulded ovarian tumor xenograft models to determine tumor formation after the changes of ROR2 expression. Western blot and Immunofluorescence staining were used to detect some protein related with EMT, and association with WNT5A/ROR2 signaling pathway correlation.Results:1.Establishment of shROR2 and shNC stably transfected cell linesAfter 4 weeks, We successfully established shROR2 stably transfected SKOV3 cell line and shNC stably transfected SKOV3 cell line by G418 screening, named SKOV3-shROR2 and SKOV3-NC respectively. With the fluorescence microscope,We observed that >90% stably transfected SKOV3 cells had green fluorescence expression with the plasmids. We detected the expression of ROR2 gene in negative control groups and shROR2 plasmids groups by Western blot, the results showed that the expression of ROR2 protein decreased significantly (p< 0.01).2.The influence of stable knockdown of ROR2 gene on the cell morphological and digestive state of SKOV3 cell lineDuring the process of stable screening, using Inverted Microscope, we observed interestingly that SKOV3-shROR2 cells lost the fibroblast-like morphology with spindle shapes and acquired an epithelium-like appearance with blunt shapes.Moreover, during the process of trypsinization, we observed interestingly that SKOV3-shROR2 cells showed less resistance to trypsinization than the negative control cells, and the cells that were suspended in the medium were made into larger cell masses, presumably indicating less adhesion to the extracellular matrix (ECM),while the cells were more tightly attached to the cells.3.The influence of stable knockdown of ROR2 gene on the proliferation of SKOV3 cell lineThe CCK8 assay showed that silencing of ROR2 significantly suppressed the proliferation of theSKOV3-shROR2 cells (p < 0.01); While at 24h, there was no significantly change of proliferation in SKOV3 cell line (p> 0.05). Using the colonyformation assay to detect the influence of ROR2 gene on the dependence and proliferation of SKOV3 cell line, the results showed that SKOV3-shROR2 cells showed less colonies than SKOV3-NC cells (p < 0.01).4.The influence of stable knockdown of ROR2 gene on the migration and invasion of SKOV3 cell lineWound healing assay was used to determine the influence of silencing of ROR2 gene on the cell migration of SKOV3-shROR2 and SKOV3-NC cell lines, the results showed that after silencing of ROR2 gene by stable transfection, the migration of the stable-transfected SKOV3 cell line was significantly decreased (p< 0.01). Transwell assay was used to detect the influence of silencing of ROR2 gene on the cell invasion of SKOV3 cell line, the results showed that after silencing of ROR2 gene by stable transfection, the invasion of the stable-transfected SKOV3 cell line was significantly decreased (p< 0.01).5.The influence of stable knockdown of ROR2 gene on the EMT of SKOV3 cell lineUsing Western blot to detect the influence of stable knockdown of ROR2 gene on the EMT of SKOV3 cell line, we found that, consistent with instantaneous transfection, ROR2 stable knockdown in SKOV3 cell line led to the upregulation of epithelial markers (E-cadherin and Keratin) and downregulation of mesenchymal markers (N-cadherin and vimentin). Besides, EMT key transcription factors, Snail,Slug and ZEB1 were also significantly decreased. Immunofluorescence assay also confirmed that the intensity of E-cadherin increased significantly in SKOV3-shROR2 cells, while the intensity of N-cadherin, Snail and Slug decreased significantly.6.The influence of stable knockdown of ROR2 gene on the non-canonical Wnt pathway of SKOV3 cell lineBased on Western blot analysis to detect downstream targets of non-canonical Wnt pathway, we found that,consistent with instantaneous transfection, stable knockdown of ROR2 in SKOV3 cell line led to a substantial decrease in ?-catenin-independent targets, including Racl, RhoA, JNK, p-PKC?, and NF-?Bp65 (p<0.01).Immunofluorescence assay also confirmed that the intensity of Racl and RhoA decreased significantly in SKOV3-shROR2 cells.7.Establishment of ovarian tumor xenograft modelsWe successfully established ovarian tumor xenograft model, and the volume and weight of tumors derived from SKOV3-shROR2 cells group were significantly smaller than the negative control group (p< 0.01), indicating that silencing of ROR2 gene resulted in inhibition of tumor formation. Western blot analysis was used to detect the expression of ROR2, VEGFA,MMP9 and MMP13, the results showed that the level of ROR2, VEGFA and MMP9 expression in SKOV3-shROR2 xenografts was much lower. While there was no change of low MMP 13 expression of in these tumor xenografts (p > 0.05).Conclusions:1.Silencing of ROR2 induced morphological alterations of losting the fibroblast-like morphology and acquiring an epithelium-like appearance, which further suggested that EMT may be involved in the development of ovarian cancer.2.SKOV3-shROR2 cells showed less adhesion to the extracellular matrix (ECM),while the cell-cell contaction was increased, indicating that ROR2 may participate in the development of ovarian cancer through the regulation of adhesion.3.Stable knockdown of ROR2 gene not only suppressed SKOV3 cell proliferation,colony formation, migration and invasion, but also inhibited tumor formation, which may primarily be involved in reversing EMT transition by the inactivation of non-canonical Wnt signaling.