Prognostic Value Of Dickkopfs-derived DKK2 In Breast Cancer Normal-like And Preliminary Elucidation That Dickkopfs-derived DKK3 Induces Apoptosis In Ovarian Cancer Cells

Objective: Breast cancer and ovarian cancer are two common malignant tumors in female.From the perspective of morbidity,breast cancer is the most commom than ovarian cancer.However,in terms of mortality,ovarian epithelial tumor mortality exceeds breast cancer,posing a serious threat to women's lives.Based on clinical practice,the nodus of diagnosis and treatment of breast cancer and ovarian cancer can be summarized into three aspects: extensive tumor heterogeneity;lacking of high sensitivity and high specificity biomarkers for early diagnosis and prognosis;resistant to radiotherapy and chemotherapy.Recently,with the vigorous development of large-scale gene microarray,sequencing technology and corresponding bioinformatical analysis,research on tumor etiology and pathogenesis has reached the molecular level.Therefore,we are firmly convinced that cancer biomarker screening and drug discovery based on high-throughput screening and molecular docking techniques provide a theoretical basis for early diagnosis and early treatment in breast and ovarian cancer.According to related literatures,Wnt/?-catenin pathway is widely involved in normal physiological processes,such as embryogenesis,development and stem cell homeostasis.However,dysfunction of Wnt/?-catenin pathway-related proteins has been widely reported in many diseases,especially malignant tumors.Dickkopfs is widely recognized as an endogenous inhibitor of the Wnt/?-catenin pathway and its members include DKK1,2,3,4 and DKKL1.Moreover,abnormal expression and regulation of Dickkopfs members in breast cancer molecular subtypes and ovarian cancer has not been fully elucidated.Therefore,our study was to analyze m RNA expression level and prognostic value of the Dickkopfs in breast cancer PAM50 subtype by bioinformatical methods,then validating the bioinformatical results with relevant biological experiments.In addition,based on the previous work of the research team,we performed bioinformatical methods and biological experiments to elucidate the expression levels of Dickkopfs members in ovarian cancer and their effects on ovarian cancer apoptosis.Methods: The first part: GOBO(Gene expression-based outcome for breast cancer online)was used to analyze level of Dickkopfs m RNA in breast cancer PAM50 classification and its clinical prognosis.Then,METABRIC(Molecular taxonomy of breast cancer international consortium),TCGA(The cancer genome atlas)breast cancer gene expression data and q RT-PCR(Quantitative real time polymerase chain reaction)experimental technique were used to valiate the m RNA of Dickkopfs in breast cancer PAM50 subtype.Subsequently,METBRIC clinical data and the Kaplan-Meier online platform were applied to verify the prognostic relationship with Dickkopfs m RNA in normal-like subtype.The second part: Our study included TCGA ovarian cancer and GTEx(Genotype-tissue expression)normal ovarian tissue gene expression data;DESeq2 package was used to perform differential expression in ovarian cancer;Fun Rich v3.1.3(Functional enrichment analysis tool)was used to perform pathway enrichment on differentially expressed genes;GSEA v3.0(Gene set enrichment analysis)was used to perform single gene set enrichment analysis;The sensitivity and specificity of Dickkopfs m RNA expression level in the diagnosis of ovarian cancer were analyzed by using the Receiver operating characteristic curve(ROC)method using Med Calc v18 software.In addition,our study included gene expression data and chemotherapeutic drug sensitivity data related to ovarian cancer cell lines from GDSC(genomics of drug sensitivity in cancer);three-dimensional structure of chemotherapeutic drugs was downloaded from Drugbanks;The correlation between DKK3 m RNA expression level and chemotherapeutic drug sensitivity data was analyzed by SPSS 22.0;Based on protein folding method,three-dimensional structure of protein was modeled using i-TASSER software;evaluation and refinement of protein structure by SAVES v5.0 and Galaxy Refine;DKK3 protein activity pocket was predicted by POCASA;Molecular docking of protein and chemotherapeutic drugs was performed by semi-flexible docking using Autodock v4.3;Pymol v2.3 was used to visualized three-dimensional structure and docking structure of protein.Finally,human ovarian cancer cell lines CAOV3,OVCAR3 and SKOV3 were selected as experimental subjects.The expression of DKK3 protein in each ovarian cancer cell line was detected by Western blot.The DKK3 overexpressing system was constructed by using pc DNA3.1-DKK3 plasmid.The viability and drug sensitivity of ovarian cancer cell lines were detected by CCK8.Western blotting based on cleaved caspase-3 was used to detect the apoptosis of ovarian cancer cell line with DKK3 overexpressing.Results: The first part: GOBO expression module showed that excluding DKK1,other members of Dickkopfs m RNA significantly upregulated in the normal-like subtype of breast cancer.Next,GOBO survival module showed that breast cancer with DKK2 m RNA high expression has better OS and RFS than the one with DKK2 m RNA low expression.However,normal-like subtype with DKK2 m RNA high expression showed worse OS and RFS than the one with DKK2 m RNA low expression.METABRIC,TCGA,q RT-PCR,and Kaplan-Meier online platform datasets validated the expression of DKK2 m RNA and its clinical prognostic value in normal-like subtype,which is consistent with the GOBO database.The second part: after differential expression analysis and previous work of the research team,we considered DKK3 as a candidate gene for ovarian cancer prognosis;Bioinformatical analysis showed that expression of DKK3 m RNA in ovarian cancer tissues was significantly lower than that in normal ovarian tissues;That DKK3 gene promotemethylation and copy number deletion accounted for down-regulated DKK3 in ovarian cancer;Association analysis between DKK3 m RNA and clinicopathological features of ovarian cancer shows that DKK3 m RNA expression level is related to the location of ovarian cancer;ROC showed that DKK3 m RNA can distinguished between ovarian cancer and normal ovary;Analysis of ovarian cancer pathway based on cluster analysis showed that TGF signaling pathway altered significantly in ovarian cancer;GSEA showed that TGF-?/Smad4 pathway was significantly enriched in ovarian cancer with DKK3 m RNA high expression;Correlation analysis showed that DKK3 m RNA was negatively correlated with the sensitivity of 28 chemotherapeutic drugs;KEGG indicated that up-regulated differential expression genes were mainly enriched in DNA replication and cell division;By correlating the DKK3 m RNA level with the drug IC50 and screening for ovarian cancer differentially expressed genes,we obtained candidate chemotherapy drugs,including cisplatin and bleomycin.Molecular docking present that binding region of cisplatin exists in the three-dimensional structure of DKK3 protein;Western blot showed that the expression levels of DKK3 protein in ovarian cancer cell lines CAOV3,OVCAR3 and SKOV3 decreased in turn;The CCK8 viability assay showed a significant decrease in cell viability in the OVCAR3 and SKOV3 with overexpressing DKK3 compared to control group;Western blot based on cleaved caspase-3 showed that apoptosis event was significantly increased in OVCAR3 and SKOV3 overexpressing DKK3 compared to control group;The protein level of Smad4 was significantly elevated in the SKOV3 overexpressing DKK3 compared to control group;CCK8 drug sensitivity test showed that SKOV3 and OVCAR3 were more sensitive to cisplatin after overexpression of DKK3 than in the control group.Conclusion: DKK2 m RNA was significantly up-regulated in normal-like subtypes of breast cancer,and DKK2 m RNA levels were negatively correlated with overall survival and recurrence-free survival of normal-like subtypes of breast cancer.Overexpression of DKK3 can reduce the viability and promote apoptosis of ovarian cancer OVCAR3 and SKOV3 cells.In addition,compared with the control group,the protein level of Smad4 was significantly increased in the DKK3 overexpressing SKOV3 cell line,suggesting that DKK3 may regulate the ovarian cancer cell line by increasing the expression level of Smad4 and further participating in the TGF-?/Smad4 pathway activation.In addition,DKK3 overexpression improved the sensitivity of ovarian cancer cell lines OVCAR3 and SKOV3 to cisplatin.In conclusion,DKK2 can be used as a prognostic marker for normal-like breast cancer.DKK3 can be used as a marker for ovarian cancer diagnosis and drug treatment sensitivity.