The Pan-cancer Analysis Of OPCML And Preparation Of Recombinant OPCML-Fc Fusion Protein And Targeting Effect On Ovarian Cancer

Ovarian cancer is a common malignant tumor of the female reproductive system.Its incidence is second only to cervical cancer and endometrial cancer,but its mortality rate ranks first.The current treatment for ovarian cancer is mainly surgery,supplemented by systemic chemotherapy ± targeted therapy.Targeted therapy is to design a corresponding drug for the identified carcinogenic site.The drug will specifically bind to the carcinogenic site when entering the body to exert a tumor suppressor effect and specifically promote the death of tumor cells without damaging other normal tissues or cells.Therefore,targeted therapy is also called biological missile,which is precise and efficient.At present,domestic targeted therapies for ovarian cancer mainly include bevacizumab for VEGF and poly-ADP polymerase inhibitors such as olaparib and niraparib.However,like platinum-resistant patients,many patients who initially responded to PARPi or Bevacizumab will eventually develop resistance.Therefore,exploring new specific anti-tumor molecular targets is still a hot spot in precision medicine research.Opioid binding protein/cell adhesion molecule-like(OPCML)is a tumor suppressor gene,which shows normal expression in normal tissues and cells,but is missing or down-regulated in a variety of tumor tissues.Studies have shown that OPCML interacts with multiple tyrosine kinase receptors,participates in the proliferation,adhesion and metastasis in ovarian cancer cells,and has no inhibitory effect and no cytotoxicity on normal cells.Therefore,OPCML is a tumor suppressor gene with potential applications.In this study,we analyzed the multi-omics data of OPCML gene in 33 different tumors,fully revealed the expression,mutation,genetic variation of OPCML and their relationship with prognosis,in order to clarify the key role of OPCML gene in tumorigenesis and development and possible regulatory mechanisms.Then we used the pCDNA3.1-OPCML-Fc vector previously constructed to express the active recombinant OPCML-Fc fusion protein in a eukaryotic system,and further explored the anti-tumor effect and its regulatory mechanism in vitro and in vivo,especially in ovarian cancer organoid models and PDX animal models.In addition,we also This research was divided into the following four parts:Part Ⅰ: Pan-cancer analysis of OPCML geneObjective: To investigate the expression,mutation,genetic variation of OPCML gene and its relationship with prognosis,in order to clarify the key role and possible regulatory mechanism of OPCML gene in tumorigenesis and development.Methods: Download the transcriptome data,mutation data,copy number variation and clinical information of 33 tumors from http://timer.cistrome.org/ and the TCGA database.Download the transcriptome data of the GTEx database.The TIMER2.0 tool was used to identify tumor types with differential expression of OPCML.According to the median of OPCML expression level,tumor patients were divided into High expression group and Low expression group.Kaplan Meier and Long-rank test were used to analyze the difference in overall survival between the two groups.The cbioportal tool was used to analyze the mutation type,mutation frequency and copy number variation of OPCML gene,and the mutation site,phosphorylation site and frequency of OPCML protein.XCELL,EPIC,MCPCOUNTER and TIDE algorithms were used to estimate immune infiltration.Using STRING to predict the OPCML interaction network and GEPIA2,the top 200 related genes with the highest correlation coefficients with OPCML were obtained.The "tidyr" package,"Cairo" package,"stringr" package and "ggplot2" package of R software were used to perform KEGG and GO pathway enrichment analysis on interacting proteins and candidate related genes.Results: OPCML was low expressed in 18 tumors including BRCA,COAD,and GBM.Down expression of OPCML is significantly related to the later stage of KIRC,but not related to the stages of other types of tumors.Among all tumor types,the OPCML high expression group has a better overall survival.High OPCML expression is associated with prolonged OS in KIRC,OV,BLCA,LGG,and LUCA.Among different tumors,the top three tumors with the highest frequency of OPCML mutations were BLCA,COAD and LUSC.The main genetic alteration types of OPCML gene are deep deletion and mutation.The frequency of missing TCGT is as high as 9%,that of UVM is up to 5%,and that of CESC is up to 3%.In BRCA,CESE,COAD,ESCA,LIHC,PAAD and other tumors,tumor fibroblast infiltration is positively correlated with the expression of OPCML;in both LGG and GBM,tumor fibroblast infiltration is negatively correlated with the expression of OPCML.Signal pathways such as cell adhesion molecules and oxytocin may be involved in the molecular mechanism of OPCML’s role in tumors.Conclusion: OPCML exhibits the characteristics of tumor suppressor genes in a variety of tumors,and may play a molecular mechanism in tumors through signal pathways such as cell adhesion molecules and oxytocin.Part Ⅱ: Expression and purification of recombinant OPCML-Fc fusion proteinObjective: To induce the expression of OPCML-Fc fusion protein in HEK293 cells to provide an experimental basis for subsequent research.Methods: The pCDNA3.1-OPCML-Fc vector was transfected into HEK293 cells to induce HEK293 to secrete OPCML-Fc fusion protein into the culture supernatant.The supernatant was affinity purified by Ni column,identified by SDS-PAGE and Western blot.Results: Successfully induced the expression of secreted OPCML-Fc fusion protein in HEK293.After the Ni affinity purification,the purity is over 95%.Conclusion: The highly expressed OPCML-Fc fusion protein was successfully obtained.Part Ⅲ: OPCML-Fc fusion protein inhibits ovarian cancer and its mechanismObjective: To investigate the inhibitory effect and mechanism of OPCML-Fc fusion protein on ovarian cancer cells and organoid models.Methods: CCK8,colony formation,Caspase 3 apoptosis assays,gap closure,and invasion experiments were used to detect the effects of OPCML-Fc fusion protein on the proliferation,apoptosis,migration and invasion of OVCAR3 and SKOV3 ovarian cancer cells.The ATP activity was detected to analyze the effect of OPCML-Fc fusion protein on ovarian cancer organoids.Western blot was used detect the regulation of OPCML-Fc fusion protein on EGFR and its downstream RAF/MEK/ERK signaling pathway.Results: OPCML-Fc fusion protein significantly inhibited the proliferation of OVCAR3 and SKOV3 cells,and had a dose-time-dependent relationship.It induced apoptosis of OVCAR3 and SKOV3 cells,inhibit their invasion and migration,and inhibited proliferation activity of some ovarian cancers organoids;OPCML-Fc recombinant protein down-regulated the ERK pathway to inhibit the occurrence and development of ovarian cancer.Conclusion: OPCML-Fc recombinant protein can inhibit the proliferation,survival,invasion and migration of ovarian cancer cells,and inhibit the growth of ovarian cancer organoid models.Part Ⅳ: Effect of recombinant OPCML-Fc fusion protein on ovarian cancer in vivoObjective: A patient-derived xenograft model of ovarian cancer in NDG mice was established,namely PDX model,to observe the targeting effect of OPCML-Fc fusion protein on ovarian cancer xenograft tumors,and to analyze its inhibitory effect on the growth of PDX.Methods: Tumors of patients with ovarian cancer were inoculated into NDG mices and passed down to next generation.The similarity between the transplanted tumor and the original tumor tissue was identified by hematoxylin-eosin staining and immunohistochemistry.The tumor-bearing mice were divided into three groups to be intravascular injected the OPCML-Fc fusion protein.The tumor volume,tumor weight and mouse body weight were measured during treatment.Results: The PDX model of ovarian cancer was successfully constructed.The success rate of P1 generation was 61.9%,and P2 generation 75%.The hematoxylin-eosin staining and PAX8 staining of PDX were similar to the original tumor tissue.In vivo results showed that the OPCML-Fc fusion protein was mainly enriched in tumor sites,and no aggregation was seen in the normal tissues;the OPCML-Fc fusion protein had a significant inhibitory effect on the PDX tumors of ovarian cancer.The OPCML-Fc fusion protein did not affect the daily activities and weight changes of mice.Conclusion: OPCML-Fc fusion protein has good affinity for ovarian cancer xenografted tumor in vivo,and can effectively inhibit the growth of xenografted tumor.