The Study On The Antitumor Function And Mechanism Of LncRNA-ZXF1 Screened By WGCNA In Endometrioid Endometrial Carcinoma

BackgroundEndometrial cancer is a malignant epithelial malignant tumor that mainly occurs in the endometrium of women.According to the latest survey results published by the internationally authoritative cancer journal CA CANCER J CLIN,endometrial cancer is the highest incidence of gynecological tumors in the United States,and the incidence in my country is also increasing year by year.Clinically,patients with endometrial cancer are mostly postmenopausal women,and the common first symptom is abnormal vaginal bleeding and drainage.However,the cause of the disease is not yet completely clear,and more clinical and basic research is needed.At present,surgical treatment is still the first choice for endometrial cancer.However,with the popularity of precision medicine and individualized treatment,immunotherapy and molecular targeted therapy for malignant tumors have also become research hotspots.The difficulty of precise treatment at this stage is to find effective and sensitive target molecules.Bioinformatics originated in the 1950s and has developed into a cross-discipline involving life sciences,statistics,mathematics,computational biology and informatics.Researchers use programming tools to complete detailed and detailed biometric analysis and visualization.Commonly used programming languages include R,Perl,and Python.Among them,the R programming language has strong logic and is closer to natural language,which reduces the difficulty of learning and operation.Many well-developed packages(Packages)allow researchers to have multiple ways to read,analyze and visualize data.Weighted Gene Co-expression Network Analysis(WGCNA)is a computational biology method used to explore gene association modules between different samples(such as cancer tissue and normal tissue),which can identify highly coordinated changes in biological processes And identify important core molecules based on the correlation between the gene set and clinical information.P21 is an important regulatory molecule in the process of tumor formation and development,and it can participate in the regulation of various molecules and pathways.The degradation of P21 can relieve the inhibitory effect on CDK,thereby accelerating the cell cycle process.LncRNA can play a role by binding to protein,DNA,RNA,etc.The binding to protein may affect the post-translational modification of protein including ubiquitination,and then regulate protein function.Competitive endogenous RNA(ceRNA)is an important way for lncRNA to regulate downstream target molecules.The principle of ceRNA is that lncRNA binds miRNA competitively to reduce the degradation of miRNA to mRNA,so as to achieve a protective effect on mRNA.This lncRNA is called ceRNA.So far,many genes with known ceRNA interacting molecules have been identified,and they are also related to human diseases.LncRNA often acts as a ceRNA to protect the expression of target molecules and regulate more downstream molecules and pathways.This project uses the WGCNA method to modularize the RNA expression data of UCEC patients in TCGA.Through the analysis of the clinical characteristics of each module,the core molecule lncRNA-ZXF1 is screened in combination with the sequencing data of the group in the early stage.We conduct follow-up molecular biology and cell biology research on it.Studies have shown that lncRNA-ZXF1 plays a role in a variety of malignant tumors and regulates tumor biological functions such as proliferation and invasion.However,lncRNA-ZXF1 has not been studied in endometrial cancer.In order to clarify the role of lncRNA-ZXF1 in endometrial cancer,a large number of basic medical research and bioinformatics methods have been applied.We have clarified the role of lncRNA-ZXF1 in endometrial cancer and its mechanism,and provided a new target for the diagnosis and treatment of endometrial cancer.Part Ⅰ:Module analysis and molecular screening of clinical information for endometrial cancer patients based on WGCNAObjectives1.Analyze the relationship between the expression data of endometrial cancer and clinical features in the Tumor Genome Atlas(TCGA)through WGCNA,and screen the core lncRNA.2.Analyze and screen the lncRNA-ZXF1,and explore the function of LncRNA-ZXF1 in endometrial cancer through enrichment analysis of its target molecules.MethodsWGCNA was used to analyze the lncRNA data of endometrial cancer in TCGA,and the topological overlap matrix was obtained through calculation by screening the appropriate soft threshold.Then perform hierarchical clustering analysis through dynamic tree cutting,and correlate with clinical information to obtain expression modules related to specific clinical features.Through the analysis of the modules,the core expression lncRNA-ZXF1 was obtained by screening.Analyze the differential expression genes(DEGs)of cancer and normal tissues in TCGA and Genotype-Tissue Expression(GTEx)data through ’Deseq2’ package,and then use bioinformatics methods to predict the target molecules of lncRNA-ZXF1.Intersection of differentially expressed genes and target molecules of lncRNA-ZXF1 is performed,and the molecules in the intersection are analyzed based on the Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Set Enrichment Analysis(GSEA),Reactome database and Gene Ontology(GO).Results1.The soft threshold is selected by calculation to be β=5,and the TCGA clinical features are cut and associated through the dynamic tree.The brown and green modules have the highest correlation with the clinical tumor grade and are negatively correlated.Analyze the module results and previous sequencing results,and screen lncRNA-ZXF1 as a molecule for follow-up research.2.Use the "Deseq2" package to analyze the data of TCGA and GTEx,and calculate and filter 4677 DEGs.The target molecules of lncRNA-ZXF1 were predicted by bioinformatics methods,and 1130 common genes were obtained by intersecting with DEGs.Perform function enrichment analysis on common genes,and the enrichment results include cancer pathways,metabolism,biological oxidation,transcription regulation,and cell proliferation.ConclusionsThe brown and green modules are negatively correlated with TCGA tumor grade.LncRNA-ZXF1 may function in endometrial cancer by regulating cell proliferation,mitosis,important pathways in tumors,and lipid metabolism.Part Ⅱ:LncRNA-ZXF1 regulates the protein expression of P21 by managing CDC20-mediated ubiquitination degradationObjectives1.To explore the expression and prognosis of lncRNA-ZXF1 in endometrial cancer.2.To clarify the role and mechanism of lncRNA-ZXF1 in endometrial cancer cells.3.To detect the effect of lncRNA-ZXF1 on the growth of subcutaneous tumors in nude mice in an animal model of endometrial cancer.Methods1.Through the analysis of tumor samples collected by TCGA,GSE17025 and Qilu Hospital,the expression of lncRNA-ZXF1 is clarified.Use bioinformatics methods to analyze the prognostic and diagnostic value of lncRNA-ZXF1.2.The expression of lncRNA-ZXF1 in endometrial cancer cell lines was explored by qPCR,and the subcellular localization of lncRNA-ZXF1 in endometrial cancer cells was detected by fluorescence in situ hybridization.Ishikawa and HEC-1A cells were transfected with IncRNA-ZXFl overexpression virus to obtain stable cell lines.Carry out CellTiter-Glo experiment,MTT experiment,clone formation experiment,EdU staining experiment and flow cell cycle experiment to explore the effect of lncRNA-ZXF1 on the proliferation of endometrial cancer cells.To clarify the effect of lncRNA-ZXF1 on the migration and invasion of endometrial cancer cells,the scratch test and the Transwell chamber test was performed.3.Use the stably transformed cells to construct an endometrial cancer xenograft animal model,and measure the tumor size every other day to draw the subcutaneous tumor growth curve.A live animal imaging system was used to measure the size of animal tumors and detect the expression of lncRNA-ZXF1,P21 and Ki67 in animal tumors.4.Verify the combination of lncRNA-ZXF1 and P21 by RIP test,and verify the effect of lncRNA-ZXF1 on P21 ubiquitination using western blot and co-IP methods.Cycloheximide(CHX)and MG132 were used to treat endometrial cancer cells to further clarify the role of lncRNA-ZXF1 in the degradation of P21 ubiquitination.Results1.Compared with normal tissues,lncRNA-ZXF1 was lower expressed in endometrial cancer tissues.The disease-free survival(DFS)of endometrial cancer patients with high expression of lncRNA-ZXF1 was better than that of patients with low expression of lncRNA-ZXF1.Receiver operating characteristic curve(ROC)showed that lncRNA-ZXF1 had a good diagnostic value in endometrial cancer.The results of fluorescence in situ hybridization experiments showed that lncRNA-ZXF1 can be expressed in the nucleus and cytoplasm.2.CellTiter-Glo experiment,MTT experiment,clone formation experiment,EdU staining experiment and flow cytometry experiment proved that lncRNA-ZXF1 inhibited the proliferation of endometrial cancer cells.GSEA analysis results also confirmed that lncRNA-ZXF1 inhibited cell cycle and nuclear DNA replication.Wound healing and Transwell experiments also showed that after overexpression of lncRNA-ZXF1,the migration and invasion of endometrial cancer cells were inhibited,and the protein expression of MMP2 and N-Cadherin decreased,while the protein expression of E-Cadherin increased.3.In vivo experiments,the growth of subcutaneous tumors in nude mice was inhibited after overexpression of lncRNA-ZXF1.The expression of P21 in subcutaneous tumors increased,and the expression of Ki67 decreased.4.Overexpression of lncRNA-ZXF1 had no effect on the RNA expression of P21,but the protein expression increased.After treatment with CHX and MG132,lncRNA-ZXF1 increased the aggregation of P21 protein and slowed down the degradation rate.RIP proved that lncRNA-ZXF1 and P21 can bind directly.LncRNA-ZXF1 can rescue the degradation of P21 by CDC20 and hinder the binding of CDC20 and P21.Conclusions1.LncRNA-ZXF1 is low expressed in endometrial cancer,and has good diagnostic and prognostic value.2.LncRNA-ZXF1 can inhibit the proliferation,migration and invasion of endometrial cancer cells.3.LncRNA-ZXF1 inhibits the growth of subcutaneous tumors in nude mice in vivo experiments.4.LncRNA-ZXF1 binds to P21 to hinder the ubiquitination and degradation of P21 by CDC20.Part Ⅲ:LncRNA-ZXF1 regulates the expression of P21 through the miR-378a-3p/PCDHA3 axisObjectives1.To explore that lncRNA-ZXF1 regulate the expression of P21 through the ceRNA mechanism of miR-378a-3p/PCDHA3.2.To explore that the prognostic and diagnostic value of PCDHA3 through bioinformatics methods,and to clarify the role of PCDHA3 in the proliferation and invasion of endometrial cancer.Methods1.After overexpressing lncRNA-ZXFl in Ishikawa and HEC-1A cell lines,we performed whole transcriptome sequencing on stably transfected cells,and used bioinformatics methods to predict the target molecules of lncRNA-ZXF1.2.We detected the expression of PCDHA3 through the database and the endometrial cancer samples collected from Qilu Hospital.Molecular biology methods were used to clarify the regulatory relationship between lncRNA-ZXF1/miR-378a-3p/PCDHA3.3.The dual luciferase reporter experiment was used to verify that the molecules in the ceRNA mechanism can bind to each other.4.CellTiter-Glo experiment,MTT experiment,clone formation experiment,EdU staining experiment and flow cell cycle experiment are used to explore the effect of PCDHA3 on the proliferation of endometrial cancer cells.Wound healing experiment and Transwell cell experiment were used to clarify the role of PCDHA3 on migration and invasion in endometrial cancer cells.Western blot experiments were used to confirm that PCDHA3 can regulate the expression of P21.Results1.Combining the sequencing results and bioinformatics methods after overexpression of lncRNA-ZXF1,we predicted the ceRNA regulatory axis of lncRNA-ZXF1/miR-378a-3p/PCDHA3.2.The data of TCGA and GSE17025,as well as the results of endometrial cancer samples from Qilu Hospital confirmed the lower expression of PCDHA3 in endometrial cancer.The ROC curve also illustrated the potential diagnostic value of PCDHA3.3.After overexpression of lncRNA-ZXF1,the expression of miR-378a-3p decreased,while the expression of PCDHA3 increased.MiR-378a-3p inhibited PCDHA3 expression.The rescue experiment confirmed that lncRNA-ZXF1 can increase the expression of PCDHA3,and miR-378a-3p can reverse this change.RIP experiment proved the binding of miR-378a-3p and AGO2.The results of the dual luciferase report experiment showed that miR-378a-3p can significantly reduce the fluorescence of IncRNA-ZXF1 and PCDHA3 wild-type,but cannot change the fluorescence of the mutants.4.CellTiter-Glo experiment,MTT experiment,clone formation experiment,EdU staining experiment and flow cell cycle experiment proved that PCDHA3 inhibited the proliferation of endometrial cancer cells.GSEA analysis results also confirmed that PCDHA3 inhibits cell cycle and nuclear DNA replication.Wound healing and Transwell experiments also showed that after overexpression of PCDHA3,the migration and invasion of endometrial cancer cells were inhibited.And the expression of P21 can increase with the increase of PCDHA3.Conclusion1.PCDHA3 is lower expressed in endometrial cancer,and PCDHA3 has good diagnostic and prognostic value in endometrial cancer.2.PCDHA3 regulates tumor cell proliferation and cell cycle in endometrial cancer.3.LncRNA-ZXF1 can regulate the expression of P21 through the ceRNA mechanism formed by the lncRNA-ZXF1/miR-378a-3p/PCDHA3 axis.Innovations and LimitationsInnovations1.This is the first study to research lncRNA-ZXF1 on regulating proliferation and invasion in endometrial cancer.2.We have clarified that lncRNA-ZXF1 regulates the protein expression of P21 through two mechanisms that mediate ubiquitination degradation and miR-378a-3p/PCDHA3 axis.3.This is the first discovery that PCDHA3 regulates proliferation and invasion in endometrial cancer.Limitations1.The number of clinical samples of endometrial cancer and adjacent tissues included in this study is relatively small.2.In the in vivo experiment,mouse tail vein injection was not performed,and no data of lung metastasis model was obtained.3.The mechanism part does not clarify the specific regulation method of PCDHA3 on P21,and the mechanism research is not in-depth.