| Objective:Ovarian cancer is the leading cause of death in the female reproductive system,with5-year survival rate as low as 30%.In 2021,21,410 new cases of epithelial ovarian cancer were diagnosed and 13,770 deaths occurred.The early symptoms of ovarian cancer are insidious and non-specific.Meanwhile,there are no tumor biomarkers with high specificity and sensitivity,which all lead to the difficulty of early diagnosis of ovarian cancer.The initial treatment mode of ovarian cancer is tumor cytoreductive surgery and platinum-based combined chemotherapy,but about 70%of patients have chemotherapeutic resistance and the treatment effect is not good.Difficulty in early diagnosis and chemotherapy resistance lead to poor prognosis of ovarian cancer.Therefore,it is necessary to find more effective molecular targets for the diagnosis and treatment of ovarian cancer.The ANLN gene encodes an actin-binding protein that plays a role in cell growth,migration and cytokinesis.Current studies have shown that ANLN is highly expressed in a variety of tumors and is significantly associated with poor prognosis.Bioinformatics analysis revealed that ANLN is a key gene affecting the prognosis of ovarian cancer.However,the role of ANLN in ovarian cancer has not been reported.Several studies have shown that the change of signaling pathway is one of the reasons for the malignant biological behavior of cancer cells.MEK-ERK signaling pathway plays an important role in ovarian cancer and the activation of MEK-ERK pathway can promote the proliferation,invasion,migration and other processes of ovarian cancer cells.Studies have found that ANLN can affect the phosphorylation level of ERK molecules in colorectal cancer,and ANLN is enriched in MEK-ERK signaling pathway in ovarian cancer according to bioinformatics.Therefore,we hypothesized that ANLN could play a role in ovarian cancer by regulating the MEK-ERK pathway.Transcription factors are proteins that control the transcription of DNA fragments into m RNA by binding to specific DNA regions.Their function is to regulate,turn on and off gene expression to ensure the correct and orderly expression of genes throughout the life cycle of cells and organisms.Abnormal expression of transcription factors may affect the occurrence and development of tumors through a variety of ways.In this study,bioinformatics was used to predict CEBPB(CCAAT enhancer binding proteinβ)as an upstream transcription factor of ANLN.However,the exactly regulatory mechanism between the two has not been reported.In conclusion,on the basis of biogenic analysis,this study analyzed the key role of ANLN in ovarian cancer at the levels of tissue,cell and molecular,and preliminarily explored its upstream and downstream mechanisms.It was confirmed that transcription factor CEBPB regulates ANLN and activates MEK-ERK signaling pathway to promote malignant biological behavior of ovarian cancer cells.Methods:Part 1:The Gene Expression Omnibus(GEO)database GEO2R tool was used to analyze the differential Gene Expression of ovarian cancer datasets GSE27651,GSE36668,GSE54388 and GSE69428.Venn diagram was used to obtain the common differentially expressed genes(DEGs)in the four datasets.GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway enrichment were analyzed by DAVID database.The STRING database was used to construct the interaction network of proteins corresponding to the differential genes,and the Cytohubba module of Cytoscape was used to screen the top 10 key genes.GEPIA2(Gene Expression Profiling Interactive Analysis2)and KM-Plotter databases were used to preliminarily verify the expression and prognostic value of key genes.The key gene ANLN was further selected for experimental verification.The m RNA expression levels of ANLN in 20 normal ovarian tissues and 22ovarian cancer tissues were detected by q RT-PCR.Immunohistochemical(IHC)method was used to detect 81 cases of epithelial ovarian malignant tumors(Malignant group),12cases of ovarian epithelial borderline tumors(Borderline group),11 epithelial ovarian benign tumor(Benign group)and 17 cases of normal ovarian epithelial(Normal group).Comparison of ANLN positive expression rate and high expression rate between different groups were carried out by chi-square or Fisher’s exact test,and IHC score by T test.The relationship between the expression of ANLN and various clinicopathological parameters(age,grade,stage,pathological type,lymph node metastasis)of ovarian cancer was further explored by chi-square test or Fisher’s exact test in the malignant tumor group.Part 2:q RT-PCR and Western blot were used to detect the expression of ANLN in three ovarian cancer cell lines:A2780,CAOV3 and OVCAR3.Ovarian cancer cell lines with low and overexpression of ANLN and their control groups were constructed by si-RNA and overexpression lentivirus,respectively.q RT-PCR and Western blot were used to verify the knockdown and overexpression efficiency.CCK-8 assay,flow cytometry,scratch assay and Transwell assay were used to explore the effects of ANLN on the proliferation,cell cycle,migration and invasion of ovarian cancer cells.Western blot was used to detect the expression of Cyclin D1,E-cadherin and N-cadherin.The ovarian cancer standardized RNA Seq data of The Cancer Genome Atlas(TCGA)were downloaded from UCSC Xena database.Gene Set Enrichment Analysis(GSEA)was used to analyze pathways related to ANLN.Western blot was used to detect the protein changes of MEK1/2,p-MEK1/2,ERK1/2 and p-ERK1/2 in the MEK-ERK pathway after differential expression of ANLN.After adding MEK pathway inhibitor PD98059(MEKi)to oe-ANLN group,the changes of proliferation,migration and invasion ability of ovarian cancer cells were detected by CCK-8 test,scratch test and Transwell test to observe whether the promoting effect of overexpression of ANLN on malignant biological behavior of ovarian cancer cells can be reversed by MEK pathway inhibitor.In addition,the effect of ANLN on the proliferation of ovarian cancer cells in vivo was investigated by subcutaneous tumor implantation model in nude mice.Part 3:PROMO and JASPAR databases were used to predict the transcription factors that could bind to the upstream promoter region of ANLN.GEPIA2 database was used to analyze the correlation between each transcription factor and the expression of ANLN in ovarian cancer.UALCAN and KM-Plotter databases were used to detect the protein expression of transcription factors in ovarian cancer tissues and the relationship between the expression level and overall survival(OS)and progression-free survival(PFS)of ovarian cancer.The obtained transcription factors were then initially screened and the transcription factor CEBPB was obtained.IHC was used to detect the protein expression of CEBPB in ovarian malignant group,borderline group,benign group and normal group,and to explore the relationship between CEBPB and clinicopathological parameters(age,differentiation degree,stage,pathological type,lymph node metastasis)in ovarian malignant group.Spearman correlation analysis was used to analyze the correlation between the expression of ANLN and CEBPB in the same ovarian cancer tissues.Chromatin Immunoprecipitation(CHIP)assay and agarose gel electrophoresis assay were used to verify the binding relationship between CEBPB and the promoter region of ANLN.CEBPB overexpression group and its control group(oe-CEBPB and oe-NC)were constructed by plasmids.The effect of CEBPB overexpression and the changes of ANLN expression level after CEBPB overexpression were detected by q RT-PCR and Western blot to explore whether CEBPB regulates the expression of ANLN.ANLN promoter region wild-type(ANLN-WT)and mutant type(ANLN-MUT)dual luciferase reporter vectors were constructed according to results of CHIP assay,agarose gel electrophoresis assay and bioinformatics prediction.ANLN-WT or ANLN-MUT were co-transfected with oe-CEBPB or oe-NC plasmids.The effect of CEBPB overexpression on luciferase activity in each group was observed.The oe-NC,oe-CEBPB and oe-CEBPB+si-ANLN groups were constructed in CAOV3 and OVCAR3 cell lines.The expression of ANLN and MEK-ERK pathway molecules in each group was detected by Western blot.CCK-8 assay,scratch assay and Transwell assay were used to explore whether CEBPB affected the proliferation,migration,invasion of ovarian cancer cells through ANLN.Results:Part 1:Based on the analysis results of four GEO datasets,a total of 74 common differentially expressed genes were screened out,including 56 up-regulated genes and 18down-regulated genes.Common differentially expressed genes were mainly concentrated in cell division(GO:BP),spindle(GO:CC)and microtubule binding(GO:MF),and enriched in cell cycle,oocyte meiosis,P53 signaling pathway,progesterin-mediated oocyte maturation and pyrimidine metabolism(P<0.05).ECT2,BIRC5,CEP55,RACGAP1,NUSAP1,TYMS,RAD51AP1,CDKN3,ANLN and KIF2C are 10 key genes in the development of ovarian cancer.GEPIA2 database indicated that the expression of these 10key genes were up-regulated in ovarian cancer(P<0.05).KM-plotter database showed that the expression of ECT2,CEP55,RACGAP1,RAD51AP1,CDKN3 and ANLN were correlated with both OS and PFS of ovarian cancer(P<0.05),which could be used as prognostic markers of ovarian cancer.The results of q RT-PCR showed that the expression level of ANLN m RNA in ovarian cancer tissues was higher than that in normal ovarian tissues(P<0.01).IHC results showed that the positive and high expression rates of ANLN in the malignant group were significantly higher than those in the benign and normal ovarian groups(P<0.01).There was no significant difference in the positive expression rate and high expression rate of ANLN between the malignant group and the borderline group(P>0.05).IHC score indicated that the protein expression level of ANLN in malignant group was higher than that in normal ovarian group(P<0.001),benign group(P<0.001)and borderline group(P<0.05).Chi-square test and Fisher’s exact test showed that the high expression rate of ANLN was correlated with the stage and grade of ovarian cancer(P<0.05),but not with the age,pathological type and lymph node metastasis(P>0.05).Part 2:q RT-PCR and Western blot showed that the expression of ANLN decreased successively in ovarian cancer cell lines CAOV3,OVCAR3 and A2780.Therefore,ANLN knockdown group and control group were constructed in CAOV3 and OVCAR3 cell lines,ANLN overexpression group and control group were constructed in A2780 and OVCAR3cell lines.CCK-8 assay showed that knockdown of ANLN significantly inhibited the proliferation of ovarian cancer cells(P<0.001),and overexpression of ANLN significantly promoted the proliferation of ovarian cancer cells(P<0.001).Flow cytometry showed that knockdown of ANLN could arrest ovarian cancer cells in G0/G1 phase(P<0.05),and overexpression of ANLN promoted the transformation of ovarian cancer cells into S phase(P<0.05).Scratch test showed that knockdown of ANLN significantly inhibited the migration ability of ovarian cancer cells(P<0.01),and overexpression of ANLN significantly promoted the migration ability of ovarian cancer cells(P<0.05).Transwell assay showed that knockdown of ANLN significantly inhibited the invasion ability of ovarian cancer cells(P<0.01),and overexpression of ANLN significantly promoted the invasion ability of ovarian cancer cells(P<0.01).Western blot analysis showed that knockdown of ANLN could inhibit the expression of Cyclin D1 and N-cadherin,and promote the expression of E-cadherin(P<0.05).Overexpression of ANLN could promote the expression of Cyclin D1 and N-cadherin,inhibit the expression of E-cadherin(P<0.05).GSEA enrichment analysis showed that ANLN was mainly enriched in cell cycle,and may be related to P53,ERBB,NOTCH,m TOR,MAPK,WNT and TGF-βsignaling pathways(P<0.05).Western blot showed that the phosphorylation ratio of MEK1/2 and ERK1/2molecules in the ERK cascade of MAPK(mitogen-activated protein kinase)signaling pathway decreased after ANLN knockdown(P<0.05):The expression of p-MEK1/2 and p-ERK1/2 was decreased,while the expression of MEK1/2 and ERK1/2 molecules was basically unchanged.The overexpression group showed a reverse trend.After adding MEK pathway inhibitor to the cells of ANLN overexpression group,it was found that inhibiting MEK pathway could reverse the promoting effect of ANLN overexpression on the proliferation,migration and invasion of ovarian cancer cells(P<0.05).The results of subcutaneous implant tumor model in nude mice showed that the tumor volume and mass of ANLN overexpression group were(0.78±0.21cm~3;0.44±0.11g),higher than the tumor volume and mass of the control group(0.43±0.22cm~3;0.26±0.10g)(P<0.05),overexpression of ANLN significantly promoted the proliferation of ovarian cancer cells in vivo(P<0.05).Part 3:PROMO and JASPAR databases predicted that CEBPB was one of the potential upstream transcription factors of ANLN.GEPIA2 database showed that there was a positive correlation between ANLN and CEBPB m RNA level(R=0.22,P<0.001).UALCAN database showed that the protein expression of CEBPB in 100 ovarian cancer tissues was higher than that in 25 normal ovarian tissues(P<0.01).KM-plotter database showed that the expression of CEBPB was correlated with OS(P<0.01)and PFS(P<0.001)of ovarian cancer patients.CEBPB may be a molecular marker affecting the prognosis of ovarian cancer patients.IHC results showed that the positive expression rate of CEBPB in the malignant group was significantly higher than that in the benign group and the normal ovarian group(P<0.01),while there was no significant difference between the malignant group and the borderline group(P>0.05).The high expression rate of CEBPB in malignant group was significantly higher than that in normal ovarian group(P<0.001),benign group(P<0.01)and borderline group(P<0.05).The IHC score of CEBPB in malignant group was higher than that in normal ovarian group(P<0.001),benign group(P<0.01)and borderline group(P<0.01).The positive expression rate and high expression rate of CEBPB in the malignant group was not correlated with the age,pathological type,stage and lymph node metastasis(P>0.05),but correlated with the grade of ovarian cancer(P<0.01).CEBPB was positively correlated with ANLN protein expression in ovarian cancer(R=0.42,P<0.001).CHIP and agarose gel electrophoresis showed that CEBPB could bind to the promoter region of ANLN.After CEBPB expression was promoted by overexpression plasmid,the m RNA and protein levels of ANLN were increased(P<0.05).Dual luciferase assay showed that after mutation of CATGCGCCAC sequence at-1696--1687 in the upstream promoter region of ANLN,overexpression of CEBPB could enhance luciferase activity in ANLN-WT group(P<0.01),but had no effect in ANLN-MUT group(P>0.05).These results indicated that CEBPB could bind to the upstream promoter region of ANLN at-1696--1687:CATGCGCCAC sequence and enhance the transcription of ANLN.Western blot analysis showed that the phosphorylation levels of MEK1/2 and ERK1/2 molecules increased after CEBPB overexpression(P<0.05),and the MEK-ERK pathway was activated.Knockdown of ANLN could restore the activation effect of CEBPB on MEK-ERK pathway(P<0.05),which means CEBPB activates the MEK-ERK pathway through ANLN.After overexpression of CEBPB,the proliferation(P<0.001),migration(P<0.01)and invasion(P<0.001)of ovarian cancer cells were increased.Functional recovery assay showed that inhibition of ANLN expression could restore the promoting effects of CEBPB overexpression on proliferation(P<0.05),migration(P<0.01)and invasion(P<0.001)of ovarian cancer cells.CEBPB promoted the malignant biological behavior of ovarian cancer cells through ANLN.Conclusion:1.ANLN is a hub gene with prognostic value in ovarian cancer,its expression is increased in ovarian cancer,which is correlated with FIGO stage and grade.ANLN can enhance the proliferation,invasion and migration ability of ovarian cancer cells in vitro and proliferation ability in vivo.2.ANLN promotes the proliferation,invasion and migration of ovarian cancer cells through MEK-ERK signaling pathway.3.The expression of transcription factor CEBPB is increased in ovarian cancer tissues,which can bind to the promoter region of ANLN,thus enhances the transcription of ANLN,activates the MEK-ERK signaling pathway,and promotes a series of malignant biological behaviors of ovarian cancer cells. |
PDF Full Text Request