Study On The Mechanism And Clinical Significance Of Lamin-B1 Expression In The Development Of Epithelial Ovarian Cancer

Ovarian canceris a heterogeneous gynecological malignant tumor,which is the second leading cause of female death in the reproductive system.Due to the lack of typical clinical manifestations and effective diagnostic methods in its initial stage,more than 70%of ovarian cancer patients have an advanced stage at the time of initial treatment.Currently,the main treatment method for ovarian cancer in clinical practice is tantamount to cytoreductive surgery and chemotherapy based on paclitaxel and platinum.Although with the improvement of surgical methods and the progress of neoadjuvant chemotherapy,most patients will relapse and eventually develop drug resistance within 5 years after diagnosis.Therefore,clinical and scientific researchers have always been committed to exploring the progression and metastasis of ovarian cancer,the mechanism of chemotherapy resistance and tumor markers.“Nuclear atypia" is a hallmark of cancer cells,so observing the morphological changes of the nucleus is an important basis for distinguishing benign and malignant tumors.The nuclear lamina is a 10-20 nm thick protein network structure located under the NE,which is conducive to the fixation of nuclear membrane proteins and can attach chromatin around the nucleus.Together with nuclear membrane-related proteins,lamins form the nuclear skeleton inside the INM,which is the main scaffold of the nucleus and participates in many cells’ pathophysiological activities.The main genes encoding lamins are LMNA,LMNB1 and LMNB2.Lamin-B1 protein is a kind of lamins expressed by gene LMNB1,which together with other types of lamins form the nuclear lamina,and is an important member of the lamins family.In recent years,the role and pathological mechanism of Lamin-B1 protein in tumors have attracted more and more attention.Lamin-B1 protein is highly reflected in most tumors,and its expression level is significantly correlated with the prognosis of most tumor patients.However,the mechanism and signal pathway of Lamin-B1 in tumors have yet to be clarified.Lamin-B1 plays different or even contradictory roles in different tumors and different types of tumor,and its role in the development of ovarian cancer has not been explored.Therefore,it is necessary to further study the role of Lamin-B1 in ovarian cancer.This study takes the research of Lamin-B1 in epithelial ovarian cancer as a breakthrough and comprehensively uses the methods of bioinformatics analysis,clinical sample analysis,and basic experiment to clarify the expression and clinical significance of Lamin-B1,explore the biological function and molecular mechanism of Lamin-B1 protein in epithelial ovarian cancer and provide new ideas and strategies for the diagnosis and treatment of ovarian cancer.PurposeThis study intends to analyze the expression and clinical relevance of Lamin-B1 in ovarian cancer through public databases and clinical samples,study the biological function of Lamin-B1 in ovarian cancer using cell experiments and animal experiments,explore whether Lamin-B1 affects the proliferation and migration of ovarian cancer cells through the cell cycle and DNA damage repair,and clarify the mechanism and clinical significance of Lamin-B1 in the occurrence and development of EOC and its potential clinical value.Methods1.The public databases were used to analyze the expression level of the Lamin-B1 gene and protein in tumor and normal tissues,especially in ovarian cancer.Kaplan-Meier survival analysis was conducted online through the public database to evaluate the difference in survival time between ovarian cancer patients with high and low expression of Lamin-B1;2.Fresh tissue samples of 32 ovarian cancers and 16 normal ovaries,paraffin sections of 183 ovarian cancers and 36 normal ovaries,and corresponding clinical data were obtained,then the expression level of Lamin-B 1 was detected in ovarian cancer by RTPCR,western blot and immunohistochemistry;3.The correlation between Lamin-B1 expression and other clinical factors was analyzed by Chi-square test,the relationship between Lamin-B1 expression and PFS/OS of ovarian cancer patients was plotted by Kaplan-Meier curve and the impact of LaminB1 expression on the prognosis of ovarian cancer patients compared with other clinical factors were evaluated by Cox risk regression model analysis and ROC curve;4.The effect of overexpression and down-regulation of Lamin-B1 on the biological function of ovarian cancer cells was explored through experiments of cell and animal;5.R software was used to perform differential gene analysis and gene enrichment analysis on GSE9891 and GSE4997 from the GEO database according to the level of LaminB1 expression,and the STRING database website was used to analyze and visualize genes related to Lamin-B1;6.The effects of down-regulation of Lamin-B1 on cell cycle and apoptosis of ovarian cancer cells were analyzed by flow cytometry,and the effects of down-regulation of Lamin-B1 on cell cycle and DNA damage repair-related protein markers were detected by western blot;7.RNA-seq was used to explore the molecules and the downstream mechanism related to Lamin-B1 and clarified the signal pathway related to Lamin-B1.Further,validate the signal pathways that are significantly different and associated with tumor progression,and clarify whether the knockdown of Lamin-B1 has effects on the expression of key molecules.Results1.The analysis of the public database:The expression of Lamin-B1 in various types of tumors is higher than that in corresponding normal tissues,and there is a significant difference in ovarian cancer;the expression level of Lamin-B1 mRNA in ovarian cancer increased with tumor grade;the survival curve showed that the high expression of Lamin-B1 was associated with poor progression-free survival(PFS)and overall survival(OS)in patients with ovarian cancer.2.The analysis of clinical samples:The expression of Lamin-B1 mRNA and protein in tumor tissues was significantly higher than that of normal tissues;the expression level of Lamin-B1 in patients with high clinicopathologic grade was significantly higher than that in patients with low grade;the chi-square test analysis of Lamin-B1 expression and clinical data of ovarian cancer patients showed that the expression level of Lamin-B1 was closely related to pathological grade and lymph node metastasis;survival analysis showed that the prognosis of ovarian cancer patients with higher Lamin-B1 was worse,and cox risk regression showed that Lamin-B1 was a stable independent riskfactor;from the results of ROC curve,the expression of Lamin-B1 is more beneficial than other clinical parameters in classifying patients.3.Basic experiment of cell and animal:lentivirus transfection was carried out to construct two stable overexpression cell lines of Lamin-B 1 and two stable knockdown cell lines respectively.Through cell proliferation and migration experiment,it was found that Lamin-B1 overexpression had no significant effect on the proliferation and migration of cells;down-regulation of Lamin-B1 significantly reduced the proliferation and migration ability of cells;the experiment of subcutaneous tumorigenesis in nude mice showed that the volume and weight of tumor formed in nude mice by down-regulating Lamin-B1 were significantly lower than that of the control group.4.Bioinformatics analysis:The differential gene analysis of two ovarian cancer datasets GSE9891 and GSE49997 found that there were 224 up-regulated genes and 123 downregulated genes in GSE9891,and 348 up-regulated genes and 90 down-regulated genes in GSE4997.Further analysis of the 30 genes with the most significant difference found that CCNB1,TPX2,PTTG1,CDC20,and FOXM1 related to cell cycle and DNA replication were up-regulated in both datasets;GSEA(Gene Set Enrichment Analysis)showed that the expression of Lamin-B1 was significantly correlated with cell cycle,DNA replication and mismatch repair pathway in ovarian cancer;the 38 genes related to Lamin-B1 were analyzed and visualized using STRING database website,which was consistent with the results of functional enrichment analysis.5.Flow cytometry and Western blot:down-regulation of Lamin-B1 induced cell cycle G2 arrest and increased the amount of apoptosis of ovarian cancer cells,and downregulation of Lamin-B1 significantly increased the expression of Cyclin A2,p53 and yH2AX and down-regulate the expression of Cyclin B1.6.Transcriptome sequencing:principal components analysis(PCA)and Pearson correlation analysis showed that the overexpression group had a high correlation and similarity with the control group,which was consistent with the effect of Lamin-B1 overexpression on cell proliferation and migration.The enrichment analysis of the KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway emphasized that Lamin-B1 participated in the regulation of the PI3K-Akt signalling pathway.Western blot further showed that FGF1 and phosphorylated Akt were down-regulated after Lamin-B1 was knocked down.Conclusion1.The expression of LaminB1 is abnormally high in ovarian cancer tissue and is closely related to the progression of ovarian cancer;the prognosis of patients with high expression of LaminB1 is poor,and the expression of LaminBl can be a stable independent risk factor to predict the prognosis of patients.2.After the down-regulation of Lamin-B1,the proliferation and migration ability of ovarian cancer cell lines decreased,indicating that Lamin-B1 plays a key role in regulating the biological behaviour of ovarian cancer cells.3.Down-regulation of Lamin-B1 induces cell cycle G2 arrest and increased the amount of apoptosis of ovarian cancer cells.Lamin-B1 can affect the proliferation and migration of cells by affecting the cell cycle and DNA damage repair and is related to PI3K/Akt signal pathway.