Effects Of NPM1 On Proliferation, Invasion And Migration Of Triple-negative Breast Cancer Cell

Objective:The treatment of breast cancer is a very challenging problem in the world at present,because breast cancer is one of the main causes of women’s death.Compared with other types of breast cancer,triple-negative breast cancer has a high recurrence rate,metastatic potential and poor overall survival due to the negative expression of ER,PR and HER2,and there is no targeted treatment scheme at present.Nuclear protein(NPM1 or B23)is a kind of protein with abundant expression in cells,which is essential for many cellular functions,including ribosome biosynthesis,chromatin remodeling,centrosome replication,embryogenesis,apoptosis and DNA repair.This topic intends to study the expression of NPM1 in triple negative breast cancer cells and normal breast cells.To explore the effect of NPM1 expression on the biological activity of breast cancer cells;To study the mechanism of NPM1 affecting the biological activity of triple negative breast cancer.Methods:1.based on bioinformatics method,analyze the relative expression of NPM1 in various tumor tissues in TIMER database,extract normal breast tissue samples and triple-negative breast cancer samples from The Cancer Genome Atlas(TCGA)database,analyze the expression of NPM1 in clinical triple-negative breast cancer samples and normal tissues,and explore the role of NPM1 in immune microenvironment of triple-negative breast cancer;The expression of NPM1 protein was detected by immunohistochemistry in HPA database.2.The expression of NPM1 protein in breast cancer cell lines(BT-549,HCC-1937,BT-474,MDA-MB-231,MCF-7)and normal breast cells(MCF-10A)was detected by Western-blot.3.The full-length open reading frame gene of NPM1 was amplified from breast cancer cell BT-549 by reverse transcription-polymerase chain reaction(RT-PCR)technology,and then the recombinant PCDH-NPM1-3Flag-Blasticidin vector of NPM1 overexpression was constructed after double enzyme digestion.The recombinant vector was confirmed to be successfully constructed by sequencing.4.The recombinant expression vector and virus helper plasmid were used to transfect human embryonic kidney 293 T cells to package lentivirus and infect MDA-MB-231 cells.The stable transgenic MDA-MB-231 cell line with over-expression of NPM1 gene was established through blast resistance screening.5.The protein of stably transformed MDA-MB-231 cells was extracted,and the overexpression of NPM1 was verified by real-time fluorescence quantitative PCR(RT-PCR)and Western-blot.6.Transwell migration test and scratch test were used to investigate the effect of NPM1 overexpression on the migration of breast cancer cells.Clone formation test and MTT assay were used to investigate the effect of NPM1 overexpression on breast cancer cell proliferation.Transwell invasion assay was used to detect the effect of NPM1 on the invasion of breast cancer cells.Results:1.TIMER database shows that the expression of NPM1 in most tumors is higher than that in normal tissues.Analysis of TCGA and HPA database shows that the protein expression of NPM1 in triple negative breast cancer is significantly higher than that in normal tissues,and it is related to the immune microenvironment of patients.2.Westernblot results showed that the relative expression of NPM1 protein in five breast cancer cells,BT-549,HCC-1937,BT-474,MDA-MB-231 and MCF-7,was higher than that in normal breast cells.3.The constructed recombinant overexpression vector was proved to be completely correct by electrophoresis and sequencing.4.After the packaged lentivirus infected the cells,RT-q PCR and Western-blot verified that NPM1 was obviously over-expressed at m RNA and protein levels in the target cells,which proved that negative control and NPM1 stably over-expressed MDA-MB-231 cell lines were successfully screened out.4.MTT assay showed that the proliferation rate of stable cell lines overexpressing NPM1 was significantly higher than that of negative Control group and blank control group(control group).5.The clone formation experiment showed that the clone formation rate of NPM1 overexpression group was significantly higher than that of negative Control group and control group.6.Invasion test results showed that the number of cells passing through the cell in NPM1 overexpression group was significantly higher than that in negative Control group and control group.7.The results of migration experiment also showed that cells in NPM1 overexpression group had higher migration ability than those in negative Control group and control group,and the differences were statistically significant.Conclusion:1.the expression of NPM1 protein in triple negative breast cancer cells and tissues is significantly higher than that in normal cells and tissues.2.The full-length open reading frame sequence of NPM1 gene was successfully amplified in triple negative breast cancer cells.3.The recombinant expression vector PCDH-NPM1-3Flag-Blasticidin of NPM1 was successfully constructed.4.Packaging recombinant lentivirus,infecting cells,and screening to obtain NPM1 overexpression stable cell strain.5.Compared with the control group,the proliferation ability of NPM1 overexpressed stable cell line was significantly improved.6.Compared with the control group,the clone forming ability of NPM1 overexpressed stable cell line was significantly enhanced.7.Compared with the control group,the migration and invasion ability of NPM1 overexpressed stable cell lines were significantly improved.Objective:Breast cancer has overtaken lung cancer as the most common cancer in women.Tumor metabolism,especially lipid metabolism,plays an important role in the progression and metastasis of breast cancer(BC).The proliferation of cancer cells requires rapid synthesis of lipids to generate biofilms,nutrients and signal molecules,etc.,to provide energy for cancer cells when they are depleted of nutrients.In healthy organisms,lipid synthesis is tightly controlled and responds to the nutritional state of the cell.However,many human cancers exhibit abnormal lipid metabolism.The role of lipid metabolism-related genes(LMG)in the prognosis of breast cancer remains unclear.Methods:The expression profiles and clinical follow-up information of 1053 cases of breast cancer were downloaded from TCGA database.Metabolic genes were downloaded from the Gene Set Enrichment Analysis(GSEA)dataset.Univariate Cox regression and minimum absolute contraction and selection operator(LASSO)regression analysis were performed for differentially expressed metabolist-related genes.Then,the metabolism-related risk model formula was constructed to calculate the risk score of each patient.Breast cancer patients were divided into high-risk group and low-risk group with median expression value of risk score as the critical value,and the survival time of patients in the two groups were analyzed.Finally,we analyzed the expression,interaction and correlation of lipid metabolism-related gene risk models.Results:Prognostic analysis shows that the survival rate of high-risk group in TCGA is significantly lower than that of low-risk group with time,and the ROC proves that the predictive ability of the model is highly accurate.2.ss GSEA analysis showed that there were significant differences in immune cells and immune active substances between high and low risk groups according to the model.The gene expression levels of immune checkpoint proteins PD-L1(also known as CD274)and PD-L2(also known as PDCD1LG2)in high risk group were significantly lower than those in low risk group.3.The difference genes of the model are obviously different at the level of immunohistochemistry.Conclusion:Lipid metabolism-related genes may become a new prognostic indicator for BC patients,and have certain ability to predict the survival of patients.The selected genes related to lipid metabolism(n=16)may be helpful to provide new strategies for tumor treatment.