Identification And Biological Function Of MicroRNA In Cerebrospinal Fluid Associated With Leptomeningeal Metastasis Of Breast Cancer

Purpose:Leptomeningeal metastasis(LM)is a fatal complication of malignant tumors.Breast cancer is one of the most common primary tumors of LM.In recent years,with the application of comprehensive treatment,the survival time of breast cancer patients is significantly prolonged,and the incidence of LM is increasing year by year.The role of microRNAs(miRNAs)in the process of metastasis and invasion has been proved to be a fundamental player in cancer research.The aim of this study is to screen and verify the differentially expressed miRNAs in cerebrospinal fluid(CSF)associated with LM of breast cancer,to find specific CSF miRNA molecular markers with LM diagnostic value,and to preliminarily explore their biological functions.Methods:1.To analyze the differential expression of miRNAs in the CSF samples of breast cancer patients with LM and breast cancer patients with brain metastasis by miRNA chip.Samples of CSF from patients with LM before and after effective treatment were also examined and miRNAs associated with LM from breast cancer were selected.2.qRT-PCR was used to verify whether the expression changes of candidate miRNAs in the CSF of breast cancer patients with LM and breast cancer brain metastasis were consistent with those screened by gene chip.3.qRT-PCR was used to detect the expression levels of candidate miRNAs in the CSF of breast cancer patients with LM at the time of initial diagnosis and after effective treatment,and to clarify whether the candidate miRNAs are related to the occurrence and development of LM and disease changes in breast cancer patients.4.miR-4459 and miR-6090 mimics were transfected into breast cancer cells to upregulate the expression of miRNA.CCK8 assay,wound healing assay and Transwell invasion assay were used to detect the effects of target miRNAs on the proliferation,migration and invasion of breast cancer cell lines MDA-MB-231 and MCF-7.5.The downstream target genes of miR-4459 were predicted by TargetScan and miRTarBase databases.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were used to perform functional enrichment analysis of their target genes.Results:1.In the screening stage,compared with the breast cancer brain metastasis group,a total of 28 miRNAs were differentially expressed in the CSF of breast cancer LM patients,including 13 up-regulated and 15 down-regulated.In the CSF samples of breast cancer before LM treatment and after effective treatment,a total of 8 miRNAs were differentially expressed,including 1 up-regulated and 7 down-regulated.Compared with the LM group and the brain metastasis group,the differentially expressed miRNAs were intercrossed,and a total of 2 LM-related miRNAs were screened,miR-4459 and miR-6090.Compared with breast cancer patients with brain metastases,the expression of miR-4459 and miR-6090 was increased in LM patients,but decreased after effective treatment.Therefore,miR-4459 and miR-6090 were selected as candidate miRNAs for further verification.2.In the validation phase,the expression of miR-4459 in the CSF of breast cancer LM patients was higher than that of breast cancer brain metastasis patients,but the difference was not statistically significant(P=0.4121).The expression of miR-6090 was increased,which was consistent with the results of miRNA microarray screening(P=0.0424).Compared with the expression levels of miRNAs at diagnosis,miR-4459 and miR-6090 in the CSF of LM patients were significantly decreased after effective treatment(P=0.047).3.Overexpression of miR-4459 significantly promoted the proliferation,migration and transmembrane invasion of breast cancer cells in MDA-MB-231 cell line.However,in the MCF-7 cell line,none of the results were significant except for the cell proliferation assay.Overexpression of miR-6090 in MDA-MB-231 and MCF-7 cells not only promoted cell proliferation,but also had no significant effect on cell migration and transmembrane invasion.4.A total of 1909 target genes downstream of miR-4459 were predicted and analyzed,which were closely related to tumor cell migration.Conclusion:Compared with breast cancer patients with brain metastases,the expression levels of miR-4459 and miR-6090 were increased in the CSF of breast cancer patients with LM.After effective treatment,the expression of miR-4459 and miR-6090 was significantly lower than that at diagnosis,suggesting that they may be related to the occurrence and development of breast cancer LM.Overexpression of miR-4459 can promote the proliferation,migration and invasion of breast cancer cells,which may be one of the mechanisms of promoting the occurrence of breast cancer LM.The downstream target genes of miR-4459 are closely related to the migration of a variety of tumor cells,suggesting that miR-4459 may promote the occurrence of breast cancer LM by regulating its target genes.