Modulatory Effect Of MiR-485-5p On RACGAP1 In Breast Cancer Distant Metastasis

Objective:The latest global cancer data in 2020 show that the incidence of female breast cancer has surpassed lung cancer to become the most common cancer in the world.Distant metastasis is the main reason for the decrease in survival rate of breast cancer.Therefore,this study aimed at screening out the hub genes(the genes that are most closely related to other genes and play a key role in the gene network)related to distant metastasis of breast cancer from the GEO database through bioinformatics methods.The function that promoting breast cancer distant metastasis of the hub gene was further discovered through in vivo and in vitro experiments.And explored the miRNA that regulated the expression of the hub gene to provide potential targets for the treatment of distant metastasis of breast cancer.Materials and Methods:Firstly,six datasets containing distant metastasis free survival data in the GEO database were selected and the hub gene associating with distant metastasis of breast cancer was analyzed by WGCNA method.COX regression analysis and Kaplan-Meier survival analysis tested the effect of the hub gene on the prognosis of breast cancer patients.Secondly,CCK8 experiment,clone formation experiment,flow cytometry experiment,scratch experiment,Transwell invasion experiment and Western-blot experiment were used to test if the hub gene played a role on the proliferation,apoptosis,cell cycle,migration,invasion ability and epithelial-mesenchymal transition markers of MCF-7 and MDA-MB-231 breast cancer cells.In vivo imaging system was used to detect the effect of the hub gene on the invasion of breast cancer cells in vivo.Then,the miRNAs that regulating the expression of the hub gene were predicted through the Starbase database,and the correlation between the predicted miRNAs and the expression of the hub gene was tested by Q-PCR experiments in human breast cancer tissues.Once confirmed the miRNA with the strongest correlation with the hub gene,Q-PCR,Western-blot,and Dual-Luciferase Reporter Assay were used to verify whether the miRNA directly regulated the expression of the hub gene.Finally,in vitro experiments tested the effect of the miRNA on the function of breast cancer cells after directly acting on the hub gene.Results:1、The blue module was the only one that was positively correlated with distant metastasis free events in breast cancer patients in both the training set and the validation set through WGCNA analysis.DAVID gene function enrichment analysis results showed that the functions of genes in the blue module were mainly enriched in cell proliferation-related signal pathways.2、COX univariate analysis indicated that the top 30 genes in the blue module ranked from largest to smallest based on GS value and KME value significantly affected the distant metastasis free survival of breast cancer patients.Further COX multivariate analysis showed that RACGAP1 and MELK genes were independent risk factors that affected the distant metastasis free survival of breast cancer patients.3、Kaplan-Meier survival analysis showed that breast cancer patients with high expression of RACGAP1 and MELK genes had worse distant metastasis free survival and overall survival.RACGAP1 was identified as the hub gene that affected the distant metastasis of breast cancer based on the larger GS and KME value than MELK.4、The results of CCK8,clone formation and flow cytometry experiment showed that RACGAP1 promoted breast cancer cell proliferation,inhibited breast cancer cell apoptosis,regulated breast cancer cell cycle distribution,so that cells in G1 phase were reduced,and cells in S phase and G2 phase were increased.5、Transwell invasion experiment demonstrated that RACGAP1 promoted the invasion of MDA-MB-231 breast cancer cells,but almost had no effect on the invasion of MCF-7 cells.RACGAP1 had no effect on migration and the expression of EMT markers of breast cancer cells.6、The results of in vivo imaging system showed that more nude mice had lung metastasis in the MCF-7-RACGAP1 group than in the MCF-7-BLANK group,and the lung metastasis occurred earlier.Nude mice injected with MDA-MB-231-RACGAP1 cells had lung metastases earlier than nude mice injected with MDA-MB-231-BLANK cells.7、The miR-130-3p and miR-485-5p were predicted by the Starbase database that regulated the expression of RACGAP1 in breast cancer.The results of Q-PCR in human breast cancer tissues showed that miR-485-5p had a stronger negative correlation with RACGAP1.8、CCK8,clone formation and flow cytometry experiment results indicated that miR485-5p directly acted on RACGAP1 to inhibit proliferation,promote apoptosis and blocked cell cycle of breast cancer cells in G1 phase.Conclusions:1、This study found that most of the genes related to distant metastasis of breast cancer were enriched in cell proliferation-related signal pathways,and the hub gene was RACGAP1.2、This study first discovered that RACGAP1 has the effect of promoting distant metastasis of breast cancer in animal experiments.The mechanism of RACGAP1 affecting the prognosis of breast cancer patients may be through regulating the proliferation,invasion and metastasis of breast cancer cells.3、This study provides a signaling pathway related to distant metastasis of breast cancer,that is miR-485-5p-RACGAP1 pathway.The molecules in the miR-4855p-RACGAP1 signaling pathway may become targets for the treatment of distant metastasis of breast cancer,especially RNA therapy that may be carried out in the future.