The Mechanism Of Hsa_circ₀005219 Regulates The Expression Of MiRNA-142-3p And Inhibits The Proliferation Of Breast Cancer MCF-7 Cells

Micro RNAs are an evolutionarily ancient,endogenous class of non-coding RNAs implicated in numerous biological processes.They have been widely studied in cell metabolism,proliferation,apoptosis,tumour development,and many other physiological processes.Bioinformatic methods predict that approximately one-third of all human genes may be targets of mi RNAs suggesting that mi RNAs may govern vast gene regulatory networks and cell signaling pathways.mi R-142-3p has been suggested to have negative effect on cancer,including breast cancer.Numerous studies have demonstrated that mi R-142-3p inhibits the classical PI3K-AKT-m TOR and RAF-MEK-ERK signaling pathways leading to inhibition of cancer cell growth.However,the regulatory mechanism of mi R-142-3p to the PI3K-AKT-m TOR and RAF-MEK-ERK signaling pathways in breast cancer cell line MCF-7 has rarely been reported.circular RNAs（circ RNAs）are newly discovered endogenous non-coding RNAs featuring structural stability,high abundance,and tissue-specific expression.So far,most studies have focused on the role of circ RNA as a ce RNA to modulate expression of mi RNAs.It is rarely reported that circ RNA ability of mi R-142-3p by preventing mi R-142-3p from degration.In this study,we use the breast epithelial cells MCF-7 as the experimental model.We detect cell proliferation,invasion and adhesion after cells overexpred/silenced mi R-142-3p.The targets of mi R-142-3p in pathways are selected by the results of RIP and bioinformatics,then the protein expression level and activation levels of key signalling proteins are evaluated by western blots.The relative circ RNA is selected through bioinformatics.We overexpress hsa_circ₀005219 by tresfect hsa_circ₀005219 over-expression vector and knock out hsa_circ₀005219 by CRISPR-Cas9.After that we analysis expression of mi R-142-3p and the effects of mi R-142-3p in order to confirm the relationship of hsa_circ₀005219 and mi R-142-3p.Results:To investigate whether mi R-142-3p expression is relevant in breast cancer cell proliferation,invasion,and adhesion,we transfect MCF-7 cells with mi R-142-3p mimics,mi R-142-3p inhibitors,or an appropriate scrambled control.We tested the expression of mi R-142-3p,after cell transfection,with quantitative RT-PCR to check the successful transfection of mi R-142-3p mimics,inhibitors,and control.Results demonstrated that cell proliferation was significantly suppressed in mi R-142-mimic-transfected MCF-7 cells compared to that in negative control group.Further,stable overexpression of mi R-142-3p also led to a significant decrease in the ability of MCF-7 cells to invade through an extracellular FN matrix coating,but led to a significant increase in the ability of adhese.The results were opposite in the inhibitor group.We transfected the mimics mi R-142-3p in MCF-7 cells and evaluated the protein expression level and activation levels of key signalling proteins by western blots.While expression of the main proteins,such as AKT,p-AKT,p-m TOR,p-ERK,and B-raf was diminished,that of PTEN protein was up-regulated.Silencing mi R-142-3p reversed the results.RIP-Chip was applied,and PTEN and AKT were identified as the putative mi R-142-3p targets.We suppressed PTEN by transfecting MCF-7 cells with PTEN si RNA,and examined its effects using q PCR and confocal immunofluorescence microscopy.PTEN was significantly suppressed at both m RNA and protein levels.We also observed a negative correlation between m RNA expression of PTEN and expression of PI3K-AKT-m TOR and RAF-MEK-ERK pathways,based on relative m RNA expression of PTEN,AKT,ERK,B-raf,and m TOR,when PTEN was suppressed.Furthermore,m RNA expression of target genes in PI3K-AKT-m TOR pathway was found to be positively correlated with mi R-142-3p expression.We overexpress hsa_circ₀005219 by tresfecting hsa_circ₀005219 over-expression vector and knock out hsa_circ₀005219 by CRISPR-Cas9.We tested the expression of hsa_circ₀005219,after cell transfection,with quantitative RT-PCR to check the successful transfection.We transfected hsa_circ₀005219 over-expression vector in MCF-7 cells and evaluated the protein expression level and activation levels of key signalling proteins by western blots.While expression of the main proteins,such asp-AKT,p-m TOR,p-ERK,and B-raf was diminished,that of PTEN protein was up-regulated.Silencing hsa_circ₀005219 reversed the resultsConclusion: mi R-142-3p inhibit the proliferation of human breast cancer cells（MCF-7）through PI3K/AKT/m TOR and Raf/MEK/ERK pathways;A feedback loop between mi R-142-3p and PI3K-AKT-m TOR and RAF-MEK-ERK signal pathways;hsa_circ₀005219 improve mi R-142-3p’s ability of regulation.