MiR-142-3p Targets The GNB2-AKT-mTOR Pathway To Inhibit Autophagy To Alleviate Paclitaxel Resistance In Breast Cancer

Background: Breast cancer has replaced lung cancer as the number one cancer worldwide,and the efficacy of paclitaxel as a first-line chemotherapeutic agent for breast cancer is inhibited by the development of advanced drug resistance.Autophagy and microRNAs(miRNAs)play a key role in cancer chemoresistance.However the role and mechanism of miRNA regulation of autophagy in paclitaxel resistance in breast cancer is not well understood.Methods: Firstly,the results of RNA and protein microarrays were used to screen out miR-142-3p and GNB2 that were significantly differentially expressed in drugresistant cells.1.CCK-8 assay was performed to identify the response of the constructed drug-resistant cells to paclitaxel compared to the parental cells of breast cancer,and Western Blot was performed to detect the differential expression of drug resistance indicators(BCRP,Pg-p).2.miR-142-3p mimics and inhibitor were transfected in resistant and parental cells,respectively.q RT-PCR was performed to detect differential expression and transfection efficiency of miR-142-3p;Western Blot was performed to detect changes in expression of resistance markers(BCRP,Pg-p);Wound healing,Transwell and flow cytometry were performed to detect the effect of miR-142-3p overexpression on cell migration ability and apoptosis rate.3.Western Blot and immunofluorescence assays were performed to verify the difference in autophagy levels in drug-resistant cells compared to parental cells,and the effect of paclitaxel treatment on autophagy levels.4.Autophagy inhibitors CQ and Baf A1 were co-treated with paclitaxel in drug-resistant cells,and the change in apoptosis rate induced by paclitaxel in drug-resistant cells was detected by flow cytometry.5.Western Blot and immunofluorescence assays were performed to detect the effect of miR-142-3p expression level on the effect of autophagic viability.6.Implantation of resistant cell lines into nude mice,intratumoral overexpression of miR-142-3p,isolation of tumors for immunohistological analysis,and extraction of tumor proteins and RNA to verify the effect of in vivo overexpression of miR-142-3p on drug resistance indicators and autophagy indicators;7.Bioinformatics analysis of the correlation and binding sites of miR-142-3p and GNB2,and dual luciferase assay to analyze the two targeting relationships.8.Combined knockdown of GNB2 and inhibition of miR-142-3p,Western Blot,scratch and Transwell assays detected the effect of miR-142-3p targeting GNB2 on drug resistance indicators,autophagy levels and migration rate;9.KEGG enrichment analysis of GNB2 enrichment in signalling pathways,Western Blot detected the effect of knockdown of GNB2 on autophagy levels and AKT/m TOR phosphorylation levels.Results: 1.Compared to parental cells,drug-resistant cells were significantly less sensitive to paclitaxel treatment and had significantly higher levels of resistancerelated proteins;2.High expression of miR-142-3p in drug-resistant cells significantly reduced the levels of resistance indicator(BCRP,Pg-p)proteins and increased sensitivity to paclitaxel,with the opposite result of inhibition of miR-142-3p in parental cells;3.Overexpression of miR-142-3p significantly inhibited drug-resistant cell viability,migration and apoptosis rates.4.Paclitaxel resistance was significantly correlated with autophagy levels: relative to parental cells,autophagy levels were significantly elevated in resistant cells,and elevated autophagy levels were positively correlated with paclitaxel concentrations;co-treatment with paclitaxel and autophagy inhibitors enhanced the response of resistant cells to paclitaxel;5.Overexpression of miR-142-3p in resistant cells markedly suppressed autophagy levels,in contrast to inhibition of miR-142-3p had opposite results;6.In vivo overexpression of miR-142-3p dramatically suppressed intratumoral drug resistance indicator and autophagy indicator protein levels and curbed tumor malignant progression.7.GNB2 has a binding site with miR-142-3p and their expression is correlated negatively;8.Knockdown of GNB2 expression reverses miR-142-3p low expression-induced drug resistance and autophagy levels;9.Knockdown of GNB2 restrains autophagy levels by promoting AKT/m TOR pathway phosphorylation.Conclusion: Overexpression of miR-142-3p in paclitaxel-resistant cells can increase sensitivity to paclitaxel treatment and ameliorate malignant progression by inhibiting GNB2 expression,which can restrain autophagy levels by promoting AKT/m TOR phosphorylation.