MiR-1268b Confers Chemosensitivity In Breast Cancer By Targeting ERBB2-mediated PI3K-Akt Pathway

Backgroud:Breast cancer remains the most common malignant tumour and the leading lethal cancer-related mortality in female.Chemotherapy is an efficiency treatment for patients with breast cancers,and neoadjuvant chemotherapy has been extensively applied to clinical practice.However,there are a lot of patients experience recurrence due to chemoresistance,In recent years,more and more non-coding RNAs especially microRNAs are found to be associated with the development and treatment of cancers.MiRNAs are a species of endogenous short non-coding RNAs,acting as mediators of gene expression by posttranscripionly repressing mRNAs and targeting genes by binding to their 3'-untranslated regions(UTRs),leading to chemoresistance.MiR-1268b has never been deeply researched in any cancers.We demonstate that miR-1268b is involved in the regulation of breast cancer chemoresistance.Methods:MiR-1268b was selected from miRNA microarray screening related with chemoresistance of breast cancer.And then tissue samples of breast biopsy with their resected tissues were selected and divided into drug-sensitive and drug-resistant group based on the chemotherapy sensitivity,the clinical data was collected by telephone follow-up.Confirmed by reverse transcription amd treal time-quantitative PCR,miR-1268b was selected as the object of the research.Two cell lines of breast cancer were transfected with miRNA mimics(over expression)and inhibitor(down expression),The Proliferation assays were monitored using CCK-8 and EdU according to the manufacturer's instructions.Tanswell chambers with polycarbonate membrane inserts were used to conduct cell migration and invasion.Cell apoptosis was performed using an Annexin V-FITC apoptosis detection.The target gene of miRNA was detected by bioinformatics databases miRWalk,and verified by dual-luciferase reporter assay,western blot,immunohistochemistry,it showed that there were several evolutionary conserved binding sites of miRNA in the 3'UTR of target gene.Furthermore,the dowmstream signalling pathway PI3K-Akt was confirmed by research and database,PI3KCA,Akt,BCL2 were found to be downstream effectors of miRNA by RT-qPCR and western blot.Results:Analysis of RT-qPCR and clinicopathologic parameters:Our data revealed that miR-1268b was found to be significantly upregulated in drug sensitive and low clinnical stage patients with breast cancers.MiR-1268b had a higher expression in chemosensitive breast cancer cell lines,compared with chemoresistant cell line(MCF-7/ADM).And the endogenous expression of miR-1268 was upregulated in less aggressive tumours compared with more aggressive cell lines.Cell functional experiments in vitro:To investigate the role of miR-1268b in cell proliferation,CCK8 and EDU assay were conducted.Besides the ability of cell proliferation,other malignant phenotypes of breast cancer cells such as the invasive and motility ability were determined.Transwell assays were performed to detect the role of miR-1268b in cell'migration and invasion.However,CCK8,EDU,and Transwell assays demomstrated that the expression of miR-1268b had no relationship with the cell' proliferation,migration or invasion ability of breast cancer.A FCM assay was performed to verified that miR-1268b could induce apoptosis of breast cancer cells.The results of chemosensitivity assay revealed that high expression of miR-1268b conferred adrinamyin sensitivity to breast cancer cells compared with the miR-NC group,while down regulation of miR-1268b increased the resistance of cancer cells to adrinamyin.The data indicated that miR-1268b could increase breast caner's sensitivity to adrinamyin.Prediction and validation of target genes:The target gene of miR-1268b was detected by bioinformatics databases miRWalk(including ten database),it demonstrated that miR-1268b could be binded to the 3'-UTR of ERBB2.Furthermore,the dowmstream signalling pathway PI3K-Akt was confirmed by research and database,PI3KCA,Akt,BCL2 were found to be downstream effectors of miRNA by RT-qPCR and western blot.Conclusion:we demonstrate for the first time that miR-1268b is involved in the regulation of breast cancer chemoresistance and apoptosis by targeting ERBB2-mediated PI3K-Akt pathway...