MicroRNA-101 Inhibits Growth,Proliferation And Migration And Induces Apoptosis Of Breast Cancer Cells By Targeting Sex-determining Region Y-box 2

Background and objectiveWorldwide,breast cancer(BC)is the most frequently diagnosed tumor in women,for whom it is the second leading cause of malignancy-related deaths.Although BC treatments are varied and developed,the mortality rate still ranges from 20%to 40%in patients treated according to the National Comprehensive Cancer Network guidelines,due to local recurrence and distant metastasis.Therefore,the development of breast cancer has been the attention focus of clinicians and scientists all over the world.Previous studies on the pathogenesis of breast cancer have focused on the mutation,amplification,rearrangement and deletion of protein coding genes and the activation of related signal pathways.Moreover the molecular targeted drugs used in clinical for breast cancer are mainly designed for the change of protein coding genes.However,With the deepening of research,it is recognized that a non-coding RNA gene(miRNAs)can also affect the development of malignant tumors.MicroRNAs(miRNAs)are a class of small(19-24 bp)non-coding single stranded RNA molecules encoded by endogenous genes whose expression profiles change in various tumors.They regulate the expressions of downstream genes by binding to the 3'-UTR of the target gene mRNA,affecting the biological function of the tumor.The aim of this study was to explore the role of a miRNA-target gene axis in breast cancer and to examine its role in the development and progression of breast cancer.Materials and Methods1?BC and paired adjacent tissue samples were obtained from 74 patients who underwent modified radical mastectomy at Jinling Hospital from 2013 to 2016.Real-time quantitative PCRi immunohistochemisty and Western blot were performed to detect the expression differences of miR-101 and SOX2 between BC tissues and paired adjacent tissue samples;2?74 cases of breast cancer were divided into high and low(positive and negative)groups according to the expression level of miR-101 or SOX2.The clinical pathological features of these patients were statistically analy-zed.3?In vitro,RT-PCR was used to detect the expression of miR-101 in breast cancer cell lines and normal breast cell lines,and appropriate were selected to perform functional experiments;qRT-PCR was performed to validate the overexpression efficiency.MTT assay,clone formation assay,Transwell assay,scratch test and flow cytometry apoptosis assay were performed to evaluate the changes of breast cancer cell proliferation,colony formation,migration,invasion and cell apoptosis after upregulating miR-101;4?Xenograft tumorigenicity assays were performed to demonstrate the effect of miR-101 on tumorigenesis and apoptosis in vivo.After stable transfection of miR-101 mimics,BC cells were subcutaneously injected into the right hind limb of nude mice.Tumor volumes were measured and recorded every 4 d.After 32 d,pictures of mice with subcutaneous tumors were taken,and then tumors were removed for immunohistochemistry and TUNEL.5?To confirm the presence of miR-101 binding sites in the SOX2 3'-UTR,SOX2-wild-type 3'-UTR(wt-SOX2)and SOX2-mutant 3'-UTR(mut-SOX2)Luciferase reporter vectors were constructed.Then,Luciferase reporter assays were performed.;6?RT-PCR and western blot were used to detect the expression of SOX2 in breast cancer cell lines and normal breast cell lines,and appropriate were selected to perform functional experiments;qRT-PCR was performed to validate the interfere efficiency.MTT assay,clone formation assay,Transwell assay,scratch test and flow cytometry apoptosis assay were performed to evaluate the changes of breast cancer cell proliferation,colony formation,migration,invasion and cell apoptosis after downregulating SOX2;7?Three groups of control experiments were carried out(NC,miR-101,miR-101+NC,miR-101+SOX2)in breast cancer cells.MTT assay,clone formation assay,Transwell assay,scratch test and flow cytometry apoptosis assay were performed to evaluate the differences of breast cancer cell proliferation,colony formation,migration,invasion and cell apoptosis among the three groupsResults1?miR-101 was significantly dowiregulated in 64(86.5%)BC tumor tissues compared with the paired tumor-adjacent tissues(P<0.05);2?SOX2 was significantly upregulated in BC tumor tissues compared with the paired tumor-adjacent tissues;and the expression of miR-101 and SOX2 were negative correlated in BC tissues;3?Patients with low expressions or high expressions of SOX2 had higher lymph node metastasis rate,pathological grade and TMN stage.The expression of miR-101 and SOX2 was not correlated with age,T stage,ER status,HER2 status and molecular subtype;4?Compared with normal mammary epithelial cells(MCF-10A),the expression of miR-101 in MCF-7,MDA-MB-231 and SK-BR-3 was downregulated,but there was no significant difference in the expression of HBL-100;5?We performed an MTT assay and observed that miR-101 overexpression significantly inhibited BC cell proliferation.Similarly,we also found that miR-101 upregulation decreased colony formation compared with the control group.The wound healing rate of cells overexpressing miR-101 was significantly slower than the control group.Likewise,miR-101 upregulation strikingly inhibited the invasion ability of BC cells through Matrigel.Furthermore,to investigate the influence of miR-101 on apoptosis,we performed an apoptosis assay by co-staining with Annexin V and PI and found that ectopic miR-101 expression dramatically promoted apoptosis compared with the miR-NC group;In vivo experiments we found that over expression of miR-101 could effectively inhibit the tumor formation of breast cancer cell lines in nude mice,and promote cell apoptosis;6?By using starBase v2.0(http://http://starbase.sysu.edu.cn/mirMrna.php),we found that SOX2,which had a putative miR-101 binding site,may be a candidate target of miR-101.The Luciferase activity of cells transfected with miR-101 mimics and the wt-SOX2 reporter was notably lower than the control group,whose Luciferase expression was released by the mutant putative binding site.By using western blot we found miR-101 overexpression significantly decreased SOX2 expression in BC cells;7?Compared with normal mammary epithelial cells(MCF-10A),the expression of SOX2 in MCF-7,MDA-MB-231 and SK-BR-3 was unregulated,but there was no significant difference in the expression of HBL-100;8?We performed an MTT assay and observed that SOX2 suppression significantly inhibited BC cell proliferation.Similarly,we also found that SOX2 suppression decreased colony formation compared with the control group.The wound healing rate of cells downregulating SOX2 was significantly slower than the control group.Likewise,SOX2 suppression strikingly inhibited the invasion ability of BC cells through Matrigel.Furthermore,to investigate the influence of miR-101 on apoptosis,we performed an apoptosis assay by co-staining with Annexin V and PI and found that ectopic SOX2 expression dramatically promoted apoptosis compared with the miR-NC group;9?To demonstrate the potential molecular mechanisms related to the miR-101-SOX2 regulation of BC cell growth,proliferation,migration and apoptosis,we performed MTT,colony formation,transwell misrration,wound healing and apoptosis assays in cells transfected with miR-NC,miR-101,miR-101?NC or miR-101+SOX2.The results of these assays all indicated that SOX2 overexpression could reverse the effect of miR-101 overexpression.We also detected expression of the apoptosis-related proteins Bcl2,Bax and cleaved caspase3 by western blotting.The results showed that both miR-101 upregulation and SOX2 knockdown significantly reduced Bcl2 expression,while increasing Bax and cleaved caspase3 levels.Moreover,SOX2 overexpression mitigated these changes in Bcl2,Bax and cleaved caspase 3 levels that were induced by miR-101 upregulation.Additionally,SOX2 overexpression mitigated the expression changes of EMT related proteins.Conclusion1?The expression of miR-101 in breast cancer tissues and breast cancer cell lines were downregulated;The expression of SOX2 in breast cancer tissues and breast cancer cell lines were downregulated;Patients with low expression of miR-101 or high expression of SOX2 had higher lymph node metastasis rate,pathological grade and TMN stage;2?miR-101 overexpression suppresses BC growth and proliferation and enhances apoptosis in vivo and vitro;3?SOX2 is a direct target of miR-101 in BC and can be regulated by miR-101;4?Silencing SOX2 significantly inhibits BC cell growth,proliferation and migration,and enhance apoptosis in vivo;5?SOX2 overexpression mitigates the miR-101-induced inhibition of malignant BC phenotypes.