MiR-34a Suppresses Breast Cancer And Increases The Chemosensitivity By Targeting Notch1Pathway

BackgroundBreast cancer is the most common cancer among women worldwide and is alsoone of the main threats to the lives of women around the world. Despite the excitingdevelopment in early detection and systemic therapy, it remains a major cause ofcancer-related death because of metastasis, relapse and treatment resistance. Growingevidence indicated that one of the most important reasons behind this was thepresence of a small population of stem-like cells called cancer stem cells. Breastcancer stem cells are endowed with stem cell properties including the capacity of self-renewal and multi-lineage differentiation. They play important roles in tumorformation, progression, recurrence and therapy resistance in breast cancer.MicroRNAs (MiRNAs) are a class of small non-coding RNAs with18~24nt in length.They can bind imperfectly to the3’-untranslated region of target mRNA to regulate geneexpression at post-transcriptional level. MiRNAs have been implicated in a wide range ofcell functions such as cell proliferation, migration, apoptosis and differentiation.Dysregulation of miRNAs have been observed in all malignant tumors and plays animportant role in the tumor development. Recent studies suggest that miRNAs also play animportant part in proliferation and differentiation of cancer stem cells. According to theirroles in tumor, miRNAs can be divided into two groups. One group is known as oncogenicmiRNAs that is upregulated in cancer cells and contributes to carcinogenesis by inhibitingtumor suppressor genes, the other is know as tumor suppressor miRNAs which normallyprevent cancer development by inhibiting the expression of proto-oncogenes. As one of thetumor suppressor miRNAs, miR-34a plays a tumor suppressing roles in multiple cacinomassuch as colon cancer, prostate cancer and pancreatic cancer. Recent studies have found thatit exhibit the characteristics of tumor suppressor in a variety of cancer stem cells. But itsroles and mechanisms in the regulation of breast cancer and breast cancer stem cells remainto be elucidated. The Notch signaling pathway is an evolutionarily conserved pathway. It plays animportant regulatory role in embryonic development, organ maturation and tumorprogression. Notch1is one of the Notch receptors that play essential parts in breastcancer development, metastasis, drug resistance and maintainence of breast cancerstem cells. Notch1is one of the target genes of miR-34a，which suggested that miR-34a/Notch1may play important regulatory roles in breast cancer cells and breastcancer stem cells.In this study, based on clinical assays and in vitro experiments, we examined theeffects of miR-34a/Notch1on breast cancer cell proliferation, invasion, metastasis, aswell as maintainence of breast cancer stemness and drug resistance. All the resultsprovide references for clinical diagnosis and treatment of breast cancer.MethodsWe detected the expression levels of miR-34a and Notch1in in breast cancertissues and normal breast tissues using qRT-PCR and examined the Notch1proteinexpressions with immunochemistry. The relationship between miR-34a and tumormetastasis, tumor stages, Notch1expression were analyzed. Then we transfectedMCF-7cells with miR-34a mimics, Notch1siRNA and their negative controlsrespectively and examined the expression of Notch1mRNA and protein with qRT-PCR and Western blot. Cell proliferation was detected with CCK-8. The migrationand invasion abilities were measured with Transwell assays. Then we detected thepercentage of CD44+/CD24-cells with flow cytometry and examined the expressionof ALDH1with Western blot. Then we constructed pcDNA3.1-Notch1plasmid thatencoded the sequence of Notch1intracellular domain. After co-transfecting it withmiR-34a mimics, cell proliferation, migration, the percentage of CD44+/CD24-cellsand the expression of Notch1, Hes1and ALDH1were also detected. Then we treatedMCF-7cells with paclitaxel, miR-34a mimics and the combination. CCK-8assay wascarried out to detecte the cell proliferation. The percentages of CD44+/CD24-cellswere measured with flow cytometry. The expression of Notch1, Hes1, ABCG2, andALDH1were examined with Western blot. Mammosphere formation assays wereperformed to determine the self-renewal ability of breast cancer stem cells.ResultsThe average expression of miR-34a was significantly downregulated in breastcancer tissues compared to the normal breast tissues. MiR-34a expression level in patients with lymph node metastases was also lower than that in patients withoutlymph node metastases. Additionally, the miR-34a expression was reduced withprogression of clinical stage. Moreover, an inverse correlation between the expressionof miR-34a and Notch1mRNA was observed. In addition, IHC scores analysisshowed Notch1was negatively associated with miR-34a expression.MiRNA target searches confirmed that Notch1has a putative miR-34a-bindingsite within its3’-UTR. Overexpression of miR-34a exerted suppressive effects onNotch1mRNA and protein expression. Luciferase assay comfirmed that miR-34aregulated Notch1by directly interacting with the3’-UTR of the gene in MCF-7cells.Moreover, overexpression of miR-34a repressed proliferation, migration and invasionin MCF-7cells, reproducing the function of knockdown of Notch1. Restoration ofNotch1counteracts the inhibitory effects of miR-34a on cell proliferation, andmigration. After transfection with miR-34a mimics or Notch1siRNA, the percentageof CD44+/CD24cells was significantly downregulated compared with the negativecontrol. Moreover, downregulation of ALDH1in the two groups was also observed.Furthermore, the repression of ALDH1and CD44+/CD24-population by miR-34a wasrescued by ectopic expression of Notch1. Then we treated MCF-7cells with paclitaxel,miR-34a mimics and the combination. The expressions of Notch1, Hes1, ABCG2andALDH1were significantly reduced in the miR-34a overexpressing cells when treatedwith paclitaxel. The mammosphere formation ability of the group treated with thecombination was also reduced significantly compared with the group treated withmiR-34a mimics or paclitaxel alone.ConclusionsOur experimental results suggest that miR-34a may inhibit cell proliferation,migration and invasion by downregulating Notch1pathway. MiR-34a/Notch1pathway may play an important role in regulating breast cancer stemness andincreasing chemosensitivity. Our findings strongly suggest that miRNA targeting stemcell signaling pathways could provide an efficient approach for breast cancer therapy.