MicroRNA-34a Decreases Breast Cancer Stem Cells By Modulating SIRT1Activity

Breast cancer is the most common malignancy leading in both incidence andmortality in females worldwide. Even treated in the early-stage, approximately30%patients will eventually develop relapse and metastasis. Patients with metastasis atdiagnosis, conventional treatment is initially effective in disease control, but mostpatients will eventually relapse over time. In order to reduce mortality, developing newtherapeutic strategies, which require fully understanding the breast cancer cellsmolecular biology. Recent study have found the existence of cancer stem cells(CSCs),CSC are a relatively rare population of cancer cells with stem cell properties ofself-renewal, and differentiate potentials, and play a vital role in the initiation,angiogenesis, invasion, and metastasis of tumors. So far, it has been found the existenceof tumor stem cells in breast cancer, leukemia, glioma, prostate cancer, pancreaticcancer, lung cancer, colorectal cancer, stomach cancer, and liver cancer et al. In2003,Al-Hajj et al first isolated breast cancer stem cells in breast cancer specimens. Wheninjected into the fat pad of NOD/SCID mice, they can form tumors using as few ashundreds of Lin-CD44+CD24-/lowESA+cells. The existence of breast cancer stem cellsenhances proliferation, invasion, and metastasis of breast cancer. Meanwhile, breastcancer stem cells are currently considered to be responsible for treatment failurebecause of their resistance to conventional chemotherapy treatments. Therefore, more attentions should be paid to new therapeutic approaches to specifically eliminate CSCsMicroRNAs (miRNAs) are21~23nucleotides small non-coding RNAs which playpivotal roles in a number of biological processes. MiRNAs act as regulators of geneexpression by binding to the3’-untranslated region (3’-UTR) of target messenger RNAs(mRNAs) via inhibiting protein expression or inducing mRNA degradation.Furthermore, it has become apparent that miRNAs are important factors in cancer, andfunction as either oncogenes or tumor suppressors. Low levels of miRNA expressionwere observed in a variety of tumor tissues. A number of miRNAs such as miR-34,miR-125, miR-200, miR-205, miR-328, and miR-30are down regulated and act astumor suppressors in breast cancer. The miR-34family, including miR-34a, miR-34b/c,is important component of the p53tumor suppressor protein transcriptional network.Transactivation of miR-34a by p53induces apoptosis and cell cycle arrest.Over-expression of miR-34a suppresses breast cancer proliferation, invasion, andmigration. Recent studies showed that miR-34a play a vital role in inhibiting CSCs ofprostate, pancreatic, and gastric cancers.SIRT1has been verified to be a direct target of miR-34a. SIRT1is homologous ofyeast sirtuin2(silent information regulator2, SIR2) protein, an NAD+-dependenthistone deacetylase that deacetylates histones and non-histones, such as the tumorsuppressor gene p53. SIRT1regulates multiple signaling pathways by adjusting theacetylation status of target genes. The functions of SIRT1are involved in metabolism,aging, and cancer. SIRT1is an apoptosis inhibitory factor. SIRT1regulates p53activityby deacetylating its382lysine residues, and inhibits activation of downstream targetgenes, inducing p53-mediated apoptosis.In this present study, we first isolated the CD44+/CD24-breast cancer stem cellpopulations in MCF-7cell line. Then we detected the expression levels of miR-34a andSIRT1in BCSCs by qRT-PCR and western blotting. Second, we modulate theexpression of miR-34a in MCF-7cell line by using chemical synthetic mimics orinhibitors, and lentivirus vectors. Down-regulation of SIRT1by using shRNA-SIRT1expression vector acted as a positive control of over-expression of miR-34a. We investigated the functions of miR-34a on proliferation, invasion, metastasis, andapoptosis, as well as possible mechanisms for breast cancer stem cells inhibition inMCF-7cell line. In vivo, miR-34a over-expressed cells and shRNA-SIRT1stabletransfected cells were inoculated into nude mice, to investigate the miR-34a inhibitioneffects on breast cancer stem cells.Part I The expression levels of miR-34a and its target gene SIRT1in breast cancerstem cells.Objective: To investigate the expression levels of miR-34a and its target gene SIRT1in breast cancer stem cells.Methods: Flow cytometry and immunomagnetic beads were used to isolateCD44+/CD24-breast cancer stem cells from serum-free suspension cultured breastcancer cells. The quantitative Real Time-PCR was used to detect the expression ofmiR-34a. The quantitative Real Time-PCR and western blotting were used to detect theexpression of SIRT1.Results: The proportions of CD44+/CD24-cells separated by FACS and MACS were9.90±3.54%and7.20±4.06%, respectively. The cells separated both by FACS andby MACS have high mammosphere formation capacity. The expression levels ofmiR-34a was significantly decreased in breast cancer stem cells, compared tonon-breast cancer stem cells and un-separated MCF-7cells (p<0.05). The mRNA andprotein levels of SIRT1were significantly elevated in breast cancer stem cells thannon-breast cancer stem cells and un-separated MCF-7cells (p<0.01).Conclusion: Both FACS and MACS can successfully isolated breast cancer stem cells;each method has its advantages. The expression of miR-34a is decreased in breastcancer stem cells, and its target gene SIRT1is over-expressed. miR-34a and SIRT1may play a role in self-renewal and maintenance of breast cancer stem cells. Part II miR-34a target SIRT1and regulates its expressionObjective: To establish miR-34a over-expression or de-expression cell model, andconstruct a shRNA-SIRT1stable tranfected cell model. Methods: Modulation miR-34a expression by chemical synthesis mimics or inhibitorsin MCF-7breast cancer cells. The expression levels of miR-34a were validated byqRT-PCR analysis. lentivirus vectors with puromycin resistance were used to selectstable miR-34a over-expression cells. ShRNA-SIRT1expression vectors weredesigned to interfering SIRT1expression in MCF-7breast cancer cells. QRT-PCR andwestern blotting analysis were used to evaluate mRNA and protein expression levels ofSIRT1.Results: The expression levels of miR-34a were significantly increased more than20folds by transfecting miR-34a mimics (p<0.01). On the contrary, cells transfected withmiR-34a inhibitors, the expression level of miR-34a was0.45±0.21fold compared toinhibitors NC group,0.51±0.31fold compared to Control group (p<0.05). TheMCF-7cells infected with lentivirus-miR-34a showed a significantly up-regulatedmiR-34a expression compared to Control groups (p<0.01). Both mRNA and proteinlevels of SIRT1were significantly down-regulated in ShRNA-SIRT1-3andShRNA-SIRT1-4tranfected cells (p<0.05). The SIRT1mRNA levels were notinfluenced by modulating miR-34a expression levels. miR-34a mimics couldeffectively inhibit the protein expression of SIRT1(p<0.01). SIRT1protein expressionshowed a significant elevation in miR-34a inhibitors transfect (p<0.05).Conclusion: The expression levels of miR-34a could effectively modulate by miR-34amimics or inhibitors, and effectively altering SIRT1protein expression levels.Lentivirus vector can effectively regulate the expression of miR-34a.pGPH1/GFP/Neo-shRNA-SIRT1expression vectors were successfully constructed,shRNA-SIRT1-3and4showed better interference effects on MCF-7cells. Part III The inhibition effects of miR-34a on MCF-7breast cancer celltransplanted to nude miceObjective: To investigate the inhibition effects of over-expression of miR-34a inxenografts, and discuss the possible mechanisms of breast cancer stem cells inhibitionin vivo. Methods: miR-34a and SIRT1stable infected or transfected cell lines were inoculatedinto nude mice, and monitored xenografts growth every3days. Immunohistochemicalstaining, qRT-PCR and western blotting analysis were used to evaluate the expressionof related proteins.Results: The tumor volumes in miR-34a over-expression or SIRT1depression groupwere small, grow slowly. The expression levels of SIRT1and ALDH1were reduced inthe miR-34a over-expression or SIRT1depression groups related to the control groups.Conclusion: Over-expression of miR-34a can inhibit tumor growth in nude mice, andthe inhibition effects may because of miR-34a inhibits breast cancer stem cells bydepressing SIRT1protein. Part IV The mechanisms of miRNA-34a inhibits breast cancer stem cellsObjective: To investigate the effects of miR-34a on proliferation, invasion, metastasis,and apoptosis in MCf-7cells, as well as the possible mechanism of breast cancer stemcells inhibition.Methods: CCK8assay and colony formation assay were analyzed for MCF-7cellproliferation. Cell migration and invasion assays were used to evaluate the invasionand metastasis ability of miR-34a over-expressed MCF-7cells. The proportion ofCD44+/CD24-breast cancer stem cells was detected by flow cytometry. Mammosphereformation assay was used to detect the self-renewal ability of breast cancer stem cell.Hochest33342and AV/PI staining were used to detect apoptosis in MCF-7cells.Western blotting and immunofluorescence were used to detect the protein expression.Results: Over-expression of miR-34a inhibits proliferation, invasion, and metastasis ofMCF-7cells; Deexpression of miR-34a can promote MCF-cell proliferation, invasion,and metastasis. The proportions of breast cancer stem cells were declined in miR-34amimics and shRNA-SIRT1transfected cells. The numbers of mammsphere werereduced by48.93±7.48%and57.78±6.59%in miR-34a mimics tranfected cells andshRNA-SIRT1transfected cells, respectively. The protein expression levels of SIRT1,ALDH1, Nanog, and BMI1were decreased in miR-34a group and shRNA-SIRT1 group cells. The protein expressions of Nanog were detected by immunofluorescence,the fluorescence in miR-34a group and shRNA-SIRT1group were darker than thecontrol groups. The proportions of early apoptotic cells in miR-34a group, miR-NCgroup, shRNA-SIRT1group, and shRNA-NC group were9.24±2.34%,1.32±1.73%,5.04±1.69%, and2.22±2.06%, respectively. The statistical analysis showed thatthere were significantly difference between miR-34a group and miR-NC groups, alsobetween shRNA-SIRT1group and shRNA-NC group (p <0.05). miR-34a can promoteapoptosis in MCF-7cell.Conclusions: Over-expression of miR-34a inhibits MCF-7cell proliferation, invasionand metastasis. miR-34a inhibits breast cancer stem cell self-renewal by targetingSIRT1, which may be related to p53-dependent Nanog depression. Meanwhile,miR-34a inhibit breast cancer stem cells, in partly, by promoting apoptosis.