| Backgroud:Breast cancer(BC)is one of the most common malignant tumors worldwide,with about 350,000 individuals dying from BC each year,and posese a serious threat to women’s lives and health.A wide array of factors are potentially associated with BC,including age,topical steroid,being overweight,drinking alcohol,and smoking.Although novel strategies for the diagnosis and treatment of BC in recent decades has developed by leaps and bounds,since it is characterized by high incidence and mortality,BC is still a major life-threat for those women over 40 years old.Therefore,people pay more attention to the molecular mechanisms of breast cancer,attempting to investigate the mechanisms underlying the pathogenesis and development of BC.MicroRNAs(miRNAs)are a class of endogenous non-coding RNAs encompassing about only 18-25 nucleotides.In a majority of organisms,miRNAs are encoded by 1-3%genomic genes.While miRNAs cannot directly encode protein products,they are universally implicated in modulating the expression levels of target genes at post-translated level.It is well known that several miRNAs can also regulate the mRNA expression level of target genes and are involved in diverse cellular physical processes,such as cell proliferation and differentiation.Previous studies have suggested that miRNAs may serve as effective therapeutic targets in cancer treatment.MiR-34 is a large family of tumor suppressor miRNAs,serving as a cancer treatment drug in clinical trials,due to its important role in the mediation of cell cycle arrest and cell survival.MiR-34 exists in a wide array of species,and miR-34a family members are evolutionarily conserved.MiR-34 has been shown to play a key role in cell fate determination by targeting the cell cycle arrest and apoptosis-related genes,as its transcriptional level is up-regulated by p53 in mammalian when DNA damage occurs.Inactivated p53 mutations or viral p53 inhibitors can suppress the expression of miR-34,beyond that,miR-34-encoding gene itself can acquire gene mutations or be inactivated on the epigenetic level when cancer occurs.Thus,these all together indicated that the decreased expression or epigenetic inactivation of miR-34a is closely related to cancer development.Among them,microRNA-34a(miR-34a)is one of the most in-depth studied anti-cancer genes,located on the chromosome 1p36 locus.In addition to anti-cancer,miR-34a can also induce cell cycle arrest,apoptosis,senescence,as well as inhibit epithelial mesenchymal transdifferentiation(EMT),cancer stem cell proliferation,etc.More and more evidence indicated that miR-34a could target many genes in breast cancer patients,including Fra-1,LMTK3,Bcl-2,Notch.Therefore,miR-34a is a potential anti-oncogene that is associated with the onset and progression of breast cancer,but the exact mechanism has been far from elucidated.Herein,our research aims to explore this mechanism.Sectionl.miR-34a expression in BC tumors and cell linesObjectives:In this section,we aim to address the following questions:whether the expression of miR-34a is inhibited in breast cancer tissues of cell lines?Materials and Methods:We collected breast cancer and adjacent non-tumor tissue samples from a total of 123 patients with breast cancer who underwent an operation in Qilu Hospital affiliated to Shandong University were recruited during March 2014 to August 2015.In addition,human breast cancer cell lines MCF-7,MDA-MB-231,MDA-MB-435,human embryonic kidney cell line HEK293T,and normal human mammary epithelial cell line MCF-10A were also collected,before miR-34a expression levels in different tissues and cell lines were evaluated by RT-PCR and in situ hybridization.Results:In situ hybridization and RT-PCR revealed that miR-34a was significantly deregulated in BC tissues compared to normal tissues(P<0.05).Similarly,the expression level of miR-34a in BC cell lines MCF-7,MDA-MB-231,and MDA-MB-435 was obviously suppressed in comparison to that in normal mammary epithelial cell line MCF-10A(P<0.05).Conclusion:These results indicated that miR-34a was obviously suppressed in BC tissues and cell lines,suggesting the potential role of miR-34a in the development of BC.Section 2.Wntl acts as direct targent of miR-34a via Wnt/β-catenin pathway in BCObjectives:To explore what is the downstream target of miR-34a in BC.Materials and Methods:Subsequently,we used bioinformatics analysis to predicte the downstream target molecule of miR-34a,and the result indicated that Wntl is a direct target of miR-34a,which is further verified by our dual-luciferase reporter assay.The full-length 3’-UTR sequence of wild-type Wntl was fused in a pMIR-REPORT vector encoding luciferase gene,and then co-transfected into human embryonic kidney 293T cells with miR-34a gene,to study whether the luciferase activity could be enhanced upon miR-34a stimulation.The relative luciferase activity was evaluated 72 hours after cotransfection,and Renilla luciferase activity was used as an internal reference.Results:The luciferase activity drastically reduced when wild-type Wntl was co-transfected with miR-34a(P<0.05),whereas mutant Wntl with miR-34a could not result in the luciferase activity decreases.These results indicated that the Wntl is a direct downstream target of miR-34a in breast cancer cells.After miR-34a mimic transfection,Wntl expression in MCF-7 was significantly reduced(P<0.05),whereas the inhibition of miR-34a contributed to a significant increase in Wntl expression level(P<0.05).Likewise,β-catenin and cyclin-D1 expression in MCF-7 cell line transfected with miR-34a mimic was also significantly reduced(P<0.05),while inhibiting the expression of miR-34a led to an obvious increase in the expression of β-catenin and cyclin-Dl(P<0.05).Conclusion:Thus,miR-34a led to reduced expression of Wntl.These all suggested that miR-34a overexpression contributed to the inactivation of Wnt/β-catenin pathway.Section 3.miR-34a suppresses BC development through targeting Wnt1 and Wnt/β-catenin pathwayObjectives:To determine what is the mechanism underlying miR-34a affects breast cancer development through Wnt/β-catenin pathway?Materials and Methods:When transfected with miR-34a mimics and inhibitor,respectively,Wntl and other Wnt/β-catenin pathway-related genes were assessed in the breast cancer cell line MCF-7 by RT-PCR and western blot assays.Furthermore,after transfection,the proliferation,invasion and migration capacities of MCF-7 cells were evaluated with MTT,would healing,and transwell assays.As to in vivo study,mouse breast cancer xenograft model was establied to assess the impact of miR-34a on the growth of breast tumors.Tumor size and weight were measured before and after injection of miR-34a mimic and inhibitor,respectively.Additionaly,Wntl,β-catenin and cyclin-D1 expression was respectively measured by RT-PCR and western blot assays,to determine whether miR-34a affects tumor growth status and the activation of Wnt/β-catenin pathway.Results:From the would healing and Transwell assays,we found that compared with the control group,miR-34a overexpression resulted in a decrease in breast cancer cell migration and invasion(P<0.05),whereas cell migration and invasion were greatly improved when cells were transfected with miR-34a inhibitor.Likewise,co-transfection of miR-34a mimic and WntlsiRNA inhibited cell ability to migration and invasion.Tumors of the mice treated with miR-34a inhibitor exhibited a larger appearance(P<0.05),with tumor size reaching 834.3 ± 159.6 cubic mm and weight 891.8 ± 115.2 mg.In addition,miR-34a mimic transfection inhibited tumor growth.Injection of miR-34a inhibitor contributed to an obvious increase in Wntl,(3-catenin,cyclin-D1 expression(P<0.05),whereas the expression levels were suppressed by injection of miR-34a mimic(P<0.05).Therefore,in vivo experiment showed that miR-34a inhibited the growth of breast cancer by interacting with Wnt/p-catenin signaling pathway.Conclusion:All these data indicated that MiR-34a can inhibit the growth,migration and invasion of breast cancer,therefore it may act as a potential hallmark in the BC diagnosis and a drug target for BC therapy.On the other hand,by virtue of targeting Wntl,P-catenin,and cyclin-D1 proteins miR-34a inactivates Wnt/beta-catenin pathway,thereby inhibiting breast cancer development.Our results provide an important theoretical basis for the molecular mechanism underlying miR-34a repressing the onset and progression of BC. |
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