Abstract
BackgroundMicroRNAs (miRNAs) have been implicated in cancer initiation and progression via their ability to affect expression of genes and proteins that regulate cell proliferation and/or cell death. Transcription of the three miRNA miR-34 family members was recently found to be directly regulated by p53. Among the target proteins regulated by miR-34 are Notch pathway proteins and Bcl-2, suggesting the possibility of a role for miR-34 in the maintenance and survival of cancer stem cells.Methodology/Principal FindingsWe examined the roles of miR-34 in p53-mutant human pancreatic cancer cell lines MiaPaCa2 and BxPC3, and the potential link to pancreatic cancer stem cells. Restoration of miR-34 expression in the pancreatic cancer cells by either transfection of miR-34 mimics or infection with lentiviral miR-34-MIF downregulated Bcl-2 and Notch1/2. miR-34 restoration significantly inhibited clonogenic cell growth and invasion, induced apoptosis and G1 and G2/M arrest in cell cycle, and sensitized the cells to chemotherapy and radiation. We identified that CD44+/CD133+ MiaPaCa2 cells are enriched with tumorsphere-forming and tumor-initiating cells or cancer stem/progenitor cells with high levels of Notch/Bcl-2 and loss of miR-34. More significantly, miR-34 restoration led to an 87% reduction of the tumor-initiating cell population, accompanied by significant inhibition of tumorsphere growth in vitro and tumor formation in vivo.Conclusions/SignificanceOur results demonstrate that miR-34 may restore, at least in part, the tumor suppressing function of the p53 in p53-deficient human pancreatic cancer cells. Our data support the view that miR-34 may be involved in pancreatic cancer stem cell self-renewal, potentially via the direct modulation of downstream targets Bcl-2 and Notch, implying that miR-34 may play an important role in pancreatic cancer stem cell self-renewal and/or cell fate determination. Restoration of miR-34 may hold significant promise as a novel molecular therapy for human pancreatic cancer with loss of p53–miR34, potentially via inhibiting pancreatic cancer stem cells.
Highlights
MicroRNAs are a conserved class of non-coding 20– 22 nt small RNAs that regulate gene and protein expression by binding to mRNA leading to mRNA degradation or inhibition of translation [1,2]. miRNAs likely regulate diverse biological processes, including tissue differentiation and maintenance, and have contributing roles in varied disease processes, including cancer [2,3,4]
We examined a series of human pancreatic cancer cell lines, MiaPaCa2, BxPC3, Capan1, Capan2, Panc-1, and the normal human lung fibroblast cell line WI-38, for miR-34a,b,c expression
Our results show that miR-34 restoration in human pancreatic cancer MiaPaCa2 and BxPC3 cells inhibited the expression of target genes, Bcl-2, Notch1 and Notch2; significantly inhibited clonogenic cell growth and invasion; induced apoptosis and G1 and G2/M arrest; and sensitized the cells to chemotherapy and radiation
Summary
MicroRNAs (miRNAs) are a conserved class of non-coding 20– 22 nt small RNAs that regulate gene and protein expression by binding to mRNA leading to mRNA degradation or inhibition of translation [1,2]. miRNAs likely regulate diverse biological processes, including tissue differentiation and maintenance, and have contributing roles in varied disease processes, including cancer [2,3,4]. The three members of the miR-34 family were found to be directly regulated by p53 and the functional activity of miR-34 indicated a potential role as a tumor suppressor [6,7,8,9,10,11,12]. We previously reported that the Bcl-2 protein is regulated directly by miR-34 [10] The report from He et al indicated that ectopic expression of miR-34 induces cell cycle arrest in both primary and tumor-derived cell lines, which is consistent with the ability of miR-34 to downregulate a program of genes promoting cell cycle progression [11]. Among the target proteins regulated by miR-34 are Notch pathway proteins and Bcl-2, suggesting the possibility of a role for miR-34 in the maintenance and survival of cancer stem cells
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