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. 2008 Sep 21:8:266.
doi: 10.1186/1471-2407-8-266.

Restoration of tumor suppressor miR-34 inhibits human p53-mutant gastric cancer tumorspheres

Qing Ji  1 , Xinbao Hao, Yang Meng, Min Zhang, Jeffrey Desano, Daiming Fan, Liang Xu
Affiliations

Restoration of tumor suppressor miR-34 inhibits human p53-mutant gastric cancer tumorspheres

Qing Ji et al. BMC Cancer. .

Abstract

Background: MicroRNAs (miRNAs), some of which function as oncogenes or tumor suppressor genes, are involved in carcinogenesis via regulating cell proliferation and/or cell death. MicroRNA miR-34 was recently found to be a direct target of p53, functioning downstream of the p53 pathway as a tumor suppressor. miR-34 targets Notch, HMGA2, and Bcl-2, genes involved in the self-renewal and survival of cancer stem cells. The role of miR-34 in gastric cancer has not been reported previously. In this study, we examined the effects of miR-34 restoration on p53-mutant human gastric cancer cells and potential target gene expression.

Methods: Human gastric cancer cells were transfected with miR-34 mimics or infected with the lentiviral miR-34-MIF expression system, and validated by miR-34 reporter assay using Bcl-2 3'UTR reporter. Potential target gene expression was assessed by Western blot for proteins, and by quantitative real-time RT-PCR for mRNAs. The effects of miR-34 restoration were assessed by cell growth assay, cell cycle analysis, caspase-3 activation, and cytotoxicity assay, as well as by tumorsphere formation and growth.

Results: Human gastric cancer Kato III cells with miR-34 restoration reduced the expression of target genes Bcl-2, Notch, and HMGA2. Bcl-2 3'UTR reporter assay showed that the transfected miR-34s were functional and confirmed that Bcl-2 is a direct target of miR-34. Restoration of miR-34 chemosensitized Kato III cells with a high level of Bcl-2, but not MKN-45 cells with a low level of Bcl-2. miR-34 impaired cell growth, accumulated the cells in G1 phase, increased caspase-3 activation, and, more significantly, inhibited tumorsphere formation and growth.

Conclusion: Our results demonstrate that in p53-deficient human gastric cancer cells, restoration of functional miR-34 inhibits cell growth and induces chemosensitization and apoptosis, indicating that miR-34 may restore p53 function. Restoration of miR-34 inhibits tumorsphere formation and growth, which is reported to be correlated to the self-renewal of cancer stem cells. The mechanism of miR-34-mediated suppression of self-renewal appears to be related to the direct modulation of downstream targets Bcl-2, Notch, and HMGA2, indicating that miR-34 may be involved in gastric cancer stem cell self-renewal/differentiation decision-making. Our study suggests that restoration of the tumor suppressor miR-34 may provide a novel molecular therapy for p53-mutant gastric cancer.

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Figures

Figure 1
Figure 1
Western blot analysis of the expression of Bcl-2 family proteins in human gastric cancer cell lines.
Figure 2
Figure 2
qRT-PCR analysis of the relative expression levels of miR-34s and target genes in human gastric cancer cell lines. The cells were lyzed to extract total RNA for qRT-PCR. Data were normalized to that of Actin and the relative levels are shown (Actin = 1000).
Figure 3
Figure 3
Restoration of miR-34 by miR-34 mimic transfection downregulates target gene Bcl-2 expression. Western blot analysis of the potential miR-34 target protein Bcl-2 48 hours after miR-34 mimic transfection of Kato III cells (100 pmol per well in 6-well plates).
Figure 4
Figure 4
Quantitative real-time PCR shows that restoration of miR-34 by miR-34 mimic transfection downregulates target gene expression. 24 hours after miR-34 mimic transfection of Kato III cells (100 pmol per well in 6-well plates), potential target gene mRNA levels were measured by qRT-PCR with SYBR Green PCR system (TaqMan). The comparative threshold cycle CT method was used to calculate relative gene expression levels compared with actin, then normalized with the value from NC mimic as 1.
Figure 5
Figure 5
Bcl-2 3'UTR Luciferase Reporter Assay shows that the miR-34 mimics transfected are functional. KATO3 cells were transfected in all wells of 6-well plates with 2 ug of Bcl-2 3'UTR luciferase reporter plasmid or its mutant, and 2 ug of the control β-galactosidase plasmid per well. Cells in each well were also co-transfected with 100 pmol of each miR-34 mimic or NC mimic as indicated, using Lipofectamine 2000. Luciferase activity was normalized relative to β-gal activity. Data are presented as mean ± s.e. **P < 0.01, ***P < 0.01, versus that of negative control miRNA mimic (NC mimic), two-tailed t-test, n = 3.
Figure 6
Figure 6
Restoration of miR-34 in Kato III cells resulted in G1 block and caspase-3 activation. A. Cell cycle analysis of Kato III cells after miR-34 restoration. Cell cycle analysis was performed 24 hours after transfected with miR-34 mimics or negative control mimic (NC mimic). Cells were stained with propidium iodide after ethanol fixation and analyzed by flow cytometry. **P < 0.01, ***P < 0.001, versus that of NC mimic, one-way ANOVA, n = 2. B. Caspase-3 activation in Kato III cells after miR-34 restoration. 24 hours after transfection, cells were lysed for measurement with the Caspase-3 activation assay kit (BioVision). Relative increase of fluorescence signal was calculated by dividing the normalized signal in each treated sample with that of NC mimic as 100. **P < 0.01 versus that NC mimic, two-tailed t-test, n = 3.
Figure 7
Figure 7
Restoration of miR-34 chemosensitizes Kato III cells with high Bcl-2, but not MKN-45 cells with low Bcl-2. MTT-based cytotoxicity assay of Kato III (A) and MKN-45 (B) cells transfected with miR-34a mimic or control mimic. The cells were transfected with miR-34a or NC mimics by Lipofectamine 2000. 24 hr later, the cells were trypsinized and plated in 96-well plates and treated with the indicated chemotherapeutic agents in triplicate. The MTT-based WST-1 assay was carried out on Day 4. Restoration of miR-34a in Kato III cells with high Bcl-2 rendered the cells 2-3-fold more sensitive to chemotherapies, but had no effect on MKN-45 cells with low Bcl-2. 34a, miR-34a mimic; NC, non-specific control miRNA mimic.
Figure 8
Figure 8
Restoration of miR-34 in Kato III cells delays cell growth. Kato III cells were infected with feline immunodeficiency virus (FIV) lentiviral system expressing miR-34a (miR-34a-MIF) or vector control (MIF), and stable cells were obtained by antibiotic selection (Zeocin 50 μg/mL) and validated for miR-34a expression. The stable cells were plated in a 24-well plate with equal cell density. Cells in triplicate were collected by trypsinization and viable cells were counted by Trypan Blue exclusion at 24-h intervals over 4 days, using a Coulter cell counter (Beckman). ***P < 0.001 versus MIF vector control, two-way ANOVA, n = 3.
Figure 9
Figure 9
Restoration of miR-34 by MIF lentiviral system inhibits Kato III tumorspheres. Kato III cells infected by miR-34a MIF or control vector MIF were plated for tumorsphere formation as described in Materials and Methods. 7–10 days later, tumorspheres were observed under microscope (A) and quantified (B). C. Quantification of cell numbers per tumorsphere. Tumorspheres were collected and filtered through a 40 um sieve (BD Biosciences), and dissociated with trypsin for single cell suspension. Cells were counted with trypan blue exclusion and the data are presented as number of cells per tumorsphere. **P < 0.01, Student's t-test, n = 3.
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References

    1. Hatfield S, Ruohola-Baker H. microRNA and stem cell function. Cell Tissue Res. 2008;331:57–66. - PMC - PubMed
    1. Corsten MF, Miranda R, Kasmieh R, Krichevsky AM, Weissleder R, Shah K. MicroRNA-21 knockdown disrupts glioma growth in vivo and displays synergistic cytotoxicity with neural precursor cell delivered S-TRAIL in human gliomas. Cancer Res. 2007;67:8994–9000. - PubMed
    1. Yu F, Yao H, Zhu P, Zhang X, Pan Q, Gong C, Huang Y, Hu X, Su F, Lieberman J, et al. let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell. 2007;131:1109–1123. - PubMed
    1. Tarasov V, Jung P, Verdoodt B, Lodygin D, Epanchintsev A, Menssen A, Meister G, Hermeking H. Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest. Cell Cycle. 2007;6:1586–1593. - PubMed
    1. Cho WC. OncomiRs: the discovery and progress of microRNAs in cancers. Mol Cancer. 2007;6:60. - PMC - PubMed

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