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


CONCLUSIONS
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.
Ji
Qing
Q
State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, PR China. qingj@umich.edu
Hao
Xinbao
X
Meng
Yang
Y
Zhang
Min
M
Desano
Jeffrey
J
Fan
Daiming
D
Xu
Liang
L
D000970:Antineoplastic Agents / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D016193:G1 Phase / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D016147:Genes, Tumor Suppressor / Q000502:physiology; D019254:Genes, bcl-2; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000502:physiology*; D018874:Spheroids, Cellular / Q000378:metabolism / Q000473:pathology*; D013274:Stomach Neoplasms / Q000235:genetics / Q000473:pathology*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism
D000970:Antineoplastic Agents; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D016159:Tumor Suppressor Protein p53; D053148:Caspase 3