MicroRNAs (miRNAs) participate in diverse biological functions and carcinogenesis by inhibiting specific gene expression. We previously reported that suppression of adenine nucleotide translocase 2 (ANT2) by using the short hairpin RNA (shRNA) approach has an antitumor effect in several cancer cells. We here examined the influence of ANT2 on expression of miRNAs in hepatocellular carcinoma (HCC) to further elucidate the tumor-suppressive mechanism of ANT2 shRNA. We first carried out screening for miRNAs, whose expression is regulated by ANT2 suppression in the Hep3B HCC cell line using miRNA microarrays. Validation of candidate miRNAs was done by incorporating clinical samples, and their effects on the tumorigenesis of HCC were studied in vitro and in vivo. miR-636 was one of the miRNAs whose expression was highly upregulated by ANT2 suppression in miRNA microarray analysis, as confirmed by real-time reverse transcription-polymerase chain reaction. Notably, miR-636 was markedly downregulated in HCC tissues compared with matched non-neoplastic liver in clinical samples. Restoration of miR-636 in Hep3B cells led to significant reduction of cell proliferation and colony formation. miR-636 restoration resulted in a decreased level of Ras, one of the putative targets of miR-636, and inactivation of its signaling pathway. Moreover, tumorigenesis was efficiently suppressed by miR-636 in an in vivo tumor xenograft model of HCC. The data suggest that miR-636 might function as a tumor suppressor miRNA affecting HCC tumorigenesis via downregulation of Ras, and that ANT2 suppression by shRNA could exert an anticancer effect by restoring miR-636 expression in HCC.
Adenine Nucleotide Translocator 2/*metabolism ; Animals ; Carcinoma, Hepatocellular/*genetics/pathology ; Cell Line, Tumor ; Cell Proliferation ; Cell Transformation, Neoplastic/*genetics/pathology ; Down-Regulation/*genetics ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Humans ; Liver Neoplasms/genetics/pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; MicroRNAs/*genetics/metabolism ; Phosphatidylinositol 3-Kinases/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; RNA, Small Interfering/*metabolism ; Signal Transduction/genetics ; Transcription, Genetic ; Tumor Stem Cell Assay ; Up-Regulation/genetics ; ras Proteins/*genetics/metabolism

Cancer stem cells (CSCs) are resistant to chemo- and radio-therapy, and can survive to regenerate new tumors. This is an important reason why various anti-cancer therapies often fail to completely control tumors, although they kill and eliminate the bulk of cancer cells. In this study, we determined whether or not adenine nucleotide translocator-2 (ANT2) suppression could also be effective in inducing cell death of breast cancer stem-like cells. A sub-population (SP; CD44+/CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. We utilized the adeno-ANT2 shRNA virus to inhibit ANT2 expression and then observed the treatment effect in a SP of breast cancer cell line. In this study, MCF7, MDA-MB-231 cells, and breast epithelial cells (MCF10A) mesenchymally-transdifferentiated through E-cadherin knockdown were used. ANT2 expression was high in both stem-like cells and non-stem-like cells of MCF7 and MDA-MB-231 cells, and was induced and up-regulated by mesenchymal transdifferentiation in MCF10A cells (MCF10AEMT). Knockdown of ANT2 by adeno-shRNA virus efficiently induced apoptotic cell death in the stem-like cells of MCF7 and MDA-MB-231 cells, and MCF10AEMT. Stem-like cells of MCF7 and MDA-MB-231, and MCF10AEMT cells exhibited increased drug (doxorubicin) resistance, and expressed a multi-drug resistant related molecule, ABCG2, at a high level. Adeno-ANT2 shRNA virus markedly sensitized the stem-like cells of MCF7 and MDA-MB-231, and the MCF10AEMT cells to doxorubicin, which was accompanied by down-regulation of ABCG2. Our results suggest that ANT2 suppression by adeno-shRNA virus is an effective strategy to induce cell death and increase the chemosensitivity of stem-like cells in breast cancer.
ATP-Binding Cassette Transporters/*genetics/metabolism ; Adenine Nucleotide Translocator 2/antagonists & inhibitors/genetics ; Adenoviridae/*genetics ; Antineoplastic Agents/pharmacology ; Apoptosis/drug effects/genetics ; Breast Neoplasms ; Cadherins/antagonists & inhibitors/genetics ; Cell Line, Tumor ; Cell Survival/drug effects/genetics ; Cell Transdifferentiation/drug effects ; Doxorubicin/pharmacology ; Drug Resistance, Neoplasm/drug effects/*genetics ; Epithelial-Mesenchymal Transition/drug effects ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockdown Techniques ; Humans ; Neoplasm Proteins/*genetics/metabolism ; Neoplastic Stem Cells/drug effects/*metabolism/pathology ; RNA, Small Interfering/*genetics ; Signal Transduction/drug effects