Exp Mol Med.  2013 Jan;45(1):e3.

ANT2 suppression by shRNA restores miR-636 expression, thereby downregulating Ras and inhibiting tumorigenesis of hepatocellular carcinoma

• Jang JY
• Lee YS
• Jeon YK
• Lee K
• Jang JJ
• Kim CW

Abstract

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.

Keyword

adenine nucleotide translocase 2; hepatocellular carcinoma; miR-636; Ras signaling pathway; RNA interference

MeSH Terms

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
Adenine Nucleotide Translocator 2
MicroRNAs
RNA, Small Interfering
Phosphatidylinositol 3-Kinases
Proto-Oncogene Proteins c-akt
ras Proteins