ABSTRACT
MicroRNA, an endogenous noncoding RNA modulating gene expression, is a key molecule that by its dysregulation plays roles in inflammatory-driven carcinogenesis. This study aimed to investigate the role of oncomiR miR-21 and its target, the programmed cell death 4 (PDCD4) in tumor growth and metastasis of the liver fluke Opisthorchis viverrini-associated cholangiocarcinoma (CCA). The expression levels of miR-21 and PDCD4 were analyzed using the TaqMan miRNA expression assay and immunohistochemistry in liver tissues of both O. viverrini plus N-nitrosodimethylamine (NDMA)-treated hamsters and human CCA samples (n=23 cases). The functional assay for miR-21 was performed in CCA cell lines by the anti-miR-21 and pre-miR-21 transfection procedures. The peak of miR-21 levels were reached at 2 (hyperplastic lesions) and 6 (CCA) months of the O. viverrini plus NDMA-induced group and had a reverse response with its target PDCD4 proteins. In human CCA, miR-21 was overexpressed in tumor tissues when compared with nontumor tissues (P=0.0034) and had a negative correlation with PDCD4 protein (P=0.026). It was also found that high expression of miR-21 was significantly correlated with shorter survival (P<0.05) and lymph node metastasis (P=0.037) of CCA patients. Transient transfection of pre-miR-21 reduced the PDCD4 level and resulted in an increase of M213 CCA cell growth and wound-induced migration ability. These results indicated that miR-21 plays a role in the carcinogenesis and metastasis of O. viverrini-associated CCA by suppressing the function of PDCD4. Modulation of aberrantly expressed miR-21 may be a useful strategy to inhibit tumor cell phenotypes or improve response to chemotherapy.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Bile Duct Neoplasms/etiology , Bile Ducts, Intrahepatic/metabolism , Cell Proliferation , Cholangiocarcinoma/etiology , Fascioliasis/complications , MicroRNAs/genetics , RNA-Binding Proteins/metabolism , Animals , Apoptosis , Apoptosis Regulatory Proteins/genetics , Bile Duct Neoplasms/metabolism , Bile Duct Neoplasms/pathology , Bile Ducts/parasitology , Bile Ducts/pathology , Bile Ducts, Intrahepatic/parasitology , Bile Ducts, Intrahepatic/pathology , Blotting, Western , Cell Movement , Cholangiocarcinoma/metabolism , Cholangiocarcinoma/secondary , Cricetinae , Fasciola hepatica/pathogenicity , Fascioliasis/genetics , Fascioliasis/parasitology , Female , Humans , Immunoenzyme Techniques , Male , Mesocricetus , Middle Aged , Opisthorchiasis/genetics , Opisthorchiasis/parasitology , Opisthorchiasis/pathology , Opisthorchis/pathogenicity , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
Altered miRNA expression could be a determinant of cancer development and/or progression. We aimed to study the role of oncomir miR-21 and tumor suppressor let-7a in the genesis of Opisthorchiasis-associated cholangiocarcinoma (CCA). The results showed that miR-21 was up-regulated while let-7a was down-regulated during cholangiocarcinogenesis in the hamster model and also in human CCA samples. The expression level of miR-21 had an inverse correlation with the mRNA level of its target RECK, a metastasis suppressor, in human CCA. Knockdown of miR-21 of KKU100 CCA cells significantly increased the mRNA level of RECK and suppressed the wound-induced migration of CCA cells. Our data suggest that miR-21 is one key molecule playing crucial roles in the CCA growth and metastasis. Manipulation of miRNA expression offers a potential avenue of CCA therapy.
Subject(s)
Bile Ducts, Intrahepatic , Opisthorchiasis , Animals , Bile Duct Neoplasms , Cholangiocarcinoma , Humans , MicroRNAs/genetics , Opisthorchiasis/geneticsABSTRACT
BACKGROUND: Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) has been implicated in the attenuation of tumor metastasis by negatively regulating metalloproteinase (MMP) levels. RECK gene expression is downregulated in many solid tumors, with this downregulation being associated with poor prognosis. This study evaluated the role of RECK in cholangiocarcinoma (CCA). METHODS: The expression of RECK, MMP-2, and MMP-9 in paraffin sections of hamster and human CCA specimens was analyzed by immunohistochemistry. Functional analysis of RECK was performed in RECK small interfering (si) RNA knockdown CCA cell lines. The effect of aspirin on RECK status and function was evaluated using Western blotting, gelatin zymography, invasion and proliferation assays, and PhosphoELISArray analysis of Ras downstream mediators. RESULTS: Hamster tissues showed high RECK expression in hyperplastic biliary duct epithelia, low RECK expression in precancerous lesions, and no RECK expression in CCA. In human specimens, RECK was highly expressed in normal biliary cells, whereas intrahepatic CCA showed low levels of expression. Downregulation of RECK was correlated with tumor metastasis (P < 0.01) and shorter patient survival (P < 0.02). RECK expression levels were inversely correlated with MMP-2 and MMP-9 expression (P < 0.05). SiRNA RECK-depleted M139 CCA cells exhibited increased MMP-2/-9 gelatinase activities and invasiveness. Aspirin (500 µM) demonstrated myriad effects in human CCA cell lines, including growth suppression, reduced phosphorylation of Akt/Erk/c-Jun, elevation of RECK expression, inhibition of MMP-2/MMP-9 activity, and enhanced invasiveness. CONCLUSIONS: RECK functions as a metastasis suppressor in CCA; upregulation of RECK expression could provide a potential therapy to improve the prognosis of this type of cancer.
