Genetic and epigenetic down-regulation of microRNA-212 promotes colorectal tumor metastasis via dysregulation of MnSOD.

Altered functions of microRNAs (miRNAs) have been associated with colorectal cancer (CRC). miR-212 is transcribed from a stable intron of a non-protein coding gene, and is reportedly down-regulated in different tumor types. We investigated the role of miR-212 in colorectal carcinogenesis and progression. We analyzed the expression of miR-212 by real-time polymerase chain reaction (PCR) analysis of colorectal cell lines and 180 paired tumor samples and surrounding healthy tissue. We overexpressed and knocked down miR-212 in CRC cell lines and assessed the in vitro effects. We also studied the effects of miR-212 overexpression on metastasis of tumors grown from HCT116 cells in nude mice. Overexpression of miR-212 inhibited CRC cell migration and invasion in vitro and formation of intrahepatic and pulmonary metastasis in vivo. We identified manganese superoxide dismutase (MnSOD) messenger RNA as a direct target of miR-212, and observed an inverse correlation between the level of miR-212 and MnSOD protein in colorectal tumor samples. MnSOD was required for down-regulation of epithelial markers and up-regulation of mesenchymal markers in CRC cells, indicating that it promoted the epithelial-mesenchymal transition. Overexpression of miR-212 reduced the levels of MnSOD to block the epithelial-mesenchymal transition process. Loss of heterozygosity and promoter hypermethylation each contributed to the down-regulation of miR-212. Reduced levels of miR-212 were associated with a more aggressive tumor phenotype and short disease-free survival times of patients (P = .0045; overall survival, P = .0015). miR-212 is down-regulated in human CRC tissues via genetic and epigenetic mechanisms. miR-212 might prevent tumor progression by targeting MnSOD messenger RNA; reduction of miR-212 could be a prognostic marker for patients with CRC. miR-212 and MnSOD might also be therapeutic targets for cancer.

Meng X ，Wu J ，Pan C ，Wang H ，Ying X ，Zhou Y ，Yu H ，Zuo Y ，Pan Z ，Liu RY ，Huang W ... -  《-》

MicroRNA-137, an HMGA1 target, suppresses colorectal cancer cell invasion and metastasis in mice by directly targeting FMNL2.

Formin-like (FMNL)2 is up-regulated in colorectal tumors and has been associated with tumor progression, but little is known about regulatory mechanisms. We investigated whether microRNAs regulate levels of FMNL2 in colorectal cancer (CRC) cells. We used real-time polymerase chain reaction and immunoblot analyses to measure levels of miR-137, high-mobility group AT-hook (HMGA)1, and FMNL2 in CRC cells and tissue samples from patients (n = 50). We used luciferase reporter assays to determine the association between miR-137 and the FMNL2 3' untranslated region, and HMGA1 and the miR-137 promoter. Chromatin immunoprecipitation assays were used to assess direct binding of HMGA1 to the miR-137 promoter. miR-137 and miR-142-3p were predicted to bind FMNL2 based on bioinformatic data. Only the level of miR-137 had a significant inverse correlation with the level of FMNL2 protein in CRC cell lines and tissues. FMNL2 messenger RNA was targeted by miR-137; expression of miR-137 inhibited proliferation and invasion by CRC cells in vitro, and metastasis to liver and intestine by CRC xenografts in nude mice. HMGA1 bound to the promoter of miR-137 and activated its transcription, which reduced levels of FMNL2 in CRC cells. Ectopic expression of miR-137 in CRC cells inhibited phosphorylation of mitogen-activated protein kinase (MAPK) and Akt, which reduced levels of matrix metalloproteinase 2, matrix metalloproteinase 9, and vascular endothelial growth factor; it also reduced invasiveness of CRC cells, inhibiting signaling via phosphatidylinositol-4,5-bisphosphate 3-kinase, Akt, and MAPK. Levels of miR-137 and HMGA1 are reduced, and levels of FMNL2 are increased, in CRC samples compared with adjacent normal mucosa. In CRC cells, miR-137 targets FMNL2 messenger RNA and is regulated by the transcription factor HMGA1. Expression of miR-137 reduces CRC cell invasion in vitro and metastasis of tumor xenografts in mice. FMNL2 appears to activate phosphatidylinositol-4,5-bisphosphate 3-kinase, protein kinase B (Akt), and MAPK signaling pathways.

Liang L ，Li X ，Zhang X ，Lv Z ，He G ，Zhao W ，Ren X ，Li Y ，Bian X ，Liao W ，Liu W ，Yang G ，Ding Y ... -  《-》

Identification of microRNA-214 as a negative regulator of colorectal cancer liver metastasis by way of regulation of fibroblast growth factor receptor 1 expression.

The purpose of this study was to identify microRNAs (miRNAs) involved in the pathology of colorectal cancer (CRC) liver metastasis and investigate their underlying mechanisms. A total of 39 miRNAs were identified to be differentially expressed between 16 primary CRC tissues with liver metastases and 16 CRC tissues without liver metastases from 32 patients by Affymetric miRNA microarrays. A panel of eight miRNAs were confirmed to be significantly and differentially expressed between CRC tissues with and without liver metastases through quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis in the 32 patients. In a validated cohort of 99 CRC patients (44 with and 55 without liver metastases), only miR-214 was validated to be significantly down-regulated in CRC with liver metastases, which was associated with an unfavorable prognosis. Ectopic expression of miR-214 suppressed proliferation, migration, and invasion in vitro, tumor growth and liver metastasis in an in vivo xenograft mouse model, whereas miR-214 knockdown promoted proliferation, migration, and invasion in CRC cell lines. Further studies indicated that fibroblast growth factor receptor 1 (FGFR1) was a potential target of miR-214. Restoring miR-214 expression in CRC cells decreased endogenous FGFR1 messenger RNA (mRNA) and protein levels. FGFR1 knockdown mimicked the tumor suppressive effect of miR-214 on CRC cells, while reintroduction of FGFR1 abolished the tumor suppressive effect of miR-214 on CRC cells. Moreover, miR-214 expression levels were inversely correlated with FGFR1 in CRC patients. Down-regulation of miR-214 expression was correlated with increased FGFR1 expression levels, which may contribute to increased CRC liver metastasis. miR-214 may serve as a potential marker to predict survival, and the miR-214-FGFR1 axis may be a therapeutic target in CRC patients.

Chen DL ，Wang ZQ ，Zeng ZL ，Wu WJ ，Zhang DS ，Luo HY ，Wang F ，Qiu MZ ，Wang DS ，Ren C ，Wang FH ，Chiao LJ ，Pelicano H ，Huang P ，Li YH ，Xu RH ... -  《-》

MicroRNA-221 promotes colorectal cancer cell invasion and metastasis by targeting RECK.

MicroRNAs (miRNAs) have recently emerged as regulators of metastasis. We provide insight into the behavior of miR-221 in colorectal cancer (CRC) metastasis by showing that miR-221 is significantly upregulated in metastatic CRC cell lines and tissues. miR-221 overexpression enhances, whereas miR-221 depletion reduces CRC cell migration and invasion in vitro and metastasis in vivo. We identify RECK as a direct target of miR-221, reveal its expression to be inversely correlated with miR-221 in CRC samples and show that its re-introduction reverses miR-221-induced CRC invasiveness. Collectively, miR-221 is an oncogenic miRNA which may regulate CRC migration and invasion through targeting RECK.

Qin J ，Luo M 《-》

OVOL2, an Inhibitor of WNT Signaling, Reduces Invasive Activities of Human and Mouse Cancer Cells and Is Down-regulated in Human Colorectal Tumors.

Activation of WNT signaling promotes the invasive activities of several types of cancer cells, but it is not clear if it regulates the same processes in colorectal cancer (CRC) cells, or what mechanisms are involved. We studied the expression and function of OVOL2, a member of the Ovo family of conserved zinc-finger transcription factors regulated by the WNT signaling pathway, in intestinal tumors of mice and human beings. We analyzed the expression of OVOL2 protein and messenger RNA in CRC cell lines and tissue arrays, as well as CRC samples from patients who underwent surgery at Xiamen University in China from 2009 to 2012; clinical information also was collected. CRC cell lines (SW620) were infected with lentivirus expressing OVOL2, analyzed in migration and invasion assays, and injected into nude mice to assess tumor growth and metastasis. Tandem affinity purification was used to purify the OVOL2-containing complex from CRC cells; the complex was analyzed by liquid chromatography, tandem mass spectrometry, and immunoprecipitation experiments. Gene promoter activities were measured in luciferase reporter assays. We analyzed mice with an intestine-specific disruption of Ovol2 (Ovol2(flox/+) transgenic mice), as well as Apc(min/+) mice; these mice were crossed and analyzed. Analysis of data from patients indicated that the levels of OVOL2 messenger RNA were significantly lower in colon carcinomas than adenomas, and decreased significantly as carcinomas progressed from grades 2 to 4. Immunohistochemical analysis of a tissue array of 275 CRC samples showed a negative association between tumor stage and OVOL2 level. Overexpression of OVOL2 in SW620 cells decreased their migration and invasion, reduced markers of the epithelial-to-mesenchymal transition, and suppressed their metastasis as xenograft tumors in nude mice; knockdown of OVOL2 caused LS174T cells to transition from epithelial to mesenchymal phenotypes. OVOL2 bound T-cell factor (TCF)4 and β-catenin, facilitating recruitment of histone deacetylase 1 to the TCF4-β-catenin complex; this inhibited expression of epithelial-to-mesenchymal transition-related genes regulated by WNT, such as SLUG, in CRC cell lines. OVOL2 was a downstream target of WNT signaling in LS174T and SW480 cells. The OVOL2 promoter was hypermethylated in late-stage CRC specimens from patients and in SW620 cells; hypermethylation resulted in OVOL2 down-regulation and an inability to inhibit WNT signaling. Disruption of Ovol2 in Apc(min/+) mice increased WNT activity in intestinal tissues and the formation of invasive intestinal tumors. OVOL2 is a colorectal tumor suppressor that blocks WNT signaling by facilitating the recruitment of histone deacetylase 1 to the TCF4-β-catenin complex. Strategies to increase levels of OVOL2 might be developed to reduce colorectal tumor progression and metastasis.

Ye GD ，Sun GB ，Jiao P ，Chen C ，Liu QF ，Huang XL ，Zhang R ，Cai WY ，Li SN ，Wu JF ，Liu YJ ，Wu RS ，Xie YY ，Chan EC ，Liou YC ，Li BA ... -  《-》