MicroRNA (miR)-597-5p Inhibits Colon Cancer Cell Migration and Invasion by Targeting FOS-Like Antigen 2 (FOSL2).

Our previous work had shown that FOS-like antigen 2 (FOSL2) is regulated by miR-143-5p in colorectal cancer (CRC). Given that it has been shown by others that FOSL2 is also a target of miR-597-5p in breast adenocarcinoma, the objective of the current work was to determine whether FOSL2 is regulated by miR-597-5p in CRC and the role of miR-597-5p in CRC. MiR-597-5p expression was determined in RNA obtained from 30 paired samples of colon cancer and tumor adjacent normal tissue, as well as in the LoVo (CRC cell line) and FHC (normal colonic epithelial cells) by quantitative real time polymerase chain reaction (qRT-PCR). MiR-597-5p expression was significantly downregulated in both CRC tissue and LoVo cells. Reporter assays using wild-type and miR-597-5p seed mutant FOSL2 confirmed that FOSL2 is a bona fide target of miR-597-5p. Modulating miR-597-5p expression levels in FHC and LoVo cells using antagomir and mimic, respectively, impacted expression of epithelial and mesenchymal cell markers as well as in vitro migration and invasion, without any effect on cell proliferation, showing that miR-597-5p functions as a suppressor of epithelial to mesenchymal transition. Restoration of FOSL2 expression rescued pro-metastatic functional properties of LoVo cells conforming that effect of miR-597-5p was being mediated by targeting FOSL2. Xenograft assays in athymic nude mice showed that miR-597-5p mimic did not reduce tumor incidence or growth in LoVo cells. However, using a hepatic metastasis model showed that miR-597-5p mimic can significantly prevent hepatic metastatic nodule formation as well as FOSL2 expression in these metastatic nodules. Importantly, FOSL2 mRNA and miR-597-5p expression was found to be inversely correlated in an independent cohort of 21 CRC patients Cumulatively our results show that miR-597-5p functions as a suppressor of metastatic progression in CRC by targeting FOSL2. Replenishment of miR-597-5p can be a potential therapeutic target where its expression along with FOSL2 can serve as potential diagnostic markers in CRC.

Fos-like antigen 2 (FOSL2) promotes metastasis in colon cancer.

Among different cancers, incidence and mortality of colorectal cancer (CRC) is one of the highest. KRAS mutation is one of the underlying features in the pathogenesis of CRC with CRC tumors harboring mutant KRAS exhibiting a more aggressive behavior compared to CRC tumors with wild type KRAS. We had earlier shown that the microRNA-143 (miR-143) replenishment not only chemosensitizers CRC cell line with mutant KRAS instead of wild-type KRAS gene, to paclitaxel-mediated cytotoxicity, but also inhibits cell migration and invasion ability. Hence, the study aimed to determine how miR-143 replenishment is inhibiting pre-metastatic behavior in CRC cells with mutant KRAS. Top ten mRNA targets of miR-143 as predicted by TargetScan were evaluated by qRT-PCR in LoVo cells which were performed mock transfection or miR-143 mimic transfection. Evaluation of the changes in cognate mRNA target(s) was done in 30 paired CRC tissue and tumor adjacent normal tissue specimens and in LoVo cells by western blot. Effect of the mRNA target on pro-metastatic behavior was assayed by gain- and loss-of-function studies using a combination of western blotting and in vitro cell proliferation and transwell migration/invasion assay in LoVo cells and in the normal colonic epithelium cell line FHC. In vivo effect of the cognate mRNA target on CRC metastasis was assayed by xenograft assay. Of the 10 predicted mRNA targets, FOSL2 (P < 0.05) and IGFBP5 (P > 0.05) was down regulated in LoVo cells transfected with the miR-143 mimic. FOSL2 mRNA levels were significantly downregulated in CRC tissue specimens compared with adjacent normal tissue (P < 0.05). Immunoblot analysis showed that FOSL2, but not IGFBP5, protein expression is down regulated in LoVo cells after the miR-143 mimic transfection. FOSL2 overexpression in the normal colonic epithelial cell line FHC or siRNA-mediated silencing in LoVo cells induced and repressed, respectively, pro-mesenchymal cell features. Whereas manipulation of FOSL2 expression did not have any effect on cell proliferation rates, silencing its expression inhibited cell migration and invasion ability in vitro. In addition, silencing of FOSL2 expression in the LoVo cells can significantly inhibited invasion of hepatic, while no effect was found for tumorigenic potential. Our results suggest that FOSL2 is a critical regulator of CRC metastasis and might be an important marker for prognostic in CRC patients.

MicroRNA-143 replenishment re-sensitizes colorectal cancer cells harboring mutant, but not wild-type, KRAS to paclitaxel treatment.

Colorectal cancer (CRC) global incidence is one of the highest among cancers. The KRAS gene has been shown as a robust biomarker for poor prognosis and drug resistance. MicroRNA-143 (miR-143) and let-7 are families of tumor suppressor microRNAs that are often downregulated in CRC, especially with coexistent KRAS mutations. In order to evaluate if miR-143 and/or let-7b replenishment would re-sensitize CRC cells to paclitaxel treatment, we investigated in effect of miR-143 and let-7b replenishments on sensitivity to paclitaxel treatment in KRAS mutant LoVo and wild-type SW48 CRC cell lines. Our results showed that miR-143, but not let-7b, increased sensitization of KRAS mutant tumor cells to paclitaxel. Furthermore, transfection of miR-143, but not let-7b, mimic negatively regulated the expression of mutant but not wild-type KRAS. Combination of miR-143 mimic and paclitaxel induced the onset of apoptosis, and reverted in vitro metastatic properties (migration and invasion) in KRAS mutant tumor cells. MiR-143 thus can be used as a chemosensitizer for the treatment of KRAS mutant tumors and warrants further investigations in in vitro and pre-clinical in vivo models.

MiR-27a promotes hepatocellular carcinoma cell proliferation through suppression of its target gene peroxisome proliferator-activated receptor γ.

BACKGROUND: MicroRNAs (miRNAs) function as essential posttranscriptional modulators of gene expression, and are involved in a wide range of physiologic and pathologic states, including cancer. Numerous miRNAs are deregulated in hepatocellular carcinoma (HCC). This study aimed to investigate the role of miR-27a in the development of HCC. METHODS: The expression of MiR-27a was measured by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to examine changes in the viability of HepG2, Bel-7402, Bel-7404 hepatoma cell lines associated with up-regulation or down-regulation of miR-27a. A dual-luciferase activity assay was used to verify a target gene of miR-27a. Immunohistochemistry, qRT-PCR, Western blotting analysis, and cell cycle and apoptosis flow cytometric assays were used to elucidate the mechanism by which miR-27a modulates liver cancer cell proliferation. RESULTS: The expression of miR-27a was significantly increased in HCC tissues and HepG2, Bel-7402, Bel-7404 hepatoma cell lines (P < 0.05). We also found that the down-regulation of miR-27a in HepG2 cells dramatically inhibited proliferation, blocked the G1 to S cell cycle transition and induced apoptosis (P < 0.05). In addition, miR-27a directly targeted the 3'- untranslated region of peroxisome proliferator-activated receptor γ (PPAR-γ), and ectopic miR-27a expression suppressed PPAR-γ expression on the mRNA and protein levels. The rosiglitazone-induced overexpression of PPAR-γ attenuated the effect of miR-27a in HCC cells. CONCLUSIONS: Our findings suggested that miRNA-27a promoted HCC cell proliferation by regulating PPAR-γ expression. MiR-27a may provide a potential therapeutic strategy for HCC treatment.

Expression and clinical significance of microRNA-376a in colorectal cancer.

The incidence of colorectal cancer (CRC) is increasing in many Asian countries and microRNAs have already been proven to be associated with tumorigenesis. Currently, microRNA-376a (miR-376a) expression and association with clinical factors in CRC remains unclear. In this study, real-time quantitative reverse transcriptase- polymerase chain reaction (qRT-PCR) was carried out on 53 matched pairs of CRC and adjacent normal mucosa to investigate the expression levels of miR-376a. According to the high or low expression of miR-376a, patients were divided into two groups. The relationship between miR-376a expression and clinicopathological factors of 53 patients was evaluated. Survival analysis of 53 CRC patients was performed with clinical follow- up information and survival curves were assessed by the Kaplan-Meier method. Immunohistochemistry (IHC) staining was performed on sections of paraffin-embedded tissue to investigate the vascular endothelial growth factor (VEGF) expression. MiR-376a showed low expression in cancer tissues compared to the adjacent normal tissues and altered high miR-376a expression tended to be positively correlated with advanced lymph node metastasis and shorter patient survival. VEGF IHC positivity was significantly more common in patients with high expression levels of miR-376a.Those results demonstrated that miR-376a may be a meaningful prognostic biomarker and potential therapeutic target in colorectal cancer.