MiR-194, commonly repressed in colorectal cancer, suppresses tumor growth by regulating the MAP4K4/c-Jun/MDM2 signaling pathway

Abstract

Tumor growth cascade is a complicated and multistep process with numerous obstacles. Until recently, evidences have shown the involvement of microRNAs (miRNAs) in tumorigenesis and tumor progression of various cancers, including colorectal cancer (CRC). In this study, we explored the role of miR-194 and its downstream pathway in CRC. We acquired data through miRNA microarray profiles, showing that the expression of miR-194 was significantly suppressed in CRC tissues compared with corresponding noncancerous tissues. Decreased miR-194 expression was obviously associated with tumor size and tumor differentiation, as well as TNM stage. Both Kaplan-Meier and multivariate survival analysis showed that downregulated miR-194 was associated with overall survival. Moreover, functional assays indicated that overexpression of miR-194 in CRC cell lines inhibited cell proliferation both in vitro and in vivo. In addition, using dual-luciferase reporter gene assay, we found MAP4K4 was the direct target of miR-194. Silencing of MAP4K4 resulted in similar biological behavior changes to that of overexpression of miR-194. We also observed through Human Gene Expression Array that MDM2 was one of the downstream targets of MAP4K4. Knockdown of MAP4K4 downregulated MDM2 expression through transcription factor c-Jun binding to the -1063 to -1057 bp of the promoter. These results suggest that miR-194, regulating the MAP4K4/c-Jun/MDM2 signaling pathway, might act as a tumor suppressor and serve as a novel target for CRC prevention and therapy.

Keywords: MAP4K4; MDM2; apoptosis; c-Jun; colorectal cancer; miR-194; proliferation.

Figure 1.

MicroRNA array analysis of miRNA expression in colorectal tissues. Hierarchical clustering of the expression values for mature miRNAs of CRN versus CRC tissues. Yellow indicates high relative expression, and blue indicates low relative expression. CRC: colorectal cancer tissues; CRN: matched adjacent noncancerous colorectal tissues.

Figure 2.

MiR-194 expression in colorectal tissues and cell lines and its relevance to overall survival. (A) MiR-194 exhibited greatest suppression effect on cell proliferation of 9 downregulated miRNAs. (B) The relative expression level of miR-194 in human CRC tissues (n = 50) and CRN tissues (n = 50), examined by qRT-PCR. (C) The relative miR-194 expression in the 6 CRC cell lines was significantly lower than that in normal colorectal cell line (NCM460). The average gene expression from NCM460 was appointed as 1. (D) Kaplan-Meier curves for overall survival analysis by miR-194 expression in CRC patients. P value was obtained by a log-rank test. *P<0.01.

Figure 3.

Tumor suppressive effects of miR-194 in RKO and SW480 CRC cell lines. (A) Relative miR-194 expression after transfected with miR-194 mimics or mimic NC, detected by SYBR qRT-PCR. The average miRNA expression from mimic NC group was appointed as 1. (B) The effect of transient transfection of miR-194 mimics or mimic NC (50 nM) for 24–120 h was examined on the proliferation of RKO and SW480 cells by CCK8 assay. (C) Colony formation assay after upregulation of miR-194 expression. (D) Overexpression of miR-194 seemed to be little effect on cell invasion of RKO and SW480 cells. Quantification was performed by counting the stained cells that invaded to the lower chamber under a light microscopy. The two CRC cells were treated as mentioned above. Flow cytometry analysis showed miR-194 induced cell cycle arrest (E) and increase of apoptosis (F). Data are presented as mean ± SD of results from 3 independent experiments. (G) Western blot analysis showed the expression levels of invasion related protein MMP2 and MMP9, cell cycle related protein cyclinD1, and apoptosis associated protein Bax and Bcl^-2 after overexpression of miR-194. *P<0.01.

Figure 4.

Relationship between expression of miR-194 and MAP4K4 in colorectal tissue samples. (A) Immunohistochemical analysis of MAP4K4 protein in representative colorectal tissue specimen is shown in ×100 magnification. Weak cytoplasmic and cytoblast staining for MAP4K4 was observed in CRN tissues while strong staining was observed in CRC tissues. (B) MAP4K4 was detected by qRT-PCR in 50 CRC tissues and matched CRN tissues. The MAP4K4 abundance was normalized against GAPDH. (C) The relationship between miR-194 and MAP4K4 expression was explored by Pearson's correlation in CRC tissues. **P<0.01.

Figure 5.

MAP4K4 is a direct target of miR-194. (A) Predicted binding site of miR-194 to the 3’UTR of human MAP4K4 by TargetScan. (B) Dual-luciferase reporter assay with cotransfection of wild-type or mutant 3’UTR vector (100 ng) and miR-194 mimics or mimic NC (50 nM) in SW480 cells. Firefly luciferase activity of each sample was normalized against Renilla luciferase activity. The effects of upregulation of miR-194 on MAP4K4 expression at mRNA level (C) and protein level (D). The effects of inhibition of miR-194 on MAP4K4 expression at mRNA level (E) and protein level (F). (G) Relative miR-194 expression after treated with siRNA-MAP4K4. (H) The expression of MAP4K4 protein after co-transfected with miR-194 inhibitor and siRNA-MAP4K4. All data from 3 separate experiments are presented as mean ± SD. *P<0.01.

Figure 6.

MAP4K4 influences MDM2 expression through regulating transcription factor c-Jun. (A) Gene expression profile array analysis in SW480 cells. Hierarchical clustering of the expression values for mRNA of siRNA-MAP4K4 cells vs. siRNA-NC cells. (B) Western blot analysis showed expression of MDM2, FAM172A, CAMLG, PLOD2 and MPEG1 protein after knockdown of MAP4K4. Silencing of transcription factor c-Jun resulted in downregulation of MAP4K4 expression in RKO and SW480 cells. (D) The human MDM2 promoter constructs containing a potential c-Jun binding motif (–1063 to –1057 bp). (E) The transcriptional activity of the MDM2 promoter. SW480 cells were co-transfected with the wild type or mutant type plasmids and c-Jun vector or blank vector for 48 h, and the luciferase activity was measured. (F) ChIP assays of c-Jun and its binding motif. Two antibodies (anti-IgG and c-Jun) were used in the ChIP assays in SW480 cells. QRT-PCR was performed to quantify the binding activity. (G) Products of qRT-PCR observed by electrophoresis method. (H) MAP4K4 knockdown influenced the expression of phosphorylated JNK and c-Jun, but had little effect on total JNK. MAP4K4-siRNA or siRNA-NC expressing SW480 transfectants were subjected to Western blot analysis. (I) Upregulation of miR-194 in SW480 cells had similar effects on expression of JNK, p-JNK and c-Jun to that of MAP4K4 knockdown. (J) The effects of overexpression of miR-194 on MDM2 expression. Representative images of 3 independent experiments with similar results are shown. **P<0.01.