MicroRNA-217 functions as a prognosis predictor and inhibits colorectal cancer cell proliferation and invasion via an AEG-1 dependent mechanism

Abstract

Background: Recent studies have indicated the possible function of miR-217 in tumorigenesis. However, the roles of miR-217 in colorectal cancer (CRC) are still largely unknown.

Methods: We examined the expression of miR-217 and AEG-1 in 50 CRC tissues and the corresponding noncancerous tissues by qRT-PCR. The clinical significance of miR-217 was analyzed. CRC cell lines with miR-217 upregulation and AEG-1 silencing were established and the effects on tumor growth in vitro and in vivo were assessed. Dual-luciferase reporter gene assays were also performed to investigate the interaction between miR-217 and AEG-1.

Results: Our data demonstrated that miR-217 was significantly downregulated in 50 pairs of colorectal cancer tissues. MiR-217 expression levels were closely correlated with tumor differentiation. Moreover, decreased miR-217 expression was also associated with shorter overall survival of CRC patients. MiR-217 overexpression significantly inhibited proliferation, colony formation and invasiveness of CRC cells by promoting apoptosis and G0/G1 phase arrest. Interestingly, ectopic miR-217 expression decreased AEG-1 expression and repressed luciferase reporter activity associated with the AEG-1 3'-untranslated region (UTR). AEG-1 silencing resulted in similar biological behavior changes to those associated with miR-217 overexpression. Finally, in a nude mouse xenografted tumor model, miR-217 overexpression significantly suppressed CRC cell growth.

Conclusions: Our findings suggest that miR-217 has considerable value as a prognostic marker and potential therapeutic target in CRC.

Fig. 1

Determining miR-217 expression in CRC tissues and cell lines and its clinical significance. (a) Relative expression level of miR-217 in human CRC tissues (n = 50) and CRN tissues (n = 50), examined by qRT-PCR. CRC: colorectal cancer tissues; CRN: matched adjacent noncancerous colorectal tissues. (b) The relative miR-217 expression level in six CRC cell lines compared with the normal colorectal cell line NCM460. The average gene expression from NCM460 was appointed as 1. (c) Kaplan-Meier survival curve for CRC patients with miR-217-high (n = 24) and miR-217-low (n = 26) character. P value was obtained by a log-rank test. *P < 0.05, **P < 0.01

Fig. 2

MiR-217 inhibits the growth of CRC cell lines in vitro and in vivo. (a) The relative expression level of miR-217 when transfected with miR-217 mimics and mimics NC measured by qRT-PCR. The average miRNA expression from mimics NC was appointed as 1. (b) The proliferation curve of SW480 and SW620 cells after transfected with miR-217 mimics by CCK8 assay. (c) Assessment of colony formation when upregulation of miR-217 expression. (d) The effects of overexpression of miR-217 on xenograft tumor growth in mice. Tumor growth curves were drew by measuring the subcutaneous tumor volumes every 4 days. Data are presented as mean ± SD. (e) Relative miR-217 expression level in excised tumors. *P < 0.05, **P < 0.01

Fig. 3

Overexpression of miR-217 enhances apoptosis and promotes G0/G1 phase arrest in CRC cells. (a) Flow cytometry analysis showed that after transfection with miR-217 mimics and mimics NC, the apoptosis rates of both SW480 and SW620 cells were markedly increased. (b) Cell cycle distribution assay was also applied using flow cytometry and treated with mimics as mentioned above. The histogram showed that miR-217 induced cell cycle arrest at G0/G1 phase. **P < 0.01

Fig. 4

Ectopic miR-217 expression inhibits invasion of SW480 and SW620 cells. (a) Transwell method was applied to assess SW480 cells’ invasion ability after transfected with miR-217 mimics and negative control. (b) The invasiveness of SW620 cells was evaluated as mentioned above. Statistics analysis was performed by counting the stained cells that invaded to the lower chamber. All measurements were repeated three times in duplicate. (c) Western blot analysis showed the expression levels of invasion related molecules MMP2 and MMP9, cell cycle related protein cyclinD1, and apoptosis associated protein Bax and Bcl-2 after overexpression of miR-217.**P < 0.01

Fig. 5

MiR-217 directly targets AEG-1 in SW480 cells. (a) Predicted binding site of human miR-217 to the 3’UTR of AEG-1 by TargetScan. (Top panel) The mutation of miR-217 binding site in the 3’UTR of AEG-1. (b) The reporter plasmids containing wild-type or mutant 3’UTR of AEG-1 was co-transfected with miR-217 mimics or mimics NC. The assay was performed twice in triplicate. The relative luciferase activity was obtained by Firefly luciferase activity normalized against Renilla luciferase activity. (c) The effects of overexpression of miR-217 on AEG-1 expression at mRNA level and protein level. (d) The effects of inhibition of miR-217 on AEG-1 expression at mRNA level and protein level. *P < 0.05, **P < 0.01

Fig. 6

AEG-1 is upregulated in CRC tissues and negatively correlated with the expression level of miR-217 in both CRC and CRN tissue samples. (a) Upregulation of AEG-1 was observed in CRC tissue samples compared with that in adjacent CRN ones by qRT-PCR. (b) The analysis (Pearson’s correlation) of relationship between expression levels of AEG-1 and miR-217 in CRC and CRN tissues, respectively. **P < 0.01