Identification and functional screening of microRNAs highly deregulated in colorectal cancer

Abstract

MicroRNAs (miRNAs) constitute a robust regulatory network with post-transcriptional regulatory efficiency for almost one half of human coding genes, including oncogenes and tumour suppressors. We determined the expression profile of 667 miRNAs in colorectal cancer (CRC) tissues and paired non-tumoural tissues and identified 42 differentially expressed miRNAs. We chose miR-215, miR-375, miR-378, miR-422a and miR-135b for further validation on an independent cohort of 125 clinically characterized CRC patients and for in vitro analyses. MiR-215, miR-375, miR-378 and miR-422a were significantly decreased, whereas miR-135b was increased in CRC tumour tissues. Levels of miR-215 and miR-422a correlated with clinical stage. MiR-135b was associated with higher pre-operative serum levels of CEA and CA19-9. In vitro analyses showed that ectopic expression of miR-215 decreases viability and migration, increases apoptosis and promotes cell cycle arrest in DLD-1 and HCT-116 colon cancer cell lines. Similarly, overexpression of miR-375 and inhibition of miR-135b led to decreased viability. Finally, restoration of miR-378, miR-422a and miR-375 inhibited G1/S transition. These findings indicate that miR-378, miR-375, miR-422a and miR-215 play an important role in CRC as tumour suppressors, whereas miR-135b functions as an oncogene; both groups of miRNA contribute to CRC pathogenesis.

Fig. 1

Different expressions of miR-215, miR-375, miR-378, miR-422a and miR-135b in 125 paired samples of CRC and adjacent mucosa. MiR-215 (A), miR-375 (B), miR-378 (C) and miR-422a (D) showed significantly lower expression in tumour tissue, whereas miR-135b (E) was overexpressed in tumour tissue (all miRNAs reached P < 0.0001, Wilcoxon paired test).

Fig. 2

Correlation of miRNA expression with clinical stage of CRC patients. (A) Expression of miR-215 correlates with stage of CRC (P < 0.0001). (B) Expression of miR-422a correlates with a stage of CRC (P = 0.0001). (C) Expression of miR-135b negatively correlates with a stage of CRC (P = 0.0003).

Fig. 3

MiRNAs expression based on lymph node positivity and pre-operative serum levels of CEA and CA19-9. (A) MiR-215 is underexpressed in lymph node positive samples (P < 0.0001). (B) MiR-378 is underexpressed in lymph node positive samples (P = 0.0415). (C) MiR-422a is underexpressed in lymph node positive samples (P = 0.0002). (D) MiR-135b is underexpressed in lymph node positive samples (P = 0.0008). (E, F) Higher levels of miR-135b are associated with elevated pre-operative serum levels of CEA (cut-off 4.6 μg/l); P = 0.0338) and CA19-9 (cut-off 40 kU/l; P = 0.0360).

Fig. 4

Efficiency of transfection and effects of anti- and pre-miR oligonucleotides on apoptosis and cell cycle. (A) Suppression of miR-135b expression by anti-miR-135b as detected by TaqMan qRT-PCR. Compared with negative control, miR-135b was reduced by 70.5 ± 16.1% in HCT-116 cells and by 60.0 ± 17.3% in DLD-1 cells. (B) Effect of miR-215 on apoptosis of HCT-116 cells. Transfection of 10 nM pre-miR-215 significantly increased the number of apoptotic cells from 14.6 ± 2.1% to 36.2 ± 5.1%. C. Staining with propidium iodide for cell cycle analysis. miR-215, miR-375, miR-378 and miR-422a significantly increased the number of cells in G0–G1 phase and reduced S-phase cells in HCT-116 (wt-p53) cell line. Results from three independent experiments (*t-test significant at P < 0.05, **P < 0.01).

Fig. 5

Viability of DLD-1 and HCT-116 cells determined with MTT assay. Cells were transfected with 10 nM precursors or 30 nM inhibitor of particular miRNAs, resp., and cell viability was measured by MTT test 48 hrs after transfection. (A) miR-375 decreased viability of DLD-1 cells by 10.0 ± 9.7% and HCT-116 cells by 32.9 ± 6.9%. (B) miR-215 decreased viability of DLD-1 cells by 19.7 ± 7.1% and HCT-116 cells by 21.7 ± 0.3%. (C) Inhibition of miR-135b expression led to decrease in viability of DLD-1 cells by 27.4 ± 19.2 % and HCT-116 by 16.0 ± 11.7%. Results from three independent experiments (*t-test significant at P < 0.05, **P < 0.01).

Fig. 6

Effect of transfection of pre-miR-215 on cell migration of DLD-1 cells. Scratch wound assay proved that higher levels of miR-215 decreased migration ability of DLD-1 cells by 46.1 ± 20.5% 24 hrs after transfection. Results from three independent experiments (*t-test significant at P < 0.05).