miR-491-5p functions as a tumor suppressor by targeting IGF2 in colorectal cancer

Figures & data

Table 1 Correlation between miR-491-5 p expression and different clinical characteristics

Characteristics	No of patients (%) (n=80)	miR-491-5p expression		P-value
		Low, n (%) (n=40)	High, n (%) (n=40)
Age (years)				0.352
<60	29 (36.3)	17 (42.5)	12 (30.0)
≥60	51 (63.7)	23 (57.5)	28 (70.0)
Gender				0.370
Female	37 (46.3)	16 (40.0)	21 (52.5)
Male	43 (53.7)	24 (60.0)	19 (47.5)
Tumor location				0.367
Colon	45 (56.3)	25 (62.5)	20 (50.0)
Rectum	35 (43.7)	15 (37.5)	20 (50.0)
Tumor size (cm)				0.650
<5	33 (41.3)	15 (37.5)	18 (45.0)
≥5	47 (58.7)	25 (62.5)	22 (55.0)
Differentiation				0.008
Well	8 (10.0)	4 (10.0)	4 (10.0)
Moderate	51 (63.8)	19 (47.5)	32 (80.0)
Poor	21 (26.2)	17 (42.5)	4 (10.0)
Serum CEA level (ng/mL)				0.650
<10	33 (41.3)	18 (45.0)	15 (37.5)
≥10	47 (58.7)	22 (55.0)	25 (62.5)
Local invasion				0.770
T1–T2	14 (17.5)	6 (15.0)	8 (20.0)
T3–T4	66 (82.5)	34 (85.0)	32 (80.0)
Lymph node metastasis				0.498
N0–N	46 (57.5)	21 (52.5)	25 (62.5)
N2	34 (32.5)	19 (47.5)	15 (37.5)
TNM stage				<0.001
I–II	44 (55.0)	12 (30.0)	32 (80.0)
III–IV	36 (45.0)	28 (70.0)	8 (20.0)

Abbreviation: CEA, carcinoembryonic antigen.

Figure 1 miIR-491-5p was downregulated in CRC tissues and cell lines.

Notes: (A) miIR-491-5p expression in CRC tissues and their matched ANTs from 80 OS patients. (B) miIR-491-5p expression in five CRC cell lines (HCT116, HCT8, HT29, SW480 and SW620) and FHC. (C) Survival analysis indicated that the CRC patients with low miIR-491-5p levels had shorter OS time when compared to those with high miIR-491-5p levels in this study. Data are shown as mean ± SD. ***P<0.001.

Abbreviations: ANTs, adjacent normal tissues; CRC, colorectal cancer; OS, overall survival.

Table 2 Univariate and multivariate analyses for OS in patients with CRC

Characteristics	Multivariate analysis for OS		Univariate analysis for OS
	HR (95% CI)		HR (95% CI)
Age (<60/≥60 years)	–	–	1.013 (0.781–1.312)	0.462
Gender (female/male)	–	–	0.819 (0.516–1.232)	0.615
Tumor location (colon/rectum)	–	–	0.718 (0.561–1.313)	0.551
Tumor size (<5/≥5 cm)	–	–	0.981 (0.671–1.234)	0.214
Differentiation (well/moderate/poor)	1.878 (1.212–3.132)	0.019	3.245 (2.142–5.781)	0.005
CEA (<10/≥10 ng/mL)	–	–	0.891 (0.526–1.216)	0.121
Location invasion (T1+T2/T3+T4)	–	–	1.034 (0.802–1.321)	0.221
Lymph node metastasis (N0–N1/N2)	–	–	0.919 (0.701–1.342)	0.395
TNM stage (I–II/III–IV)	1.742 (0.965–3.145)	0.002	3.342 (2.311–5.095)	<0.001
miIR-491-5p (low/high)	0.479 (0.245–0.939)	0.032	0.314 (0.226–0.524)	0.004

Abbreviations: CRC, colorectal cancer; OS, overall survival.

Figure 2 miIR-491-5p inhibited CRC cell proliferation in vitro.

Notes: (A) qRT-PCR analysis of miIR-491-5p expression in HCT116 and HCT8 cells after transfection of miIR-491-5p agomir/antagomir or their negative control (agomiR-NC and antagomiR-NC). (B) Cell proliferation was determined in HCT116 and HCT8 cells after transfection with miIR-491-5p agomir/antagomir or agomiR-NC/antagomiR-NC using CCK-8 assay. (C) Colony formation assay was performed to detect the ability of proliferation in HCT116 and HCT8 after transfection with miIR-491-5p agomir/antagomir or agomiR-NC/antagomiR-NC. Data are shown as mean ± SD. *P<0.05, ***P<0.001.

Abbreviations: CCK-8, cell counting kit-8; CRC, colorectal cancer; NC, negative control; OS, overall survival; qRT-PCR, quantitative real-time PCR.

Figure 3 miIR-491-5p suppressed OS cell growth in vivo.

Notes: (A) Representative images of tumors from implanted mice. (B) Quantitative analysis of xenografted tumor volume. Data are shown as mean ± SD. ***P<0.001.

Abbreviation: OS, overall survival.

Figure 4 IGF2 is a direct target gene of miIR-491-5p.

Notes: (A) The luciferase activity was measured after cotransfecting with WT/Mut IGF2 3′-UTR plasmid and agomiIR-491-5p/agomiR-NC in 293 T cells. qRT-PCR (B) and Western blot (C) analyses were performed to detect the expression of IGF2 in HCT116 and HCT8 after transfection with agomiIR-491-5p and antagomiIR-491-5p or agomiR-NC and antagomiR-NC. Data are shown as mean ± SD of three independent experiments. *P<0.05, **P<0.01, and ***P<0.001.

Abbreviations: Mut, mutant; NC, negative control; qRT-PCR, quantitative real-time PCR; WT, wild type.

Figure 5 Overexpression of IGF2 reverses the effects induced by miIR-491-5p overexpression in HCT116 and HCT8 cells.

Notes: (A) IGF2 protein expression was detected in HCT116 and HCT8 cells cotransfected with agomiIR-491-5p and IGF2 expression plasmid or blank vector (vector). (B and C) Cell proliferation was determined in HCT116 and HCT8 cells cotransfected with agomiIR-491-5p and IGF2 expression plasmid or blank vector (vector). Data are shown as mean ± SD of three independent experiments. *P<0.05, **P<0.01, and ***P<0.001.

Figure 6 The relationship between miIR-491-5p and IGF2 in CRC tissues.

Notes: (A) Representative IHC staining (×200) for IGF2 from CRC tissues and matched ANTs. (B) IHC scores of IGF2 in low miIR-491-5p CRC tissues were markedly higher than those in high miIR-491-5p CRC tissues. (C) The correlation between the levels of miIR-491-5p expression and IHC scores of IGF2 in CRC tissues. (D) qRT-PCR was employed to measure the expression of IGF2 mRNA in 80 CRC specimens and corresponding ANTs. (E) The correlation analysis was performed between IGF2 mRNA and miIR-491-5p expression levels. Data are shown as mean ± SD.***P<0.001.

Abbreviations: ANTs, adjacent normal tissues; CRC, colorectal cancer; IHC, immunohistochemical; NC, negative control; qRT-PCR, quantitative real-time PCR.

Figure 7 Plasma levels of miIR-491-5p were downregulated in CRC patients.

Notes: (A) The relative levels of miIR-491-5p in patients with CRC and healthy controls. The data are shown as log₁₀ and normalized to cel-miR-39. (B) ROC curves for 491-5 p in 80 CRC patients and 40 healthy controls. Data are shown as mean ± SD. ***P<0.001.

Abbreviations: CRC, colorectal cancer; ROC, receiver operating characteristic.