MicroRNA-17 promotes cell proliferation and migration in human colorectal cancer by downregulating SIK1

Figures & data

Figure 1 miR-17 is upregulated in CRC tumor tissue and implies poor prognosis. (A) The miR-17 expression in the Oncomine database including TCGA Colorectal, Gaedcke Colorectal, Kaiser Colon, Skrzypczak Colorectal, and Hong Colorectal. (B) The relative expression of miR-17 in CRC cancerous tissue (n=51) and adjacent non-tumor tissue (n=50) was measured by quantitative real-time PCR. (C) Kaplan-Meier analysis of the 5-year overall survival rate of patients with high (n=51) and low (n=50) expression of miR-17.

Figure 2 miR-17 is upregulated in tumor cell lines and promotes cell proliferation and invasion in CRC. (A) The expression of miR-17 in three CRC cell lines (HCT116, SW480, and HT29) and in normal colon epithelial cells (FHC). (B) The relative expression of miR-17 was measured by quantitative real-time PCR analysis of HCT116 and SW480 cells transfected with miR-17 mimics and NC for 48 hrs. (C) The cell proliferation rate measured by 24 h CCK-8 assay of SW480 and HCT116 cells transfected with mimic NC, miR-17 mimic, inhibitor NC, and miR-17 inhibitor. (D) Flow cytometry was performed to detect cell apoptotic rate of SW480 and HCT116 cells. (E) Transwell assay was performed to evaluate the invasion of SW480 and HCT116 cells. All values are presented as mean ± SD of three replicates. *P<0.05, **P<0.01, ***P<0.001, compared with FHC cells, mimic NC group, or inhibitors NC group.

Figure 3 Bioinformatic analysis of gene targets of miR-17. (A) Venn chart of common genes from 3 publicly available databases, including TargetScan, miRWalk, and miRTarbase. (B) The analysis of 8 possible target genes from TCGA database and the human protein atlas database. (C) Analysis of OS based on SIK1 expression from the human protein atlas database.

Figure 4 SIK1 may be a direct target of miR-17. (A) The potential binding sequences of SIK1 3ʹUTR and miR-17 based on bioinformatics analysis. The wild-type (WT) and mutated binding sequences are shown. Quantitative real-time PCR analysis (B) and Western blot analysis (C) of SIK1 mRNA and protein in SW480 and HCT116 cells transfected with mimic NC, miR-17 NC, inhibitor NC, and miR-17 inhibitor NC. Relative luciferase activity in SW480 (D) and HCT116 (E) cells transfected with reporter vector containing WT binding sequence (psiCheck-SIK1-3ʹUTR-WT) or mutated binding sequence (psiCheck-SIK1-3ʹUTR-mut) along with miR-17 mimic and negative control. All values are presented as mean ± SD of three replicates. *P<0.05, **P<0.01, compared with mimic NC group, or inhibitors NC group.

Table 1 The comparison of SIK1 expression in cancerous tissue and adjacent non-tumor tissue

Groups	Case	Expression of SIK1 protein				P-value
		Low expression		High expression
-	+	++	+++
Cancerous tissue	101	17	56	23	5	<0.001
Normal tissue	101	6	18	26	51

Table 2 Correlations between SIK1 expression and clinical characteristics of patients with CRC

Groups	Case	%	SIK1 expression in cancer tissue
Low expression	%	High expression	%	x^2	P-value
Gender
Male	57	56.44	40	54.79	17	60.71	0.288	0.591
Female	44	43.56	33	45.21	11	39.29
Age (y)
≤65	62	61.39	43	58.90	19	67.86	0.684	0.408
>65	39	38.61	30	41.10	9	32.14
Tumor location
Colon	56	55.45	35	47.95	21	75.00	7.119	0.008
Rectum	45	44.55	38	52.05	6	21.43
Differentiation
Well and Moderate	84	83.17	65	89.04	19	67.86	6.488	0.011
Worse	17	16.83	8	10.96	9	32.14
Initial CEA (ng/mL)
<20	70	69.31	52	71.23	18	64.29	0.459	0.498
≥20	31	30.69	21	28.77	10	35.71
Tumor invasion
T1 and T2	35	34.65	20	27.40	15	53.57	6.123	0.013
T3 and T4	66	65.35	53	72.60	13	46.43
Lymph node metastasis
Negative	47	46.53	29	39.73	18	64.29	4.906	0.027
Positive	54	53.47	44	60.27	10	35.71
Clinic stages (TNM)
I and II	45	44.55	28	38.36	17	60.71	4.095	0.043
III and IV	56	55.45	45	61.64	11	39.29

Figure 5 Expression of SIK1 protein in colorectal cancer tissue and adjacent normal tissue by IHC. (A) Weak SIK1 staining in tumor tissue (40x). (B) Weak SIK1 staining in tumor tissue (100x). (C) Weak SIK1 staining in tumor tissue (200x). (D) Strong SIK1 staining in adjacent non-tumor tissue (40x). (E) Strong SIK1 staining in adjacent non-tumor tissue (100x). (F) Strong SIK1 staining in adjacent non-tumor tissue (200x).

Figure 6 SIK1 is downregulated in colorectal cancer tissue in comparison to adjacent normal tissue. Western blot (A) and quantitative real-time PCR (B) analysis of SIK1 protein and mRNA in surgical specimens. All values are presented as mean ± SD of three replicates. *P<0.05, compared with non-tumor tissue. C, cancerous tissue; ANT, adjacent non-tumor tissue.

Table 3 Univariate and multivariate survival analysis of patients with colorectal cancer

Factors	Univariate analysis		Multivariate analysis
HR (95%CI)	P-value	HR (95%CI)	P-value
Age (years)
≤65 vs >65	1.16 (0.62–2.16)	0.6703	—	—
Gender
Male vs female	1.59 (0.84–3.00)	0.2282	—	—
Histology type
Well, moderate vs poor	0.94 (0.43–2.06)	0.7700	—	—
Clinical stages (TNM)
I, II vs III, IV	0.51 (0.30–0.88)	0.0140	0.45 (0.23–0.89)	0.021
Tumor location
Colon vs rectal	1.01 (0.55–1.87)	0.2936	—	—
SIK1 expression
High vs low	0.32 (0.17–0.60)	<0.001	0.33 (0.16–0.67)	0.002