UHRF1 promotes proliferation of gastric cancer via mediating tumor suppressor gene hypermethylation

Figures & data

Table 1. Association of UHRF1 expression in the tumor tissues with demographic and clinicopathologic characteristics in 238 patients with GC

Category	n	Expression of UHRF1 (n)				P value^a
		−	+	++	+++
	238	42	83	78	35
Gender						0.475
Male	151	24	58	48	21
Female	87	18	25	30	14
Age						0.096
< 60	131	23	54	39	15
≥ 60	107	19	29	39	20
Location of tumor						0.345
Cardia	13	4	4	2	3
Body	77	11	22	30	13
Antrum	148	27	57	46	19
Tumor size, cm						0.134
<5	139	30	51	40	18
≥5	99	12	32	38	17
Depth of invasion						0.016
T1-T2	40	11	19	8	2
T3-T4	198	31	64	70	33
Stages						0.001
I–II	76	20	34	16	6
III–IV	162	22	49	62	29
Differentiation						0.049
Well and moderate	121	24	50	34	13
Poorly and not	117	18	33	44	22
Lymph node metastases						0.013
0	43	10	22	9	2
≥ 1	195	32	61	69	33
Metastases to other organs						0.001
Present	15	0	1	8	6
Absent	223	42	82	70	29

^a P values were calculated through the Kruskal–Wallis H-test and the Mann–Whitney U test to analyze the relationship between UHRF1 expression and clinicopathologic characteristics.

Table 2. Univariate Analysis and Multivariate analysis of the Correlation Between Clinicopathological Parameters and Survival of Patients With Gastric Cancer

Clinicopathological Parameters	Cumulative Survival Rates, %		Mean Survival Time, mo	Univariate Analysis			Multivariate analysis
	3-Year	5-Year		HR	95% CI	P	HR	95% CI	P
Gender
Male	38	22	37.49	1.000	reference
Female	37	23	35.28	1.106	0.836–1.463	0.479
Age
< 60	50	29	45.15	1.000	reference	1.000	reference
≥ 60	23	14	26.32	1.91	1.458–2.523	< 0.001	1.791	1.327–2.417	< 0.001
Location of tumor
Cardia	69	15	48.23	1.000	reference	1.000	reference
Body	26	13	26.53	1.949	1.032–3.680	< 0.05	1.665	0.847–3.275	0.140
Antrum	41	28	40.95	1.189	0.640–2.207	0.584	1.185	0.617–2.277	0.610
Tumor size, cm
< 5	43	27	40.70	1.000	reference	1.000	reference
≥ 5	30	16	31.04	1.382	1.049–1.820	< 0.05	0.821	0.606–1.111	0.202
Depth of invasion
T1-T2	80	65	70.03	1.000	reference	1.000	reference
T3-T4	29	14	29.94	3.824	2.438–5.998	< 0.001	1.803	1.100–2.953	< 0.05
Stages
I–II	49	34	47.33	1.000	reference	1.000	reference
III–IV	33	17	31.69	1.941	1.219–3.088	< 0.01	1.130	0.765–1.669	0.539
Differentiation
Well and moderate	42	27	41.15	1.000	reference	1.000	reference
Poorly and not	33	17	32.06	1.358	1.033–1.785	< 0.05	0.957	0.662–1.382	0.814
Lymph node metastases
0	77	67	70.67	1.000	reference	1.000	reference
≥1	29	12	29.18	4.285	2.725–6.738	< 0.001	2.965	1.777–4.948	< 0.001
Metastases to other organs
Present	7	7	18.93	1.000	reference	1.000	reference
Absent	40	23	37.87	2.245	1.297–3.884	< 0.01	2.277	1.223–4.240	< 0.01
UHRF1 expression
−	64	38	52.26	1.000	reference	1.000	reference
+	45	30	43.37	1.287	0.849–1.951	0.235	1.421	0.920–2.194	0.113
++	28	13	28.33	2.164	1.431–3.273	< 0.001	1.711	1.093–2.680	< 0.05
+++	11	6	20.71	3.085	1.8905.035	< 0.001	2.553	1.506–4.327	< 0.001

CI indicates confidence interval; HR, hazard ratio.

Table 3. Proliferation related genes upregulated after UHRF1 downregulation in MKN45 cells.

Accession	Gene name	Full name	value in MKN45-NC	value in MKN45-shUHRF1
NM_001265	CDX2	caudal type homeobox 2	0.6994	0.2876
NM_000077	CDKN2A(p16 ^INK4)	cyclin-dependent kinase inhibitor 2A	0.1387	0.0643
NM_001031680	RUNX3	runt-related transcription factor 3	0.2603	0.1421
NM_00117093	FOXO4	forkhead box O 4	0.1216	0.0705
NM_005037	PPARG(PPARγ)	peroxisome proliferator-activated receptor gamma	0.0178	0.0114
NM_007294	BRCA1	breast cancer 1, early onset	0.1166	0.0761
NM_002675	PML	promyelocytic leukemia	0.8048	0.6693

Figure 1. Kaplan–Meier survival curves of GC patients with different level of UHRF1 expression stratified by the TNM stage of the tumor (log-rank test). (A) Correlation of UHRF1 expression with overall survival (cum survival) in all stages. (B) Correlation of UHRF1 expression with overall survival in Stage I–II. (C) Correlation of UHRF1 expression with overall survival in Stage III–IV.

Figure 2. Expression of UHRF1 in GC cell lines. (A) The expression level of UHRF1 mRNA in 3 GC cell lines and one immortalized normal gastric mucosal epithelial cell line (GES) was measured using qRT-PCR. GAPDH was used as an internal control and the fold change was calculated by 2^−ΔΔCt. (B) The expression of UHRF1 in 3 GC cell lines and GES cell line was examined through western blot analysis. β-actin was used as an internal control.

Figure 3. UHRF1 promotes GC cell proliferation in vitro. (A) Western blot analysis of UHRF1 expression in MKN45 cells infected with UHRF1 shRNA (shUHRF1) or negative control (shNC) and parental cells. β-actin was used as an internal control. (B) Western blot analysis of UHRF1 in SGC7901 cells transfected with UHRF1 plasmid or vector control (NC) and parental cells. (C) XTT assay of MKN45, MKN45-shNC and MKN45-shUHRF1 cells. (D) XTT assay of SGC7901, SGC7901-NC and SGC7901-UHRF1 cells. (E) Colony formation assay of MKN45 cells infected with shUHRF1 and SGC7901 cells transfected with UHRF1 plasmid. Colonies were evaluated and values were reported as the ratio. (F) Apoptosis assay of MKN45 cells infected with shUHRF1 and SGC7901 cells transfected with UHRF1 plasmid. (G) Flow cytometry cell cycle analysis of MKN45, MKN45-shNC and MKN45-shUHRF1 cells. (H) Flow cytometry cell cycle analysis of SGC7901, SGC7901-NC and SGC7901-UHRF1 cells. Data are shown as mean ± SEM (n = 3) of one representative experiment. Similar results were obtained in 3 independent experiments.

Figure 4. UHRF1 reduces tumor suppressor expression via enhancing methylation. (A) Effects of UHRF1 silencing on gene methylation of 7 tumor suppressor genes in MKN45 cells infected with UHRF1 shRNA (shUHRF1) assayed by Methylation-specific PCR (MSP). (B) Effects of UHRF1 upregulation on gene methylation of 7 tumor suppressor genes in SGC7901 cells transfected with UHRF1 plasmid assayed by MSP. (C) Effects of UHRF1 silencing on mRNA expression of 7 tumor suppressor genes in MKN45 cells infected with shUHRF1 assayed by qRT-PCR. GAPDH was used as an internal control and the fold change was calculated by 2^−ΔΔCt. (D) Effects of UHRF1 upregulation on mRNA expression of 7 tumor suppressor genes in SGC7901 cells transfected with UHRF1 plasmid assayed by qRT-PCR.

Figure 5. Down-regulation of UHRF1 inhibits GC growth in vivo. (A) MKN45-shUHRF1 and MKN45-shNC cells were injected subcutaneously into nude mice. At 4 weeks after implantation, MKN45-shUHRF1 cells produced smaller tumors than control cells. (B) Growth curve of tumor volumes. Each data point represents the mean ±SEM of 5 mice. (C) Average weight of tumors in nude mice. (D) The mRNA expression of UHRF1 in transplanted tumors formed by MKN45-shUHRF1 and MKN45-shNC cells was measured using qRT-PCR. GAPDH was used as an internal control and the fold change was calculated by 2^−ΔΔCt. (E) Representative photographs of H&E staining and immunohistochemical analysis of Ki-67 antigen in tumors of nude mice (left). Comparison of proliferation index is shown on the right.