Metallothionein-like 5 expression is correlated with poor prognosis and promotes proliferation of cervical squamous cell carcinoma

Figures & data

Table 1. Correlations between MTL5 and clinicopathologic characteristics

Variables	Cases (n=133)	MTL5 protein level		P value
		Low (n=50)	High (n=83)
Age (year)				0.407
< 45	62	21	41
≥ 45	71	29	42
Horizontal diffusion diameter				0.002*
< 4.0 cm	90	42	48
≥ 4.0 cm	43	8	35
Stromal invasion depth				0.089
< 2/3	78	34	44
≥ 2/3	55	16	39
Vagina invasion				0.214
Negative	101	35	66
Positive	32	15	17
Parametrial invasion				0.445
Negative	96	38	58
Positive	37	12	25
Lymphovascular invasion				0.111
Negative	90	38	52
Positive	43	12	31
Pathological differentiation				0.432
Well	21	8	13
Moderate	99	35	64
Poor	13	7	6
Lymph node metastasis				0.005*
Negative	78	37	41
Positive	55	13	42
FIGO stage				<0.001*
Stage I	72	39	33
Stage II	61	11	50

* indicates P<0.05 with statistical significance.

Figure 1. mRNA and protein expression of MTL5 in CESC.

(a) mRNA level of MTL5 was evaluated by RT-qPCR in 17 pairs of fresh-resected CESC tumor tissues and nontumorous ovarian tissues, indicating that CESCs possess higher MTL5-mRNA levels compared to normal cervical tissues. Data was analyzed by paired Student’s t-test (P = 0.002). (b) Representative high protein level of MTL5 in CESC tissues, showing predominant nucleus localization. (c) Representative low-protein immunostaining of MTL5 in CESC tissues. (d-f) Box plots showed the differential MTL5 protein expression in CESC tissues with different horizontal diameters (d), lymph node status (e), and FIGO stages (f). The Y-axis was presented as the IHC score of MTL5 in CESC tissues. Data was shown as mean ± SD and compared by unpaired Student’s t-test.

Table 2. Disease-free survival (DFS) analyses by Kaplan–Meier method and log-rank test

Variables	Cases (n=133)	5-year DFS rate (%)	Mean DFS (months)	P value
Age (year)				0.070
< 45	62	32.9%	56.0 ± 4.6
≥ 45	71	55.9%	63.6 ± 4.8
Horizontal diffusion diameter				0.041*
< 4.0 cm	90	49.0%	63.0 ± 4.1
≥ 4.0 cm	43	27.6%	42.3 ± 4.9
Stromal invasion depth				0.034*
< 2/3	78	49.9%	64.3 ± 4.4
≥ 2/3	55	32.6%	47.4 ± 4.2
Vagina invasion				0.357
Negative	101	42.5%	59.8 ± 3.8
Positive	32	42.5%	53.4 ± 6.7
Parametrial invasion				<0.001*
Negative	96	52.3%	68.0 ± 4.1
Positive	37	20.5%	39.9 ± 4.3
Lymphovascular invasion				0.013*
Negative	90	48.9%	64.8 ± 4.1
Positive	43	30.3%	46.0 ± 4.8
Pathological differentiation				0.273
Well	21	53.0%	60.4 ± 5.8
Moderate	99	41.9%	58.5 ± 4.0
Poor	13	25.2%	35.7 ± 4.9
Lymph node metastasis				0.005*
Negative	78	50.7%	67.4 ± 4.5
Positive	55	30.6%	45.7 ± 4.0
FIGO stage				<0.001*
Stage I	72	57.2%	69.3 ± 4.5
Stage II	61	19.2%	43.7 ± 3.8
MTL5 expression				<0.001*
Low	50	63.0%	74.0 ± 4.9
High	83	28.3%	42.9 ± 2.6

* indicates P<0.05 with statistical significance.

Figure 2. Disease-free survival analyses of CESC cohort.

Kaplan–Meier method was used to analyze the prognostic significances of retrieved clinicopathological characteristics, such as MTL5 protein level (a), age at diagnosis (b), horizontal diffusion diameter (c), stromal invasion depth (d), parametrial invasion (e), lymphovascular invasion (f), lymph node metastasis (g), and FIGO stage (h). Data was analyzed by log-rank test.

Figure 3. The mRNA level of MTL5 and its clinical significance in TCGA database.

(a) The mRNA level of MTL5 in CESC tissues was extracted from TCGA database. By analyzing the transcripts per million (TPM) level of MTL5 in CESC tissues (n = 306) and normal cervix tissues (n = 13), we found that MTL5-mRNA level was significantly higher in CESC tissues (P < 0.001). (b) Relationship between MTL5-mRNA level and overall survival of CESC patients was plotted by Kaplan–Meier method, which indicated an unfavorable effect of MTL5 on CESC prognosis.

Table 3. Multivariate analysis by Cox regression model

Variables	Hazard ratio	95% CI	P value
Age	0.649	0.383-1.101	0.109
Horizontal diffusion diameter	1.028	0.578-1.827	0.926
Stromal invasion depth	1.378	0.814-2.334	0.232
Parametrial invasion	1.866	1.030-3.380	0.040*
Lymphovascular invasion	1.481	0.872-2.514	0.146
Lymph node metastasis	1.361	0.765-2.422	0.294
FIGO stage	1.829	1.040-3.217	0.036*
MTL5 expression	1.961	1.034-3.720	0.039*

* indicates P<0.05 with statistical significance.

Figure 4. The effects of MTL5 on CESC proliferation and growth.

(a, b) Two shRNAs targeting MTL5 (MTL5-sh#1 and MTL5-sh#2) as well as scrambled shRNA (Scram-sh) were transfected into C33A and SiHa cell lines, respectively. The knockdown efficiencies were tested by Western blotting. (c, d) CCK-8 assays reflected that MTL5-shRNA can significantly attenuate the proliferation of both C33A and SiHa cell lines. (e, f) The growth curves of xenografts generated by MTL5-sh#1 cells and Scram-sh cells showed that MTL5 interference remarkably impaired CESC tumor growth in vivo. (g, h) Images showed significantly different xenograft size after tumor isolation.