Overexpression of COL5A1 promotes tumor progression and metastasis and correlates with poor survival of patients with clear cell renal cell carcinoma

Figures & data

Figure 1 Expression of COL5A1 in tissue and cell lines.

Notes: (A) Representative COL5A1 immunohistochemical images (200×) in adjacent normal tissue and ccRCC tissue. COL5A1 protein expression in the ccRCC tissue was significantly higher than that in the adjacent normal tissue. (B) Western blot detection of COL5A1 protein expression in HK-2, 786-O, and Caki-1 cells. COL5A1 protein was upregulated in ccRCC cell lines in comparison with HK-2 cells. GAPDH was used as loading control. (C) COL5A1 mRNA expression in HK-2, 786-O, and Caki-1 cells. The ccRCC cell lines showed a significantly higher COL5A1 mRNA expression in comparison with HK-2 (all P<0.05).

Abbreviation: ccRCC, clear cell renal cell carcinoma.

Table 1 The expression of COL5A1 protein in ccRCC tissue and paired adjacent normal tissue

Type of tissue	No.	COL5A1 immunostaining (%)
		−	1+	2+	3+
ccRCC tissue	256	16 (6.3)	166 (64.8)	70 (27.3)	4 (1.6)
Adjacent normal tissue	256	237 (92.6)	19 (7.4)	0	0

Abbreviation: ccRCC, clear cell renal cell carcinoma.

Table 2 The relationship of COL5A1 with the clinicopathological characteristics of patients with ccRCC

Characteristics	No.	COL5A1 mRNA expression (fold: T/N)	P-value	COL5A1 protein expression			P-value
				0/1+	2+	3+
Age (years)			0.710				0.657
<60	136	1.72±0.59		100	34	2
≥60	120	1.75±0.56		82	36	2
Gender			0.819				0.156
Male	151	1.74±0.56		113	37	1
Female	105	1.74±0.60		69	33	3
Tumor diameter (cm)			0.001				<0.001
≤7	213	1.66±0.49		162	51	0
>7	43	2.14±0.76		20	19	4
TNM stage			<0.001				<0.001
I + II	200	1.60±0.44		162	38	0
III + IV	56	2.21±0.72		20	32	4
Fuhrman grade			<0.001				<0.001
G1 + G2	164	1.61±0.45		130	34	0
G3 + G4	92	1.96±0.69		52	36	4
Distant metastasis			<0.001				<0.001
Absent	199	1.60±0.44		161	38	0
Present	57	2.20±0.72		21	32	4
Recurrence (after surgery)			<0.001				0.002
Absent	144	1.51±0.41		124	20	0
Present	55	1.83±0.45		37	18	0
Necrosis			<0.001				<0.001
Absent	185	1.61±0.43		149	36	0
Present	71	2.07±0.74		33	34	4
Sarcomatoid			0.001				<0.001
Absent	173	1.63±0.48		135	38	0
Present	83	1.96±0.68		47	32	4
ECOG PS			0.184				0.003
0	208	1.71±0.56		157	49	2
≥1	48	1.85±0.64		25	21	2
UISS category			0.002				<0.001
Low risk	136	1.61±0.48		112	24	0
Medium risk	105	1.83±0.59		64	39	2
High risk	15	2.22±0.88		6	7	2
SSIGN score			<0.001				<0.001
0–3	162	1.57±0.43		138	24	0
4–7	57	1.69±0.46		40	17	0
≥8	37	2.52±0.66		4	29	4

Abbreviations: ccRCC, clear cell renal cell carcinoma; ECOG PS, Eastern Cooperative Oncology Group performance status; N, adjacent normal tissue; SSIGN, Mayo clinic stage, size, grade, and necrosis; T, ccRCC tissue; UISS, University of California Los Angeles Integrated Staging System.

Figure 2 COL5A1 mRNA is correlated with survival of ccRCC patients.

Notes: (A) COL5A1 mRNA expression in patients with localized and metastatic ccRCC. Patients with metastatic ccRCC had significantly higher COL5A1 mRNA expression compared to patients with localized ccRCC (P<0.05). (B) Kaplan–Meier curves for OS after surgery. Patients with high COL5A1 mRNA expression had poor OS (log-rank test, P<0.001). (C) COL5A1 mRNA expression in ccRCC patients with and without recurrence. Patients with recurrence had significantly higher COL5A1 mRNA expression compared to patients without recurrence (P<0.05). (D) Kaplan–Meier curves for RFS after surgery. Patients with high COL5A1 mRNA expression had poor RFS (log-rank test, P<0.001).

Abbreviations: ccRCC, clear cell renal cell carcinoma; OS, overall survival; RFS, recurrence-free survival.

Table 3 Univariate and multivariate analyses for predicting overall survival of patients with ccRCC (n=256)

Characteristics	Univariate analysis		Multivariate analysis
	HR (95% CI)	P-value	HR (95% CI)	P-value
Age (<60 years vs ≥60 years)	1.037 (0.653–1.647)	0.879	–	–
Gender (female vs male)	1.209 (0.758–1.930)	0.426	–	–
Tumor diameter (≤7 cm vs >7 cm)	1.656 (0.971–2.824)	0.064	–	–
Tumor stage (I + II vs III + IV)	3.633 (2.284–5.779)	<0.001	1.837 (1.063–3.175)	0.029
Tumor grade (1+2 vs 3+4)	3.432 (2.120–5.555)	<0.001	–	–
Metastasis (absent vs present)	3.416 (2.145–5.441)	<0.001	–	–
Necrosis (absent vs present)	3.087 (1.942–4.907)	<0.001	2.214 (1.360–3.607)	0.001
Sarcomatoid (absent vs present)	2.707 (1.702–4.306)	<0.001	1.871 (1.130–3.097)	0.015
ECOG PS (0 vs ≥1)	1.636 (0.983–2.722)	0.058	–	–
COL5A1 expression (low vs high)	2.577 (1.523–4.359)	<0.001	1.876 (1.073–3.279)	0.027

Abbreviations: ccRCC, clear cell renal cell carcinoma; ECOG PS, Eastern Cooperative Oncology Group performance status.

Table 4 Comparison of the prognostic accuracy of the prognostic models and COL5A1 expression for predicting overall survival of patients with ccRCC

Model	Overall survival
	C-index	AIC
COL5A1 expression	0.645	664.244
TNM	0.664	654.684
TNM + COL5A1	0.715	650.497
UISS	0.689	644.157
UISS + COL5A1	0.736	634.873
SSIGN	0.715	644.780
SSIGN + COL5A1	0.742	642.472

Abbreviations: AIC, Akaike information criteria; ccRCC, clear cell renal cell carcinoma; SSIGN, Mayo clinic stage, size, grade, and necrosis; UISS, University of California Los Angeles Integrated Staging System.

Table 5 Univariate and multivariate analyses for predicting recurrence-free survival of patients with localized ccRCC (n=199)

Characteristics	Univariate analysis		Multivariate analysis
	HR (95% CI)	P-value	HR (95% CI)	P-value
Age (<60 years vs ≥60 years)	1.164 (0.682–1.968)	0.577	–	–
Gender (female vs male)	1.152 (0.674–1.969)	0.605	–	–
Tumor diameter (≤7 cm vs >7 cm)	2.679 (1.478–4.857)	0.001	–	–
Tumor stage (I vs II)	2.838 (1.583–5.089)	<0.001	–	–
Tumor grade (1+2 vs 3+4)	3.137 (1.828–5.383)	<0.001	4.843 (2.714–8.641)	<0.001
Necrosis (absent vs present)	2.314 (1.326–4.036)	0.003	–	–
Sarcomatoid (absent vs present)	2.550 (1.477–4.402)	0.001	3.554 (2.010–6.283)	<0.001
ECOG PS (0 vs ≥1)	1.892 (0.952–3.759)	0.069	–	–
COL5A1 expression (low vs high)	3.549 (2.002–6.293)	<0.001	4.751 (2.613–8.637)	<0.001

Abbreviations: ccRCC, clear cell renal cell carcinoma; ECOG PS, Eastern Cooperative Oncology Group performance status.

Table 6 Comparison of the prognostic accuracy of the prognostic models and COL5A1 expression for predicting recurrence-free survival of patients with localized ccRCC

Model	Recurrence-free survival
	C-index	AIC
COL5A1 expression	0.662	501.258
TNM	0.609	511.762
TNM + COL5A1	0.709	492.767
UISS	0.656	506.115
UISS + COL5A1	0.739	482.557
SSIGN	0.673	504.193
SSIGN + COL5A1	0.740	481.659

Abbreviations: AIC, Akaike information criteria; ccRCC, clear cell renal cell carcinoma; SSIGN, Mayo clinic stage, size, grade, and necrosis; UISS, University of California Los Angeles Integrated Staging System.

Figure 3 Effect of COL5A1 knockdown on cell proliferation and apoptosis in vitro.

Notes: (A) Effect of COL5A1 knockdown in Caki-1 cells was determined by real-time PCR and Western blot analysis. (B) Cell viability was assessed by MTT assay at 0, 24, 48, 72, and 96 hours after treatment with siRNA. Knockdown of COL5A1 inhibited Caki-1 cells proliferation. (C) Effect of COL5A1 on colony formation of Caki-1 cells. Knockdown of COL5A1 caused significant reduction of colony number (P<0.05). (D) Apoptosis assay with Caki-1 was done 48 hours post-siRNA transfection by flow cytometric analyses. Knockdown of COL5A1 significantly increased Caki-1 cells apoptosis (P<0.05).

Figure 4 Effect of COL5A1 knockdown on cell migration and invasion in vitro.

Notes: (A) Representative images of wound healing assay. Knockdown of COL5A1 significantly inhibited Caki-1 cells migration. (B) Representative images of crystal violet-stained migrated cells. Knockdown of COL5A1 significantly inhibited Caki-1 cells invasion.

Figure 5 Effect of COL5A1 knockdown on tumor growth in vivo.

Notes: (A) Knockdown of COL5A1 by siRNA inhibited the growth of Caki-1-derived xenografts in nude mice. (B) Knockdown of COL5A1 inhibited COL5A1 protein expression in vivo. (C) The tumor growth curves of the tumor xenografts. (D) The tumor weight of siRNA-control group and siRNA-COL5A1 group (P<0.05).