HSPA4 Knockdown Retarded Progression and Development of Colorectal Cancer

Figures & data

Figure 1 HSPA4 is highly expressed in CRC. (A and B) Expression levels of HSPA4 in CRC tumor tissues and adjacent normal tissues were detected by qRT-PCR (A) and IHC staining (B). (C) Kaplan-Meier survival analysis of HSPA4 expression and overall survival in CRC patients. Data are presented as mean ± SD (n = 3) of three triplicates or independent experiments.

Table 1 Expression Patterns in Colorectal Cancer Tissues and Para-Carcinoma Tissues Revealed in Immunohistochemistry Analysis

HSPA4 Expression	Tumor Tissue		Para-Carcinoma Tissue		P value
	Cases	Percentage	Cases	Percentage
Low	42	48.3%	62	89.9%	0.000
High	45	51.7%	7	10.1%

Table 2 Relationship Between HSPA4 Expression and Tumor Characteristics in Patients with Colorectal Cancer

Features	No. of Patients	HSPA4 Expression		P value
		Low	High
All patients	87	42	45
Age (years)				0.336
< 70	43	23	20
≥ 70	44	19	25
Gender				0.068
Male	43	25	18
Female	44	17	27
Naked eye typing				0.131
Mass type	23	8	15
Ulcerative type	13	5	8
Infiltrative type	50	29	21
Differentiation				0.657
Undifferentiated/ low differentiated	14	6	8
Medium differentiation/high differentiation	73	36	37
Tumor size				0.260
< 5cm	39	16	23
≥ 5cm	47	25	22
Tumor site				0.410
Left colon	24	13	11
Right colon	61	27	34
Stage				0.019
Stage 1/2	51	30	21
Stage 3/4	36	12	24

Table 3 The Correlation Between HSPA4 Expression and Multivariate Cox Regression

Variables		Univariate Analysis		Multivariate Analysis
		HR (95%CI)	P value	HR (95% CI)	P value
HSPA4 expression	High	1		1
	Low	0.526(0.292–0.948)	0.033	0.473(0.233–0.959)	0.038
Gender	Female	1		1
	Male	0.889(0.502–1.576)	0.688	0.770(0.410–1.445)	0.415
Naked eye type	Mass type	1		1
	Ulcerative type	3.504(1.418–8.655)	0.007	2.937(1.132–7.619)	0.027
	Infiltrative type	1.608(0.755–3.426)	0.218	1.51(0.679–3.354)	0.312
Differentiation	Undifferentiated /low	1		1
	Middle/ high	0.378(0.191–0.745)	0.005	0.396(0.189–0.829)	0.014
Tumor size	< 5	1		1
	≥5	1.263(0.702–2.271)	0.436	1.358(0.716–2.576)	0.348
Tumor site	Left colon	1		1
	Right colon	0.99(0.521–1.881)	0.975	0.753(0.378–1.502)	0.421
Stage	Stage 1/2	1		1
	Stage 3/4	2.341(1.318–4.16)	0.004	2.034(1.029–4.019)	0.041

Figure 2 Knockdown of HSPA4 inhibits cell proliferation and promotes apoptosis in CRC cells. (A) Cell proliferation of HCT116 and RKO cells with or without knockdown of HSPA4 was evaluated in Celigo cell counting assays. Flow cytometry analysis based on Annexin V-APC staining was utilized to detect cell apoptotic ratio (B) and cell cycle distribution (C) for HCT116 and RKO cells. Data are presented as the mean ± SD (n = 3) of three triplicates or independent experiments. **P<0.01, ***P<0.001.

Figure 3 Knockdown of HSPA4 inhibits cell migration in CRC cells. (A and B) Cell migration of HCT116 and RKO cells with or without knockdown of HSPA4 was evaluated in wound healing assay (A) and Transwell assay (B). Data are presented as the mean ± SD (n = 3) of three triplicates or independent experiments. **P<0.01, ***P<0.001.

Figure 4 Knockdown of HSPA4 inhibits tumor growth in mice xenograft models. (A) The total bioluminescent intensity of tumors in shCtrl and shHSPA4 RKO cell groups. (B) Tumor volume measurements comparing shCtrl and shHSPA4 RKO cell groups. (C) Average tumor weights in shCtrl and shHSPA4 RKO cell groups. Images of mice and tumors in shCtrl and shHSPA4 groups. (D) Ki67 staining of tumor tissues in shCtrl and shHSPA4 groups. (A–C) data represent the mean ± SD of ten mice with 3 representative images shown. *P<0.05, ***P<0.001.

Figure 5 Exploration of molecular mechanisms downstream of HSPA4. (A) Human apoptosis antibody array analysis was performed in RKO cells with or without HSPA4 knockdown. (B) Western blotting analysis of the indicated signaling proteins in RKO cells with or without HSPA4 knockdown. Data are presented as the mean ± SD (n = 3) of three triplicates or independent experiments. *P<0.05, **P<0.01, ***P<0.001.