SPAG9 is involved in hepatocarcinoma cell migration and invasion via modulation of ELK1 expression

Figures & data

Figure 1 SPAG9 and ELK1 expression in HCC tissue specimens and cell lines.

Notes: (A) IHC staining of SPAG9 and ELK1 in hepatocarcinomas and adjacent noncancerous liver tissues. Weak SPAG9 and ELK1 staining can be observed in the adjacent noncancerous liver tissues (left panel) compared to the strong SPAG9 cytoplasmic staining and ELK1 nuclear staining in cancerous tissues (right panel) (magnification: 200×). (B) Protein and (C) mRNA levels of SPAG9 and ELK1 were examined in HCCLM3, HuH7, and HepG2 cell lines using Western blot and qRT-PCR analyses, respectively. (D) Mann–Whitney U-testing of IHC scores (***P<0.001) (the bars show standard error), and (E) Spearman rank correlation analysis of SPAG9 and ELK1 expression in HCC tissues.
Abbreviations: SPAG9, sperm-associated antigen 9; IHC, immunohistochemistry; qRT-PCR, quantitative real-time polymerase chain reaction; ELK1, ETS-like gene 1, tyrosine kinase; HCC, hepatocellular carcinoma.

Table 1 The relationship between SPAG9 expression and the clinical features of HCC

Parameters	Total	SPAG9 expression		P-value
		Positive n (%)	Negative n (%)
Age (year)
≤50	20	13 (65.0)	7 (35.0)	0.769
>50	30	17 (56.7)	13 (43.3)
Sex
Male	39	24 (61.5)	15 (38.5)	0.736
Female	11	6 (54.5)	5 (45.5)
HBsAg
Presence	36	22 (61.1)	14 (38.9)	0.283
Absence	14	8 (57.1)	6 (42.9)
Cirrhosis
Presence	30	21 (70.0)	9 (30.0)	0.089
Absence	20	9 (45.0)	11 (55.0)
TNM stage
I+II	31	15 (48.4)	16 (51.6)	0.032*
III+IV	19	15 (78.9)	4 (21.1)
Relapse or metastasis
Presence	30	22 (73.3)	8 (26.7)	0.018*
Absence	20	8 (40.0)	12 (60.0)
ELK1 status
Positive	21	18 (85.7)	3 (14.3)	0.002*
Negative	29	12 (41.4)	17 (58.6)

Note:

* P<0.05.

Abbreviations: SPAG9, sperm-associated antigen 9; HCC, hepatocellular carcinoma; HBsAg, hepatitis B surface antigen; TNM, tumor node metastasis; ELK1, ETS-like gene 1, tyrosine kinase.

Figure 2 Reduction in SPAG9 expression suppresses cell migration and invasion of HCC cell lines.

Notes: (A and B) SPAG9 siRNA-treated cells had decreased mRNA (A) and protein (B) levels compared to the control cells. (C) Results from the wound-healing assay showed that SPAG9 depletion decreased cell migration of HCCLM3 and HuH7 cells. (D) Results from the transwell migration and matrigel invasion assay showed that SPAG9 depletion decreased cell migration and invasion of HCCLM3 and HuH7 cells; *P<0.05, **P<0.01.
Abbreviations: SPAG9, sperm-associated antigen 9; siRNA, small interfering RNA; HCC, hepatocellular carcinoma; h, hour.

Figure 3 SPAG9 regulates ELK1 expression and p38 activation in HCC cell lines.

Notes: (A) Western blot and (B) qRT-PCR analyses revealed that siRNA knockdown of SPAG9 decreased protein and mRNA levels of SPAG9 and ELK1, respectively, in HCCLM3 and HuH7 cell lines. (C) Western blot analysis showed that SPAG9 depletion decreased SPAG9 and p-p38 levels in HCCLM3 and HuH7 cells.
Abbreviations: SPAG9, sperm-associated antigen 9; siRNA, small interfering RNA; qRT-PCR, quantitative real-time polymerase chain reaction; ELK1, ETS-like gene 1, tyrosine kinase; p-p38, phosphorylated protein-38; HCC, hepatocellular carcinoma.

Figure 4 SPAG9-induced migration and invasion of HCC cells is dependent on ELK1.

Notes: (A) Transwell migration and matrigel invasion assay revealed that ELK1 transfection rescued the inhibition in cell migration and invasion induced by SPAG9 depletion. (B) The expression of ELK1 was significantly upregulated in the siCtrl + ELK group and siSPAG9+ ELK1 group compared to the controls. (C) Transwell migration and matrigel invasion assay revealed that ELK1 depletion decreased the migration and invasion in HuH7 cells. (D) Western blot showed that siRNA knockdown of ELK1 decreased the protein level of ELK1, while SPAG9 expression was not changed in HuH7 cells. *P<0.05.
Abbreviations: SPAG9, sperm-associated antigen 9; siSPAG9, SPAG9 siRNA; siCtrl, control siRNA; siRNA, small interfering RNA; ELK1, ETS-like gene 1, tyrosine kinase; HCC, hepatocellular carcinoma.