Up-regulation of SOCS4 promotes cell proliferation and migration in esophageal squamous cell carcinoma

Abstract

Background: Suppressors of cytokine signaling family member 4 (SOCS4) was shown to serve critical and multifaceted roles in the progression of numerous cancers, including hepatocellular carcinoma, thyroid cancer, breast cancer, and lung adenocarcinoma. While, the expression and the roles of SOCS4 in esophageal squamous cell carcinoma (ESCC) remain elusive. The current study is aimed to investigate the expression pattern and functions of SOCS4 in ESCC.

Methods: The relationship between SOCS4 and the clinicopathological features of ESCC was analyzed. SOCS4 expression in ESCC tissues was measured by western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemical (IHC) staining. The roles of SOCS4 in modulating ESCC cell behaviors were examined using a series of assays, including cell proliferation assay, cell counting kit-8 (CCK-8) assay, cell cycle analysis, and wound-healing assay.

Results: In human ESCC tissues, SOCS4 expression was up-regulated and correlated with tumor size and lymph node metastasis, however was not correlated with the overall survival (OS) of patients. SOCS4 silencing in ESCC cells resulted in the suppression of cell growth, which was related to the cell cycle. SOCS4 knockdown also inhibited nuclear factor-kappa B (NF-κB) signaling and decreased the migratory potential of ESCC cells.

Conclusions: These findings revealed that increased expression of SOCS4 in ESCC may promote the progression, proliferation, and migration by NF-κB signaling. Inhibition of SOCS4 may be a promising therapeutic strategy for ESCC.

Keywords: NF-κB; Suppressors of cytokine signaling family member 4 (SOCS4); esophageal squamous cell carcinoma (ESCC); migration; proliferation.

Figure 1

SOCS4 expression was up-regulated in ESCC tissues. SOCS4 mRNA (A) expressions in ESCC tissue and normal tissues in TCGA database (A), SOCS4 protein expressions in ESCC tissue (T) and normal tissues (N) were measured using western blotting (B). *, P<0.05, compared with normal tissues. SOCS4, suppressors of cytokine signaling family member 4; ESCC, esophageal squamous cell carcinoma.

Figure 2

SOCS4 expression in ESCC tissues and adjacent normal tissues by immunohistochemistry. Representative immunostaining of SOCS4 in human ESCC tissues (A,B) and adjacent normal tissues (C,D), magnification 40× (A,C), magnification 200× (B,D). SOCS4, suppressors of cytokine signaling family member 4; ESCC, esophageal squamous cell carcinoma, Scale bar: 50 µm.

Figure 3

Kaplan-Meier survival curves of ESCC patients with a high SOCS4 expression (n=56) had a similar OS compared to those with a low SOCS4 expression (n=47). P>0.05. SOCS4, suppressors of cytokine signaling family member 4; ESCC, esophageal squamous cell carcinoma.

Figure 4

SOCS4 knockdown inhibits ESCC cell proliferation. SOCS4 expression in Eca-109 cells following transfection with control siRNA (Si-ctrl), SOCS4 siRNAs (Si-SOCS4-1 and Si-SOCS4-1) were measured by RT-qPCR (A) and western blot (B), respectively. The effect of SOCS4 knockdown on cell proliferation. Eca-109 cells transiently transfected with Si-ctrl, Si-SOCS4-1, and Si-SOCS4-1 were cultured for 72 h. Cell growth was measured by counting cell numbers (C,D), and cell viability was measured using an CCK-8 assay. The effect of SOCS4 knockdown on cell cycle was measured by flow cytometry (E). ***, P<0.001, compared with Si-ctrl. SOCS4, suppressors of cytokine signaling family member 4; ESCC, esophageal squamous cell carcinoma.

Figure 5

SOCS4 knockdown inhibits ESCC cell migration. (A,B) Effects of SOCS4 knockdown on cell migration. Eca-109 cells transfected with control siRNA (Si-ctrl) or SOCS4 siRNAs (Si-SOCS4-1 and Si-SOCS4-1) were subjected to a wound closure assay. ***, P<0.001, compared with Si-ctrl (100×). SOCS4, suppressors of cytokine signaling family member 4; ESCC, esophageal squamous cell carcinoma.

Figure 6

SOCS4 knockdown inhibits NF-κB signaling. Eca-109 cells were transfected with control siRNA (Si-ctrl) or SOCS4 siRNAs (Si-SOCS4-1 and Si-SOCS4-1), and then the cells were prepared for immunoblotting against p-P65, P65, ICAM1, VCAM1, uPA, and TNFα. β-actin was used as an internal loading control. **, P<0.001, compared with Si-ctrl. SOCS4, suppressors of cytokine signaling family member 4.