INHBA knockdown inhibits proliferation and invasion of nasopharyngeal carcinoma SUNE1 cells in vitro

Abstract

Up-regulated expression of INHBA has been reported in multiple malignant tumors. However, in nasopharyngeal carcinoma (NPC), the expression pattern and clinical significance of INHBA are still unclear. This study aimed to detect the expression of INHBA and its prognostic significance in NPC, then explore the tumor-associated functions of INHBA gene and the potential mechanism. The INHBA expression of mRNA levels in tumor tissues and noncancerous nasopharyngeal tissues was investigated by RT-qPCR. The protein expression in cells were detected by western blot. Cell proliferation was detected by CCK assay and cell invasion ability was evaluated by Transwell assay. The expression of INHBA in paraffin-embedded NPC tissues was detected by immunohistochemistry (IHC). Statistical analyses were further applied to assess the clinical significance of INHBA expression. The result reveals INHBA mRNA level is elevated in NPC tissues compared to those in noncancerous nasopharyngeal epithelial tissues. In paraffin-embedded NPC tissues, immunoreactivity of INHBA was primarily detected in 53.70% (58/108) of these patients. The overexpression was notably associated with the clinical stage (UICC) (P=0.048), N classification (P=0.042), carotid sheath involvement (P=0.016), and decreased disease-free survival (DFS) (P=0.004) and overall survival (OS) (P=0.010). Multivariate analysis revealed that INHBA expression was an independent prognostic factor for DFS (P=0.028). CCK assay showed SUNE1 cells' proliferation was decreased in INHBA knockdown group than control. Transwell assay showed the invasion of SUNE1 cells was decreased in INHBA knockdown group by comparison with control. Further study showed knockdown of INHBA expression in SUNE1 cells could block the TGF-β signaling pathway. In conclusion, INHBA is up-regulated in NPC, and is significantly correlated with clinical stage (UICC), N stage, carotid sheath involvement, and survival. Knockdown INHBA in SUNE1 cells could inhibit the cells' proliferation and invasion. The underlying mechanism may be blockade of the TGF-β signaling pathway.

Keywords: INHBA; NPC; invasion; prognosis; proliferation.

Figure 1

The mRNA expression of INHBA in noncancerous nasopharynx tissues and NPC tissues. Expression levels of INHBA in 40 NPC tissues and 16 noncancerous nasopharynx tissues were detected by real-time PCR.

Figure 2

Representative IHC staining of INHBA in NPC tissue samples. INHBA expression was mainly localized in the cytoplasm. Magnification is 400×. A. Negative staining of INHBA. B. “+” (weakly positive) expression of INHBA. C. “++” (positive) expression of INHBA. D. “+++” (strongly positive) expression of INHBA.

Figure 3

Kaplan-Meier survival curves by univariate analysis (log-rank test). A. DFS rate for NPC patients with high INHBA expression compared to those with low INHBA expression. B. OS rate for NPC patients with high INHBA expression compared to those with low INHBA expression.

Figure 4

Kaplan-Meier survival curves by univariate analysis (log-rank test) in specific patient subgroups stratified by clinical stage (UICC), T stage, and N stage. A, B. DFS and OS rate for NPC patients of early stage tumor with high INHBA expression compared to those with low INHBA expression. C, D. DFS and OS rate for NPC patients of advanced stage tumor with high INHBA expression compared to those with low INHBA expression. E, F. DFS and OS rate for NPC patients of T1-2 tumor with high INHBA expression compared to those with low INHBA expression. G, H. DFS and OS rate for NPC patients of T3-4 tumor with high INHBA expression compared to those with low INHBA expression. I, J. DFS and OS rate for NPC patients of N0 tumor with high INHBA expression compared to those with low INHBA expression. K, L. DFS and OS rate for NPC patients of N1-3 tumor with high INHBA expression compared to those with low INHBA expression.

Figure 5

Knockdown INHBA in SUNE1 cells inhibit cell proliferation and invasion in vitro. A. Representative images of cell invasion evaluated by Transwell assay (200×). B. Quantification of the cell number invaded through the membrane. C. CCK assay was performed to determine the cell proliferation under each treatment. Decreased proliferation of SUNE1 cells was detected after INHBA knockdown. *P<0.05 versus the control group.

Figure 6

INHBA knockdown blocked the TGF-β signaling pathway in SUNE1 cells. A. Protein levels of TGF-β1, p-Smad2, p-Smad3 were assessed by western blot in SUNE1 INHBA-shRNA cells and control cells. B. Gray value of protein bands of A.