Knockdown of serpin peptidase inhibitor clade C member 1 inhibits the growth of nasopharyngeal carcinoma cells

Abstract

Nasopharyngeal carcinoma (NPC) is a type of cancer originating in the nasopharynx. There are no NPC‑specific treatments available at present. Serpin peptidase inhibitor clade C member 1 (SERPINC1) serves roles in anticoagulation and anti‑inflammation. The aim of the present study was to investigate the role of SERPINC1 in the proliferation and apoptosis of NPC cells. Tumor and adjacent healthy tissue samples were collected from patients with NPC. Additionally, the SERPINC1 gene was silenced in the HNE3 cell line using short interfering RNA targeted against SERPINC1 (SERPINC1‑siRNA). Cell viability was determined via a Cell Counting Kit‑8 assay; furthermore, proliferation and apoptosis were investigated via flow cytometry. Western blotting and reverse transcription‑quantitative polymerase chain reaction analysis were performed to determine the expression levels of protein and mRNA. It was revealed that the expression levels of SERPINC1 mRNA and protein were increased in NPC tumor tissues compared with in adjacent healthy tissues. The expression of SERPINC1 mRNA and protein in HNE3 cells decreased following SERPINC1‑siRNA transfection. Furthermore, knockdown of SERPINC1 promoted apoptosis and inhibited proliferation. It was also demonstrated that silencing SERPINC1 upregulated the expression of B‑cell lymphoma-2 (Bcl‑2)‑associated X protein and p53 mRNA and protein, and downregulated that of Bcl‑2, survivin and cyclin D1. Downregulation of SERPINC1 reduced the phosphorylation of phosphatidylinositol 3‑kinase (PI3K), protein kinase B (Akt) and mammalian target of rapamycin (mTOR). Thus, SERPINC1 knockdown may promote the apoptosis of HNE3 cells and inhibit proliferation via the suppression of the PI3K/Akt/mTOR signaling pathway.

Figure 1.

Expression of SERPINC1 in NPC tissues. (A and B) Expression levels of SERPINC1 protein were determined in six paired tumor and healthy adjacent tissue samples collected from patients with NPC by performing western blot analysis. (C) Expression of SERPINC1 mRNA was analyzed in 41 patients by reverse transcription-quantitative polymerase chain reaction. Data are presented as the mean ± standard deviation. *P<0.05. SERPINC1, serpin peptidase inhibitor clade C member 1.

Figure 2.

Effects of SERPINC1-siRNA transfection on the expression of SERPINC1 and the viability of HNE3 cells. HNE3 cells were treated with PBS, transfected with empty vector or SERPINC1-siRNA. (A and B) Western blot analysis was performed to determine the expression levels of SERPINC1 protein in each group. (C) Reverse transcription-quantitative polymerase chain reaction was performed to determine the mRNA expression of SERPINC1 in each group. (D) Cell viability was analyzed using a Cell Counting Kit-8 assay. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 vs. the empty vector group. Empty vector, negative control siRNA; NS, not significant; SERPINC1, serpin peptidase inhibitor clade C member 1; siRNA, small interfering RNA.

Figure 3.

Effects of SERPINC1-siRNA transfection on apoptosis and proliferation of HNE3 cells. (A and B) Flow cytometry was performed to evaluate apoptosis in non-transfected, empty vector-transfected and SERPINC1-siRNA-transfected cells. (C and D) Flow cytometry was performed to analyze the proliferation of each group following transfection. Data are presented as the mean ± standard deviation. **P<0.01 vs. the empty vector group. 7-AAD, 7-aminoactinomycin D; empty vector, negative control siRNA; NS, not significant; PE, phycoerythrin; SERPINC1, serpin peptidase inhibitor clade C member 1; siRNA, small interfering RNA.

Figure 4.

Effects of SERPINC1-siRNA transfection on the expression of apoptosis and cell cycle-associated genes in HNE3 cells. Reverse transcription-quantitative polymerase chain reaction was performed to determine the expression levels of (A) Bax, (B) Bcl-2, (C) survivin, (D) cyclin D1 and (E) p53 mRNA in non-transfected, empty vector-transfected and SERPINC1-siRNA-transfected cells. (F and G) Expression levels of Bax, Bcl-2, survivin, cyclin D1 and p53 proteins were determined via western blot analysis. Data are presented as the mean ± standard deviation. **P<0.01 vs. the empty vector group. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; empty vector, negative control siRNA; NS, not significant; SERPINC1, serpin peptidase inhibitor clade C member 1; siRNA, small interfering RNA.

Figure 5.

Effects of SERPINC1-siRNA transfection on PI3K/Akt/mTOR pathway. (A) Western blot analysis was performed to determine the expression and phosphorylation of PI3K, Akt and mTOR proteins. (B-D) p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR ratios were calculated for non-transfected, empty vector-transfected and SERPINC1-siRNA-transfected cells. Data are presented as the mean ± standard deviation. **P<0.01 vs. the empty vector group. Akt, protein kinase B; empty vector, negative control siRNA; mTOR, mammalian target of rapamycin; NS, not significant; p, phosphorylated; PI3K, phosphatidylinositol 3-kinase; SERPINC1, serpin peptidase inhibitor clade C member 1; siRNA, small interfering RNA.