PCSK9 promotes tumor growth by inhibiting tumor cell apoptosis in hepatocellular carcinoma

Abstract

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9), one of the key enzymes in the process of lipid transport, is involved in the disease progression of various types of tumors. This article is to study the role of PCSK9 in the progression of hepatocellular carcinoma (HCC).

Methods: Immunohistochemistry was used to assess the expression of PCSK9 in tumor specimens from 105 HCC patients who underwent curative resection. Western blotting and quantitative real-time PCR were used to test the protein and mRNA expression levels in HCC cell lines. Cell Counting Kit-8 (CCK-8) and clone formation assays were performed to evaluate the proliferation ability of different kinds of cells in vitro. Flow cytometry was used to analyze cell cycle distribution and apoptosis rate. A xenograft model was established to study the effect of PCSK9 on HCC growth in vivo. TUNEL and immunofluorescence assays were used to detect cell apoptosis.

Results: High expression of PCSK9 in tumor tissues was related to microvascular invasion (p = 0.036) and large tumor size (p = 0.001) in HCC patients. Overall survival and disease-free survival after surgery were poor in patients with high expression of PCSK9 (p = 0.035 and p = 0.007, respectively). In vivo and in vitro experiments showed that PCSK9 promoted the growth of HCC by inhibiting cell apoptosis. A mechanistic study revealed that PCSK9 increases FASN expression, thereby inhibiting apoptosis of HCC cells via the Bax/Bcl-2/Caspase9/Caspase3 pathway.

Conclusions: PCSK9 expression level in HCC is an indicator of poor prognosis for patients with HCC. FASN-mediated anti-apoptosis plays an important role in PCSK9-induced HCC progression.

Keywords: Apoptosis; FASN; Hepatocellular carcinoma; PCSK9.

Fig. 1

Intratumoral PCSK9 expression was associated with prognosis in HCC patients who underwent curative resection. a Immunohistochemistry staining of liver cancer tissues from different HCC patients. Typical cases with low or high PCSK9 expression are shown. Kaplan–Meier analysis showed that patients with high PCSK9 expression had poor overall survival (p = 0.035) (b) and poor disease-free survival (p = 0.007) (c)

Fig. 2

Construction of HCC cell lines with overexpression or downregulation of PCSK9. a Relative mRNA expression level of PCSK9 in HCC cell lines and a normal hepatocyte cell line (L02). b Transfection efficiency of PCSK9-overexpressing virus in the HCCLM3 cell line. c Transfection efficiency of PCSK9-knockdown virus in the HepG2 cell line. d Relative mRNA expression level of PCSK9 in the HCCLM3 cell line (p < 0.001). e Western blot images and summarized data showing that PCSK9 was successfully overexpressed in the HCCLM3 cell line (p = 0.014). f Relative mRNA expression level of PCSK9 in the HepG2 cell line (p = 0.004). g Western blot images and summarized data showing that PCSK9 was successfully downregulated in the HepG2 cell line (p = 0.001). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

PCSK9 inhibited apoptosis and promoted the proliferation of HCC cells in vitro. a CCK8 assay for cell proliferation of HCCLM3-PCSK9 cells and HepG2-shPCSK9 cells compared with their vector control. PCSK9 promoted HCC cell proliferation. b Clone formation and summarized data for HCCLM3-PCSK9 cells (p = 0.005) and HepG2-shPCSK9 cells (p = 0.001) compared with their vector controls. PCSK9 promoted clone formation. c Flow cytometry detection of the cell cycle distribution of HCCLM3-PCSK9 cells and HepG2-shPCSK9 cells compared with their vector controls. PCSK9 had no significant influence on the G2/M phase in cell cycle distribution (p > 0.05). d Flow cytometry apoptosis detection to determine the cell apoptosis rates of HCCLM3-PCSK9 cells (p < 0.001) and HepG2-shPCSK9 cells (p < 0.001) compared with their vector controls. PCSK9 decreased the apoptosis rate of HCC cells in vitro. **p < 0.01, ***p < 0.001

Fig. 4

PCSK9 promoted HCC growth in vivo. We constructed orthotopic human HCC xenograft mouse models with HCC cell lines with overexpression or downregulation of PCSK9. a Comparison of tumor volume (0.29 ± 0.26 vs. 1.13 ± 0.43 cm³, n = 6, p = 0.001) and tumor mass (0.61 ± 0.35 vs. 1.59 ± 0.25 g, n = 6, p < 0.001) of the HCCLM3-Vector and HCCLM3-PCSK9 groups. b Comparison of tumor volume (0.34 ± 0.13 vs. 0.18 ± 0.12 cm³, n = 6, p = 0.045) and tumor mass (0.61 ± 0.17 vs. 0.33 ± 0.15 g, n = 6, p = 0.013) of the HepG2-shVector and HepG2-shPCSK9 groups. c TUNEL fluorescence of liver cancer from BALB/c nude mice orthotopically implanted with HCCLM3 or HepG2 cells and their control groups. The apoptosis rate was lower in the HCCLM3-PCSK9 group than in the control group (p = 0.001). The apoptosis rate was higher in the HepG2-shPCSK9 group than in the control group (p = 0.006). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

The FASN/Bax/Bcl-2/Caspase9/Caspase3 pathway is involved in the regulation of PCSK9 on HCC cell apoptosis. a Western blot images and summarized data showing that FASN is overexpressed in the HCCLM3-PCSK9 cell line compared with its control group (p = 0.001). b Western blot images and summarized data showing that FASN is downregulated in the HepG2-shPCSK9 cell line compared with its control group (p < 0.001). c The Bax/Bcl-2/Caspase9/Caspase3 apoptosis signaling pathway-associated proteins were detected by Western blot in HCCLM3-PCSK9 cells and controls. d The Bax/Bcl-2/Caspase9/Caspase3 apoptotic signaling pathway-associated proteins were detected by Western blot in HepG2-shPCSK9 cells and controls. e Specifically blocking FASN with C75 altered the expression of Bax/Bcl-2/Caspase9/Caspase3 apoptotic signaling pathway-associated proteins in the HCCLM3-PCSK9 cell line. *p < 0.05, **p < 0.01, ***p < 0.001