Protein kinase N1 promotes proliferation and invasion of liver cancer

Abstract

Protein kinase (PK) N1, also called PKC-related protein 1, participates in the proliferation, invasion and metastasis of various malignant tumors. However, the role of PKN1 in liver cancer remains to be elucidated. The present study investigated the expression of PKN1 using immunohistochemistry in surgical specimens from 36 patients and analyzed the correlation with VEGF, microvascular density (MVD), cell proliferation index (Ki67) and clinicopathological parameters. PKN1 was highly expressed in hepatocellular carcinoma (HCC) and was positively correlated with histological grading of HCC, Ki67 expression and MVD. PKN1 expression in moderately and poorly differentiated HCC was significantly higher compared with highly differentiated HCC. Expression of PKN1 was positively correlated with Ki67 and MVD, and Ki67 expression was positively correlated with MVD. The effects of PKN1 on proliferation, invasion and apoptosis of liver cancer cells were detected in vitro. Cell viability, migration and invasion were reduced and the apoptosis rate was significantly improved when PKN1 expression was silenced in liver cancer cells. Thus, PKN1 serves an important role in the development and progression of liver cancer. Inhibition of PKN1 activity may provide a promising therapeutic target for liver cancer.

Keywords: liver cancer; microvascular density; proliferation; protein kinase N1.

Figure 1

Expression of PKN1, VEGF, Ki67 and MVD distribution in HCC tissues (original magnification, x400). (A) PKN1 immunohistochemically staining located in cytoplasm demonstrated higher intensity in moderately differentiated HCC and (B) lower intensity in highly differentiated HCC. (C) VEGF expression in the cytoplasm shows higher intensity in poorly differentiated HCC and (D) lower in highly differentiated HCC. (E) Ki67 expression in the nucleus demonstrated higher proliferation index in poorly differentiated HCC and (F) lower index in highly differentiated HCC. MVD had higher distribution in (G) moderately differentiated HCC compared with (H) highly differentiated HCC. PKN1, protein kinase N1; Ki67, cell proliferation index; MVD, microvascular density; HCC, hepatocellular carcinoma.

Figure 2

siPKN1-2 and siPKN1-3 were confirmed as efficient silencing sequences. PKN1 expression in HepG2 and Hep3B cell lines was detected by reverse transcription-quantitative PCR and western blotting following transfection with siPKN1-1, siPKN1-2 and siPKN1-3 sequences. It was determined that siPKN1-2 and siPKN1-3 were efficient silencing sequences to perform the following experiments. si, short interfering; PKN1, protein kinase N1; CON, control; NC, negative control.

Figure 3

Downregulated PKN1 expression suppresses proliferation of liver cancer cells. Silencing PKN1 inhibited proliferation of liver cancer cells. (A) Proliferation curve was generated according to optical density of proliferative cells at 0, 24 and 48 h after PKN1 transfection. (B) Colony forming ability was suppressed when cells were cultured for 10 days after transfection and stained with 0.1% crystal violet. ^**P<0.01. PKN1, protein kinase N1; NC, negative control; si, short interfering.

Figure 4

Downregulated PKN1 expression inhibits migration and invasion of liver cancer cells (original magnification, x100). (A) Wound scratch assay revealed that cell healing ability in siPKN1 group was significantly slower than the NC group. (B) Transwell invasion assay demonstrated that downregulation of PKN1 significantly reduced invasion of liver cancer cells across the Matrigel (original magnification, x400). ^**P<0.01. PKN1, protein kinase N1; si, short interfering; NC, negative control.

Figure 5

Downregulated PKN1 expression enhances apoptosis of liver cancer cells. Flow cytometry was used to analyzed apoptosis rate of HepG2 and Hep3B cells, including Q4 (early apoptosis) and Q2 (late apoptosis). The results demonstrated apoptosis was promoted when PKN1 was silenced compared with the NC groups. ^**P<0.01. PKN1, protein kinase N1; NC, negative control; si, short interfering.