Objective:

2

To investigate the effects of Pien Tze Huang (PZH) on the migration and invasion of HCC cells and underlying molecular mechanism.

Methods:

2

Cell counting kit-8 (CCK-8) was applied to evaluate the cell viabilities of SMMC-7721

SK-Hep-1

C3A and HL-7702 (6×10

3

cells/well) co-incubated with different concentrations of PZH (0

0.2

0.4

0.6

0.8 mg/mL) for 24 h. Transwell

wound healing assay

CCK-8 and Annexin V-FITC/PI staining were conducted to investigate the effects of PZH on the migration

invasion

proliferation and apoptosis of SK-Hep-1 and SMMC-7721 cells (650 μg/mL for SK-Hep-1 cells and 330 μg/mL for SMMC-7721 cells)

respectively.

In vivo

lung metastasis mouse model constructed by tail vein injection of HCC cells was used for evaluating the anti-metastasis function of PZH. SK-Hep-1 cells (10

6

cells/200 μL per mice) were injected into B-NDG mice via tail vein. Totally 8 mice were randomly divided into PZH and control groups

4 mice in each group. After 2-d inoculation

mice in the PZH group were administered with PZH (250 mg/kg

daily) and mice in the control group received only vehicle (PBS) from the 2nd day after xenograft to day 17. Transcriptome analysis based on RNA-seq was subsequently used for deciphering anti-tumor mechanism of PZH. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were applied to verify RNA-seq results. Luciferase reporter assay was performed to examine the transcriptional activity of yes-associated protein (YAP).

Results:

2

PZH treatment significantly inhibited the migration

invasion

proliferation and promoted the apoptosis of HCC cells

in vitro

and

in vivo

(

P

<

0.01). Transcriptome analysis indicated that Hippo signaling pathway was associated with anti-metastasis function of PZH. Mechanical study showed PZH significantly inhibited the expressions of platelet derived growth factor receptor beta (PDGFRB)

YAP

connective tissue growth factor (CCN2)

N-cadherin

vimentin and matrix metallopeptidase 2 (MMP2

P

<

0.01). Meanwhile

the phosphorylation of YAP was also enhanced by PZH treatment

in vitro

and

in vivo

. Furthermore

PZH played roles in inhibiting the transcriptional activity of YAP.

Conclusion:

2

PZH restrained migration

invasion and epithelial-mesenchymal transition of HCC cells through repressing PDGFRB/YAP/CCN2 axis.