Ginsenoside-Rh2suppressesproliferationandmigrationofhepatocellularcarcinomacellsbytargetingEZH2toregulateCDKN2A-2Bgeneclustertranscription资源-CSDN文库

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Ginsenoside-Rh2 suppresses proliferation and migration of

hepatocellular carcinoma cells by targeting EZH2 to

regulate CDKN2A-2B gene cluster transcription

Li Qi

1,2*

(1. Core Laboratory for clinical Medical Research, Beijing Tian Tan Hospital, Capital Medical

University, Beijing, 100050;

2. Institute of Medical Plant Development, Chinese Academy of Medical Sciences and Peking

Union Medical College, Beijing, 100091) 10

Foundations: The Specialized Research Fund for the Doctoral Program of Higher Education (20131106120033)

Brief author introduction:Li Qi (1977-),Female,Associated researcher,Main research:Tumor molecular biology.

E-mail: liqi@implad.ac.cn

Abstract: Ginsenoside-Rh2 (G-Rh2) exerts important pharmacological effects with regard to the

control of human hepatocellular carcinoma (HCC). EZH2 is a potent histone methyltransferase of

H3K27me3, which has been determined as an oncogene in many malignancies. The CDKN2A-2B gene

cluster encodes three important tumor suppressors, P14, P15 and P16. In this study, the anticancer

effect and molecular mechanism of G-Rh2 on HCC was investigated. Treatment of HCC cells with 15

G-Rh2 inhibited cell proliferation, migration and induced cell cycle arrest at the G0/G1 phase. We

demonstrate for the first time that this effect was specifically mediated by down-regulating expression

of EZH2. Further molecular mechanism study indicated that the decreased EZH2 promoted P14, P15

and P16 gene transcription through reducing H3K27me3 modification in the promoter of CDKN2A-2B

gene cluster loci. Similarly, silencing of EZH2 by siRNA down-regulated P14, P15, P16 mRNA levels 20

and inhibited HCC cell proliferation. Our results suggested that EZH2 could be a potentially

therapeutic target by G-Rh2 in HCC, which provided a rationale for the development of drugs that

inhibited histone methylase as a strategy against various cancers.

Key words: Ginsenoside-Rh2; EZH2; CDKN2A-2B gene cluster; Hepatocellular carcinoma

0 Introduction

Ginsenosides are extracted from the root of Panax ginseng, which are the major active

components of ginseng. Ginsenoside-Rh2 (G-Rh2), a plant glycoside with a dammarane skeleton,

is recognized as a major active anticancer saponin with potential therapeutic effect against

leukemia, prostate cancer, glioblastoma, lung cancer, hepatocellular carcinoma, et al. In addition, 30

G-Rh2 has been found to inhibit cell growth, migration and induce apoptosis in several cancer

cells

[1-5]

Liver cancer is the most common and the third most frequent deadly cancers worldwide.

Hepatocellular carcinoma (HCC) represents the major histological subtype of primary liver cancer,

accounting for 70 to 85 % of the total liver cancer

[6]

. Although the comprehension of the HCC 35

carcinogenesis has significantly improved in the past years, and lots of therapeutic strategies have

been improved. However, the 5-year overall survival rate remains very poor

[7]

. Generally,

hepatocarcinogenesis is a multistep process involving a few of genetic or epigenetic alterations

that eventually result in the malignant transformation of hepatocytes

[8]

. Mounting evidence

indicates that epigenetic alterations contribute significantly to the initiation and progression of 40

HCC

[9]

. As an important type of histone modification, histone methylation plays a central role in

regulating chromatin dynamics and transcription. Therefore, a histone methylase that promotes

HCC tumorigenesis could be an attractive novel target for drug discovery.

The Polycomb Repressive Complex 2 (PRC2) component enhancer of zeste homologue 2

(EZH2) is a potent histone methyltransferase that specifically catalyzes the trimethylation of 45

histone 3 lysine 27 (H3K27), H3K27me

compresses the chromatin structure and leads to the

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transcriptional repression of genes

[10]

. EZH2 is up-regulated in several cancers associated with

increased global H3K27me

and prompts silencing of tumor suppressors and developmental

regulators

[11, 12]

. In HCC, studies have demonstrated a correlation between increased EZH2

expression and inferior prognostic

[13, 14]

. Silencing EZH2 in liver cancer cells attenuated liver 50

cancer proliferation and metastasis.

As key regulators of G1 phase cell-cycle arrest, CDKN2A-2B on chromosome 9p21 is

inactivated in a wide range of human cancers through epigenetic mechanisms

[15]

. The

CDKN2A-2B gene cluster contains two cell cycle regulator genes, CDKN2A and CDKN2B, which

encode three important tumor suppressors, P14, P15 and P16

[16]

. P14, P15 and P16 genes have 55

been investigated aberrantly expressed in hepatocellular carcinomas. Recent investigations on

EZH2 with a potential to suppress CDKN2A-2B have revealed that EZH2 is a key inhibitor for p14,

p15 and p16 expression in various cancers, including HCC

[17]

In this study, we firstly clarify that G-Rh2 could inhibit HCC cells proliferation, migration and

down-regulated EZH2 expression levels. The decreased EZH2 led to reduced repressive histone 60

modifications H3K27me3 in the promoter regions of CDKN2A-2B gene cluster, which markedly

activated transcription, thereby increasing mRNA expression levels of the P16, P14, and P15

tumor suppressor genes.

1 Materials and methods

1.1 Reagents and cell culture 65

G-Rh2 was purchased from Sigma (St. Louis, MO) and dissolved in dimethyl sulfoxide

(DMSO). Human hepatocellular carcinoma HepG2 cell were obtained from American Type

Culture Collection. Cells were cultured in MEM medium (Hyclon, Logan, UT, USA) with 10%

fetal bovine serum (Hyclon) at 37℃ with 5% CO2.

1.2 Cell counting kit-8 (CCK-8) assay 70

The CCK-8 detection kit (Dojindo, Shanghai, China) was used to measure cell viability

according to the manufacturer’s instructions. Cells were seeded in a 96-well (100 µl per well)

plates at a density of 5×10

/ml. After 12 h, cells were treated with G-Rh2 for a certain period of

time. Then，CCK-8 solution (10 µl per well) was added and the plate was incubated at 37℃ for 2

h. The viable cells were counted by absorbance measurements with a monochromator microplate 75

reader at a wavelength of 450 nm.

1.3 Flow cytometry for cell cycle analysis

Cells were plated into 6-well plates and treated with G-Rh2 (20 µM) for 24 h, added BrdU

(1mM/mL) for 4 h and then harvested. Cells were stained with anti-BrdU and 7-AAD using the

BD Pharmingen™ BrdU Flow Kits Kit (BD, San Diego, CA, USA) according to the 80

manufacturer’s instructions. Cell cycle distribution was determined by flow cytometry

(FACSAria

Ⅱ, Becton Dickinson, Mountain View, CA, USA).

1.4 Cell migration assay

Cells were treated with G-Rh2 (20 µM) for 24 h and then harvested. 5×10

cells in serum-free

medium were placed into the upper chamber of an insert of the transwell assay (8 µm pore size, 85

Millipore, Darmstadt, Germany). MEM medium containing 20% FBS was added to the lower

chamber. After incubation at 37℃ for 24 h, we removed the cells remaining on the upper

membrane with cotton wool. Cells that had migrated through the membrane were stained with

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0.1% crystal violet, imaged.

1.5 RNA preparation and quantitative reverse transcription polymerase chain 90

reaction (qRT-PCR)

Total RNA was extracted from cells by Trizol reagent (Invitrogen Life Technology, Carlsbad,

CA, USA). RNA integrity was analyzed on a 1.2% agarose gel. RNA quantity was determined

using a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

RNA samples were reverse transcribed with a PrimeScript™ RT reagent Kit (Takara 95

Biotechnology, Dalian, China) for cDNA synthesis and genomic DNA removal. qPCR was carried

out in the TaKaRa real-time PCR system using SYBR® Premix Ex Taq™ II Kit (TaKaRa).

GAPDH was used as the endogenous control. The primer pairs were listed in Table 1.

Tab. 1 Primers for real-time qPCR 100

Primer Name

Sequences

EZH2

F: GGGAAGCAGGGACTGAAACG

R: CAGCACCACTCCACTCCACATT

P14

F: GAGGGTTTTCGTGGTTCACATC

R: TCAGTAGCATCAGCACGAGGG

P16

F: TTCAGGGGTGCCACATTCG

R: CCGACCCTGTCCCTCAAATC

P15

F: TGGACCTGGTGGCTACGAAT

R: GCCGTCACCCAAGTGCTAATC

GAPDH

F: GCACCGTCAAGGCTGAGAAC

R: TGGTGAAGACGCCAGTGGA

1.6 Western blot analysis

Total protein was isolated from cells with RIPA lysis buffer (Pulilai, Beijing, China). The

protein content was determined by bicinchonininc acid (BCA) kit (Pulilai). Proteins were

separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with

5% non-fat milk, incubated with primary antibodies EZH2 (dilution 1:3000, Millipore, #17-662), 105

H3k27me

(dilution 1:3000, Millipore, #17-622), and β-actin (dilution 1:5000, Sigma, #A5441) at

4℃ overnight and then incubated with secondary antibodies. Bands were visualized with ECL

reagent (Thermo Scientific, Rockford, MA, USA). β-actin was used as the loading control.

1.7 Synthetic EZH2 siRNA knockdown

EZH2 knockdown in HCC cells was performed by siRNA transfection. siRNA oligonucleotides 110

targeting EZH2 were designed and synthesized by Sigma (SASI_Hs01_00147882). The

nonspecific siRNA oligonucleotides (Genepharma, Shanghai, China) were used as negative

controls. All siRNA oligonucleotide sequences are listed in Table 2. Cells were transfected with

150 pmol siRNA/well in 6-well plates, using Lipofectamine RNAiMax Reagent (Invitrogen)

based on the manufacturer’s instructions. 115

Tab. 2 siRNA oligonucleotides

siRNA Name

Sequences

siEZH2 sense

siEZH2 antisense

CAUCGAAAGAGAAAUGGAATT

UUCCAUUUCUCUUUCGAUGTT

siControl sense

siControl antisense

UUCUCCGAACGUGUCACGUTT

ACGUGACACGUUCGGAGAATT

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