TWIST1 expressionChi Jin

www.impactjournals.com/oncotarget/

Oncotarget, 2017, Vol. 8, (No. 34), pp: 56174-56184

MicroRNA-610 suppresses osteosarcoma oncogenicity via targeting

TWIST1 expression

Chi Jin

_{, Yongjian Feng}

_{, Yongjian Ni}

_{and Zhonglin Shan}

Correspondence to: Chi Jin, email: [email protected]

Keywords: osteosarcoma, microRNAs, miR-610, Twist1, cisplatin

Received: January 16, 2017 Accepted: March 24, 2017 Published: April 11, 2017

Copyright: Jin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

ABSTRACT

Osteosarcoma is the most frequent primary bone tumor affects adolescents and young adults. Recently, microRNAs (miRNAs) are short, non-coding and endogenous RNAs that played as important roles in the initiation and progression of tumors. In this study, we try to explore the biological function and expression of miR-610 in the osteosarcoma. We showed that miR-610 expression was downregulated in the osteosarcoma tissues and cell lines. Elevated expression of miR-610 suppressed the osteosarcoma cell proliferation, cell cycle, invasion and EMT program. Moreover, overexpression of miR-610 increased sensitivity of MG-63 and U2OS cells to cisplatin. Twist1 was identified as a direct target gene of miR-610 in the osteosarcoma cell. Furthermore, we demonstrated that Twist1 was upregulated in the osteosarcoma tissues and cell lines. The expression of Twist1 was negatively associated with the expression of miR-610 expression in the osteosarcoma tissues. Ectopic expression of Twist1 inhibited the sensitivity of miR-610-overexpressing MG-63 cells to cisplatin. We also showed that overexpression of Twist1 increased the proliferation and invasion of miR-610-overexpressing MG-63 cells. These data indicated that ectopic expression of miR-610 suppressed the osteosarcoma cell proliferation, cell cylce, invasion and increased the sensitivity of osteosarcoma cells to cisplatin through targeting the Twist1 expression.

INTRODUCTION

Osteosarcoma is the most frequent primary bone

tumor affects adolescents and young adults, which is

prone to early metastasis and frequently occurs in the long

bones [1–5]. Despite the recently advancements including

adjuvant chemotherapy, radiotherapy and wide tumor

excision, the prognosis and 5 years survival rate of these

patients remains poor [6–9]. Chemotherapeutic drugs such

as cisplatin and doxorubicin are well used in osteosarcoma

and 5-year survival rate has been increased from 20% to

70% [10, 11]. However, the molecular mechanisms about

acquiring chemoresistance are still unknow [12–14]. It is

urgent need to find these molecular mechanisms to explore

therapeutic strategies.

MicroRNAs (miRNAs) are a type of short,

non-coding and endogenous RNAs that inhibit the protein non-coding

genes expression through partial complementary binding

to the 3’ UTR of mRNAs [15–19]. It has been shown that

miRNAs mediate several biology functions including cell

development, differentiation, apotosis, proliferation, and

metabolism [20–24]. Aberrantly expression of miRNAs

was found in a variety of human tumors such as gastric

cancer, glioblastoma, Ewing’s sarcoma, bladder cancer,

colon Cancer and also osteosarcoma [16, 25–29]. In

addition, mounting evidences demonstrated that miRNAs

also act crucial roles in the development of drug resistance

in different cancers [30–33].

In this study, we try to explore the biological

function and expression of miR-610 in the osteosarcoma.

We showed that miR-610 expression was downregulated

in the osteosarcoma tissues and cell lines. Elevated

expression of miR-610 suppressed the osteosarcoma cell

proliferation, cell cycle, invasion and EMT program.

Moreover, overexpression of miR-610 increased sensitivity

of MG-63 and U2OS cells to cisplatin.

RESULTS

miR-610 increased the sensitivity of

osteosarcoma cells to cisplatin

The miR-610 expression level was downregulated

in the osteosarcoma cells (HOS, SAOS-2, MG-63 and

U2OS) compared to in the hFOB cells (Figure 1A).

qRT-PCR analysis suggested that miR-610 mimic could

significantly enhance miR-610 expression both the MG-63

and U2OS cell (Figure 1B and 1C). The response of

MG-63 and U2OS cells to cisplatin enhanced after treated with

the miR-610 mimic compared to the scramble-transfected

cells (Figure 1D and 1E).

The expression of miR-610 was downregulated in

osteosarcoma tissues

We next determined the miR-610 expression level

in the osteosarcoma tissues. We showed that miR-610 was

downregulated in 21 osteosarcoma cases compare to the

adjacent non-tumor tissues (Figure 2A). The miR-610

expression was lower in the osteosarcoma tissues compare

with the adjacent non-tumor tissues (Figure 2B).

Overexpression of miR-610 suppressed

epithelial-mesenchymal transition (EMT) program

Ectopic expression of miR-610 increased the

epithelial marker E-cadherin protein expression and

decreased the mesenchymal marker N-cadherin,

Vimentin and Snail protein expression (Figure 3A).

MiR-610 overexpression promoted the E-cadherin mRNA

expression and supressed N-cadherin, Vimentin and Snail

mRNA expression (Figure 3B).

Elevated expression of miR-610 suppressed the

osteosarcoma cell proliferation

Ectopic expression of miR-610 suppressed the

MG-63 and U2OS cell proliferation (Figure 4A and 4B).

Moreover, overexpression of miR-610 decreased the

ki-67 expression in the MG-63 and U2OS cell (Figure 4

C and 4D). In addition, elevated expression of miR-610

supressed the PCNA expression both in the MG-63 and

U2OS cell (Figure 4E and 4F).

miR-610 overexpression inhibited the

osteosarcoma cell cycle and invasion

Ectopic expression of miR-610 suppressed the

MG-63 and U2OS cell cycle (Figure 5A and 5B). elevated

expression of miR-610 decreased cell invasion both in the

MG-63 and U2OS cell (Figure 5C and 5D).

Twist1 was a direct target gene of miR-610

To search the molecular mechanism involved in

the function of miR-610 in osteosarcoma cell, we used

the TargetScan databases to find potential target gene of

Figure 1: miR-610 increased the sensitivity of osteosarcoma cells to cisplatin.

miR-610. The putative binding site of miR-610 and Twist1

is shown in Figure 6A. Luciferase assay demonstrated that

miR-610 overexpression decreased luciferase activity of

wild-type (WT) 3ʹUTR of the Twist1 vector, but not in the

mutated-type (Mut) 3ʹUTR of the Twist1 construct both in the

MG-63 and U2OS cell (Figure 6B and 6C). Overexpression

of miR-610 suppressed the Twist1 protein expression both in

the MG-63 and U2OS cell (Figure 6D and 6E).

Twist1 was upregulated in osteosarcoma tissues

The Twist1 expression level was upregulated in

the osteosarcoma cells (HOS, SAOS-2, MG-63 and

U2OS) compared to in the hFOB cells (Figure 7A).

We furtherly determined the Twist1 expression level

in the osteosarcoma tissues. We showed that Twist1

was upregulated in 25 osteosarcoma cases compare to

the adjacent non-tumor tissues (Figure 7B). The Twist1

expression was higher in the osteosarcoma tissues

compare with the adjacent non-tumor tissues (Figure 7C).

The expression of Twist1 was negatively associated with

the expression of miR-610 expression in the osteosarcoma

tissues (Figure 7D).

miR-610 increased the sensitivity of

osteosarcoma cells to cisplatin and decreased

the osteosarcoma cell proliferation and invasion

through downregulating Twist1

The Twist1 mRNA and protein expression was

upregulated in the MG-63 cell after tranfected with

Twist1 vector (Figure 8A and 8B). The response of

MG-63 and U2OS cells to cisplatin enhanced after treated

with the Twist1 vector compared to the cotrol-transfected

cells (Figure 8C and 8E). Moreover, the responses of

miR-610-overexpressing MG-63 and U2OS cells to

Figure 2: The expression of miR-610 was downregulated in osteosarcoma tissues.

Figure 3: Overexpression of miR-610 suppressed epithelial-mesenchymal transition (EMT) program.

cisplatin were increased after transfection with the Twist1

vector compared with the control vector (Figure 8D

and 8F). Overexpression of Twist1 enhanced MG-63

cell proliferation (Figure 8G) and invasion (Figure 8I).

Moreover, ectopic expression of Twist1 promoted the

proliferation (Figure 8H) and invasion (Figure 8G) of

miR-610-overexpressing MG-63 cells.

DISCUSSION

Osteosarcoma is the most frequent bone tumor

occurring in adolescence and childhood, with the high

mortality[14, 34, 35]. However, the molecular mechanism

about osteosarcoma progression still remains elusive. It is

supposed that miRNA deregulated expression is involved

in the progression of human tumors[36–39]. In this study,

we explored the biological function and expression of

610 in the osteosarcoma. We showed that

miR-610 expression was downregulated in the osteosarcoma

tissues and cell lines. Elevated expression of miR-610

suppressed the osteosarcoma cell proliferation, cell cycle,

invasion and EMT program. Moreover, overexpression

of miR-610 increased sensitivity of MG-63 and U2OS

cells to cisplatin. Twist1 was identified as a direct target

gene of miR-610 in the osteosarcoma cell. Furthermore,

we demonstrated that Twist1 was upregulated in the

Figure 4: Elevated expression of miR-610 suppressed the osteosarcoma cell proliferation.

Figure 5: miR-610 overexpression inhibited the osteosarcoma cell cycle and invasion.

Figure 6: Twist1 was a direct target gene of miR-610.

osteosarcoma tissues and cell lines. The expression of

Twist1 was negatively associated with the expression of

miR-610 expression in the osteosarcoma tissues. Ectopic

expression of Twist1 inhibited the sensitivity of

miR-610-overexpressing MG-63 cells to cisplatin. We also showed

that overexpression of Twist1 increased the proliferation

and invasion of miR-610-overexpressing MG-63 cells.

These data indicated that miR-610 acted as a tumor

suppressor role in the osteosarcoma progression and

was associated to the sensitivity of osteosarcoma cell to

cisplatin.

Previous data demonstrated that miR-610 was

involved in tumor initiation and progression [40–42].

For example, Wang et al. [40]. suggested that miR-610

suppressed the gastric cancer cell migration and invasion

through inhibiting vasodilator-stimulated phosphoprotein

(VASP) expression. Zeng et al. [43]. confirmed that

miR-610 was decreased in the hepatocellular carcinoma tissues

and cell lines. Ectopic expression of miR-610 reduced

the hepatocellular carcinoma cell proliferation and

tumorigenicity through regulating the protein 1 (TBL1X)

and lipoprotein receptor-related protein 6 (LRP6)

expression. Mo et al. [42]. showed that the expression

level of miR-610 was downregulated in the glioblastoma

cell and tissues and overexpression of miR-610 suppressed

the glioblastoma cell proliferation through regulating the

CCND2 and AKT3 expression. Sun et al. [41]. found

that miR-610 expression level was downregulated in the

colorectal cancer tissues and overexpression of miR-610

decreased the colorectal cancer cell proliferation, invasion

and migration by regulating the hepatoma-derived growth

factor (HDGF) expression. Yan et al. [44]. also showed

that miR-610 expression was decreased in glioma samples

and overexpression of miR-610 suppressed the glioma

cell proliferation, invasion and migration by targeting

the MDM2 expression. In line with these data, we also

demonstrated that miR-610 expression was downregulated

in the osteosarcoma tissues and cell lines. Elevated

expression of miR-610 suppressed the osteosarcoma cell

proliferation, cell cycle, invasion and EMT program.

To study the potential mechanism about miR-610

regulated the proliferation and invasion of osteosarcoma,

we used open-target prediction programs (TargetScan

databases) to predict the target gene of miR-610, and Twist1

was focused. Twist1 is a conserved basic transcription factor

of helix-loop-helix and regulates cell migration, embryonic

morphogenesis and differentiation of myoblast, mesodermal

and osteoblast [45–48]. Recently, several studies showed

Figure 7: Twist1 was upregulated in osteosarcoma tissues.

that Twist1 played as an oncogene that increased the

tumor cell proliferation, migration and invasion, induced

the EMT progression, angiogenesis and anti-apoptosis

[49–51]. Twist1 has been found to be upregulated in a lot

of tumors such as gastric cancer, ovarian cancer, breast

cancer, bladder cancer and also osteosarcoma [52–56].

Increasing studies suggested that Twist1 was involved in

drug resistance of cancer [55, 57, 58]. In our study, we

firstly used the TargetScan databases to show that there is

a putative binding site of miR-610 and Twist1. Luciferase

assay demonstrated that miR-610 overexpression decreased

luciferase activity of WT 3ʹUTR of the Twist1 vector,

but not in the Mut 3ʹUTR of the Twist1 construct both in

the MG-63 and U2OS cell. Overexpression of miR-610

suppressed the Twist1 protein expression both in the

MG-63 and U2OS cell. Moreover, we also indicated that Twist1

expression level was upregulated in osteosarcoma tissues

and cell lines. The expression of Twist1 was negatively

Figure 8: miR-610 increased the sensitivity of osteosarcoma cells to cisplatin and decreased the osteosarcoma cell

proliferation and invasion through downregulating Twist1.

associated with the expression of miR-610 expression in

the osteosarcoma tissues. Furthermore, we showed that

Twist1 decreased the sensitivity of osteosarcoma cells

to cisplatin and ectopic expression of Twist1 promoted

the osteosarcoma cell proliferation and invasion. We

demonstrated that miR-610 overexpression increased the

sensitivity of osteosarcoma cells to cisplatin and decreased

the osteosarcoma cell proliferation and invasion through

downregulating Twist1 expression.

In summary, our study suggested that miR-610

expression level was downregulated in the osteosarcoma

samples and cell lines. Ectopic expression of miR-610

suppressed the osteosarcoma cell proliferation, cell cylce,

invasion and increased the sensitivity of osteosarcoma

cells to cisplatin through targeting the Twist1 expression.

MATERIALS AND METHODS

Tissues, cell lines cultured and transfection

Osteosarcoma and the matched adjacent

non-tumor tissues were obtained from surgical resection in

our hospital. Tissues are immediately frozen in the liquid

nitrogen and stored until used. This project was approved

with the Institutional Ethics Committee of Central Hospital

of Cangzhou City and written informed consent was get

from each patient. The Osteosarcoma cell lines (MG63,

HOS, U2OS, and Saos-2) and normal human osteoblastic

cell line hFOB were cultured in the DMEM (Dulbecco’s

modified Eagle’s medium) suppled with FBS, penicillin

and streptomyc. miR-610 mimic and oligonucleotide

scramble were obtained from the GenePharm (Shanghai,

China) and transfected into the MG-63 cells by using

Lipofectamine 2000 (Invitrogen, CA, USA) following

to the manufacturer’s protocol. These characteristics of

patients are described in Supplementary Table 1.

Quantitative RT-PCR

Total RNA was prepared from cells or tissues with

TRIzol (Life Technologies, NY). Quantification for miR-610

and TWIST1 was performed by using qRT-PCR following

to the manufacturer’s information. The expression of U6 was

used as the internal control to the expression of miR-610;

the expression of GAPDH was used as the internal control

to the expression of TWIST. TWIST, forward primer:

5ʹ-ACGAGCTGGACTCCAAGATG-3ʹ and reverse primer:

5ʹ-CACGCCCTGTTTCTTTGAAT-3ʹ; GAPDH, forward

primer: 5ʹ-GACTCATGACCACAGTCCATGC-3ʹ; and

reverse primer: 5ʹ-AGAGGCAGGGATGATGTTCTG-3ʹ.

Cell proliferation and colony formation

Cells were cultured in the 96-well and cell

proliferation was determined by MTT assay (Promega,

Madison, USA) following to the company’s instruction.

Cells proliferation was counted after 24 and 48 hours. The

absorbance was measured by the microtiter plate reader at

450 nm (Molecular Devices, CA, USA).

Cell cycle and invasion

For cell cycle analysis, cells were fixed with 70%

ethanol, re-suspended in the PBS containing Rnase, Triton

X-100 and propidium iodide (Sigma). The cell then was

analyzed by FACSArray Flow Cytometer (Becton Dickerson,

San Jose, CA, USA). For cell invasion, transwell chambers

were used. Cells were cultured on the upper chamber with

the non-coated membrane (Millipore) in the serum-free

media. The lower chamber with the media containing FBS

was used as a chemoattractant. The migration cell on the

lower chamber was determined using microscope.

Luciferase report assay

Cells were cultured in the 48-well plates with

about 60% confluence. Cell was transfected with

miR-610 mimics or scramble and pGL3-TWIST1-3’UTR

and PRL-TK by using Lipofectamine 2000 according to

manufacturer’s instructions. Renilla and firefly luciferase

activitie was determined by using the dual-luciferase

reporter (Promega, USA).

Western blot analysis

Proteins were isolated using a RIPA

(radioimmunoprecipitation) lysis buffer. Total protein was

separated by 12% SDS-PAGE and then transferred to

polyvinylidene fluoride (PVDF) membranes (Millipore,

USA). After blocked with the nonfat milk, the membrane was

incubated with antibodies against TWIST (1:1000, Santa Cruz,

USA), GAPDH (1:5000, Santa Cruz, USA). The membrane

was measured by ECL reagent (Applygen, Beijing).

Statistical analysis

Data are shown as mean ± SD. Comparisons

between more than two groups were performed with

ANOVA and different between two groups was done using

Student’s t-tests. P < 0.05 was considered statistically

significant.

CONFLICTS OF INTEREST

None.

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