Onco-microRNA miR-130b promoting cell growth in children APL by targeting PTEN

Xiang-Cui Gong, Yuan-Qin Xu, Yan Jiang, Hui Guan, Hua-Lin LiuDepartment of Hematology, Affiliated Hospital of Medical College of Qingdao University, Qingdao, Shandong, 266034, China

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Onco-microRNA miR-130b promoting cell growth in children APL by targeting PTEN

Xiang-Cui Gong, Yuan-Qin Xu, Yan Jiang, Hui Guan, Hua-Lin Liu*
Department of Hematology, Affiliated Hospital of Medical College of Qingdao University, Qingdao, Shandong, 266034, China

ABSTRACT

Objective: To study the expression of microRNA-130b (miR-130b) in children acute promyelocytic leukemia (APL) and its role for regulating PTEN expression. Methods: A total of 50 children APL marrow tissues and 15 normal marrow tissues between January and December in 2012 were collected into our study. The expression of miR-130b in APL and normal marrow tissues were detected by quantitative real-time polymerase chain reaction. MiR-130b inhibitor was transfected into HL-60 cells. Cell Counting Kit-8 assay and flow cytometry were used to measure cell proliferation and apoptosis, respectively. The expression of PTEN, a potential target of miR-130b, and its downstream genes, Bcl-2 and Bax, in transformed cells were detected by quantitative real-time polymerase chain reaction and western-blot. Results: The expression of miR-130b was signifi cantly higher in children APL marrow tissues than in normal marrow tissues (P<0.05). Down-regulation of miR-130b could signifi cantly suppress cell proliferation and induce apoptosis in HL-60 cells (P<0.05). PTEN expression was upregulated when miR-130b was knocking-down (P<0.05). As downstream genes of PTEN, the expression of Bcl-2 and Bax were regulated as well. Conclusions: MiR-130b is overexpressed in children APL marrow tissues and associated with cell growth. MiR-130b may promote children APL progression by inducing cell proliferation and inhibiting apoptosis.

ARTICLE INFO

Article history:

Received in revised form 20 January 2016

Accepted 15 February 2016

Available online 20 March 2016

MicroRNA-130b

Acute promyelocytic leukemia

PTEN

Bcl-2

Bax

Cell growth

1. Introduction

Acute promyelocytic leukemia (APL), classifi ed as M3 in French-American-British classification systems, is a subtype of acute myelocytic leukemia (AML). Globally, APL occupies 7%-27% of all AML types, while in China, this proportion enlarges to 12%-23%[1]. Compared to other AML types, APL has a higher risk of hemorrhage and causes a high mortality[2]. Nowadays, systematical chemotherapies based on all-trans-retinoic acid (ATRA) and arsenic trioxide is the major treatment method[3]. However, both single and combination drug therapies have many disadvantages such as early drug resistance, early recurrence, and serious side-eff ect[4]. Thus, the treatment outcome and prognosis of APL are still unsatisfi ed. Hence, the discovery of novel biological therapeutic target is a key for improving the survival of APL patients.

MicroRNAs (miRs) are small (19-25 nt), noncoding RNA molecules that negatively regulate gene expression by interacting with the 3’untranslated region of targeting mRNA, eventually leading to translational suppression and/or degradation of the targeting mRNA[5,6]. In adult AML patients, the aberrant expression of miR-335 was associated with chemoresistance[7]. In children APL, miR-125b could inhibit cell apoptosis and induce ATRA chemoresistance by targeting BCL2-antagonist/killer 1[8]. MiR-130b was demonstrated as an important oncogenic miR in manycancers[9]. For example, in hepatocellular carcinoma, miR-130b was up-regulated and associated with large tumor metastasis[10]. It might be suggested that PTEN, a classical tumor suppressor, was a target of miR-130b by bioinformatics study. As a member of phosphatases, PTNE can change PIP3 into PIP2 by phosphorylation, consequently inhibiting the activation of Akt signal[11].

In this study, the expression of miR-130b in children APL marrow tissues was detected and its functions and mechanisms for cell growth in vitro were investigated. Finally, we proved that miR-130b was up-regulated in APL marrow tissues and HL-60 cells, and it could suppress cell proliferation and induce apoptosis by inhibiting PTEN expression.

2. Materials and methods

2.1. Clinical specimens and cell culture

A total of 50 children APL marrow tissues and 15 normal marrow tissues were collected and stored in liquid nitrogen between January and December in 2012. The range of age was from 7 to 14 year, and the median age was 9 year. The human APL cell line, HL-60, was purchased from T Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Gibco? RPMI 1640 medium (Gibco, USA) supplemented with 10% v/v fetal bovine serum (FBS; Gibco, USA) and maintained in a humidifi ed incubator.

2.2. Quantitative real-time polymerase chain reaction (qRTPCR)

Total RNA was isolated from marrow tissues and HL-60 cells using TRIZOL? reagent (Invitrogen, USA). The relative expression of miR-130b was detected by Bulge-Loop? miR qRT-PCR Starter Kit (RiboBio, Guangzhou, China). PTEN, Bcl-2 and Bax mRNA expression levels were measured by Quant one step qRT-PCR Kit (TIANGEN, Beijing, China). MiR-130b primers were purchased from RiboBio Co., Ltd. PTEN, Bcl-2, Bax and β-actin qPCR primers were synthesized by Augct Co., Ltd and the sequences were shown as follow: PTEN sense 5’-GTAAGGACCAGAGACAAAAAG-3’, anti-sense 5’-CTTTTTTAGCATCTTGTTCTG-3’, Bcl-2 s e n s e 5’-T TC T T TG AG T TCG G TG G G G TC-3’, a n t is e n s e 5’-T G C ATAT T T G T T T G G G G C A G G-3’, B a x sense 5’-TCCACCAAGAAGC TGAGCGAG-3’, antisense 5’-GTCCAGCCCATGATGGT TC T-3’, β-actin 5’-C TCCATCC TGGCC TCGC TGT-3’, anti-sense 5’-GCTGTCACCTTCACCGTTCC-3’. The 2-△△Ctmethod was used for data calculation.

2.3. MiR transfection

HL-60 cell transfection was performed using the Lipofectamine? 2000 Transfection Reagent (Invitrogen, USA) according to the manufacturer instructions. Briefl y, 5伊105cells were cultured in sixwell culture plates containing 1.5 mL RPMI 1640 per well without FBS. The 100 pmoL miR-130b inhibitor or scrambled-miR inhibitor (RiboBio, Guangzhou, China) was mixed with 5 μL Lipofectamine? 2000 in 500 μL Opti-MEM? I Reduced Serum Medium (Gibco, USA) and incubated for 15 min at room temperature. The complex was then added to the cells and ensured full distribution over all plate surface. After 8 h of incubation, the FBS and antibiotics were added and the cells were incubated for the 24 h, 48 h and 72 h.

2.4. Cell Counting Kit-8 (CCK-8) assay

The viability of cells was assessed by the CCK-8. CCK-8 assay was performed at time intervals 24 h, 48 h and 72 h post transfection. A total of 10 μL CCK-8 solution was added to 5伊105HL-60 cells suspended in 100 μL RPMI 1640 medium and incubated for 4 h at 37℃ in dark. Absorbance was measured by using a spectrophotometer at 450 nm.

2.5. Flow cytometry (FCM) analysis

HL-60 cells were collected and diluted into 5伊105at time interval 48 h post transfection. Cell cycles and the apoptotic cells were evaluated by Annexin V-FITC and propidium iodine staining (Roche, USA) according to the manufacturer’s instruction and analyzed with a FCM.

2.6. Western blot

Cells were washed with cold PBS for twice and broken by RIPA. Lysate was centrifuged at 15 000 r/min for 15 min. The supernatant was obtained and detected the protein content via BCA Kit (Millipore, USA). Proteins were separated by vertical electrophoresis and transferred to PVDF membrane (Millipore, USA). PTEN, Bcl-2, Bax and β-actin antibodies (all purchased from Santa Cruz, USA) were used to detect the proteins expression, respectively. Secondary HRP-conjugated goat anti-rabbit or anti-mouse antibody (Abgent, USA) were used at a 1:5 000 dilution and developed by the ECLRegent (Millipore, USA).

2.7. Statistical analysis

Measurement date were presented as mean ± SD. The SPSS version 13.0 (SPSS, Chicago, USA) was used for two-tailed Student-t test and ANOVA. P<0.05 was considered statistically signifi cant.

3. Results

3.1. MiR-130b expression in APL marrow tissues

Analysis of qRT-PCR data demonstrated that the relative expression of miR-130b was significantly increased in APL marrow tissues (n=50, 2.198±0.051) compared to normal marrow tissues (n=15, 6.562±0.271) (P<0.001).

3.2. Down-regulation effect of miR-130b inhibitor on miR-130b expression in HL-60 cells

In order to elucidate the biological significance of miR-130b, negative control miR inhibitor (NC inhibitor, n=8, 1.000) or miR-130b inhibitor (n=8, 0.331±0.016) was transfected into HL-60 cells. After 48 h post-transfection, miR-130b was significantly downregulated in HL-60 cells (P<0.001).

3.3. Effects of inhibition of miR-130b on suppressing viability and inducing apoptosis of HL-60 cells

Overexpression of miR-130b could effectively inhibit viability of HL-60 cells compared to control group (P<0.01). Moreover, BrdU incubation assay showed that miR-130b could signifi cantly repress DNA synthesis of HL-60 cells (135.37±6.43 vs. 57.39±4.20, P=0.002). FCM tested the infl uence of miR-130b for cell apoptosis. Compared to NC miR group, miR-130b could eff ectively increase apoptotic cell percentage (13.352±1.981 vs. 24.482±1.943, P=0.003).

3.4. PTEN as a downstream target of miR-130b in APL

To investigate the possible molecular mechanisms, the mRNA level of PTEN, a possible target of miR-130b, predicted by bioinformatics retrieval, in APL marrow tissues, was fi rstly detected, and Person correlation test was used to analyze the association between miR-130b and PTEN mRNA. It could be seen clearly that a negative correlation existed between miR-130b and BMI-1 mRNA (r=-0.571, P<0.001). Moreover, it was also confi rmed that the expression of PTEN were up-regulated in miR-130b inhibitor group than in NC group (P<0.001). As downstream genes of PTEN, the expression of Bcl-2 and Bax were also regulated (P<0.001). Expression of Bcl-2 was decreased, and the expression of Bax was increased (P<0.001).

4. Discussion

APL is a special subtype of AML. High risk of bleeding and DIC after chemotherapy by anthracyclines are the main features of APL. Identifi cation of PML/RARααfusion gene promoted the application of ATRA and arsenic trioxide[12]. APL became the first human cancer which could be treated with its tumor specifi c antigen[13]. However, novel and safe therapeutic molecules are still needed to be found to further improve patients’ survival.

As a novel and eff ect therapeutic targets, miRs can play doubleedged sword roles in cancer, and their aberrant expressions are related to many malignant features[14]. MiR-92a was reported to suppress cell growth of HL-60 cells by silencing P63 expression[15,16]. MiR-21 is a famous onco-miR, and downregulating miR-21 could enhance HL-60 cell apoptosis induced by cytarabine[17]. MiR-130b was reckoned as a prognostic evaluation biomarker and a vital tumor promoter in many cancers. In ESCC, high expression of miR-130b was associated with higher TNM stage and poorer prognosis, moreover, miR-130b enhanced proliferation, migration, and invasion in human ESCC cells by targeting PTEN[18]. In our study, it was found that the expression of miR-130b in APL marrow tissues was significantly higher than in normal marrow tissues. That meant this diff erential expression of miR-130b in APL marrow tissues might play an important regulation function for APL progression. In vitro, the proliferation inhibiting eff ect was detected after knocking down miR-130b expression, and cell apoptosis was also induced by miR-130b inhibitor.

PTEN is located at 10q23.3 and encodes a dual-specificity phosphatase with lipid and protein phosphatase activities[19]. PTEN dephosphorylates is an important activator of Akt named PI(3,4,5) P3, which controls cell proliferation, apoptosis, and many other functions[20]. Inhibition of PTEN results in increased levels of activated p-Akt and consequently regulates Bcl-2/Bax expression[21]. Studies indicated that PTEN is a potential target of miR-221/222, miR-22 and miR-144 in diff erent malignancies[18]. In this study, it was demonstrated that PTEN was a direct target of miR-130b by qRT-PCR and western blot, thus promoting Bcl-2 expression and reducing Bax expression. This phenomenon may partly explain the eff ect of miR-130b for HL-60 cells proliferation and apoptosis.

The results of our study indicate that expression of miR-130b is up-regulated in patients with diagnosed APL. To the best of our knowledge, this is an interesting study to demonstrate that miR-130b is a tumor promoter by directly targeting PTEN in APL.

Conflict of interest statement

We declare that there is no confl ict of interest.

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Document heading 10.1016/j.apjtm.2016.01.024

IF: 1.062

Asian Pacific Journal of Tropical Medicine

journal homepage:www.elsevier.com/locate/apjtm

15 December 2015

*

Hua-Lin Liu, Department of Hematology, Affi liated Hospital of Medical College of Qingdao University, Qingdao Shandong, 266034, China.

Tel: +86-18505321068

E-mail: qyfyliuhualin@163.com

Foundation project: This study was supported by the Project of Shandong Province Higher Educational Science and Technology Program (J14LL06).