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Abstract. – OBJECTIVE: MicroRNAs have caught more attention for their role in tumor pro-gression. Retinoblastoma (RB) is one of these ordi-nary malignant tumors. This study aims to identify whether mir-138-5p can regulate the development of RB, and find out its potential mechanism.

MATERIALS AND METHODS: Mir-138-5p ex-pression in RB cells was monitored by RT-qPCR. Besides, the role of mir-138-5p in RB develop-ment was explored through function experiments in vitro. The potential mechanism was further ex-plored by RT-qPCR, luciferase assay, and West-ern blot assay.

RESULTS: In our investigation, mir-138-5p was lower-expressed in RB cells than that in retinal pig-ment epithelial cells. Moreover, overexpression of mir-138-5p repressed cell viability, migration and invasion, and induced apoptosis of RB cells, while downregulated mir-138-5p increased cell viability, migration and invasion, and reduced apoptosis of RB cells. Furthermore, pyruvate dehydrogenase kinase 1 (PDK1) could be downregulated via over-expression of mir-138-5p, while PDK1 was upregu-lated via knockdown of mir-138-5p.

CONCLUSIONS: Our results suggested that mir-138-5p could repress the development of RB via suppressing PDK1, which may offer a new vi-sion for interpreting the mechanism of RB tum-origenesis.Key Words:

MicroRNA, mir-138-5p, Retinoblastoma, PDK1.

Introduction

Retinoblastoma (RB) is a rare human malignant tumor. The incidence of RB accounts for most of children’s intraocular tumors in the whole world1. Although effective therapy is available for RB pa-tients, children with RB have greater life-threate-ning risks2. Therefore, it is urgent to find more po-tential regulators and targets for treatment of RB. Recently, aberrant non-coding RNAs expression

is identified in most cancers and closely associa-ted with patients’ prognosis. Moreover, mounting evidence indicates that microRNAs contribute to regulating oncogene expression, which further acts on progression of malignant cancers, inclu-ding RB3-5. Accumulating evidence demonstrates that mir-138-5p participates in tumor carcino-genesis. Mir-138-5p knockdown promotes cell growth and invaded ability via suppressing sur-vivin in bladder cancer6. By targeting GPR124 in non-small cell lung cancer, mir-138-5p could re-verse resistance to gefitinib7. Moreover, mir-138-5p can inhibit autophagy via suppressing SIRT1 in pancreatic carcinoma8. Mir-138-5p contributes to suppressing development and metastasis of co-lorectal carcinoma via targeting PD-L19. Howe-ver, it remains unclear how mir-138-5p regulates the tumorigenesis of RB.

Our present study firstly revealed a lower expression of mir-138-5p in RB cells. Besides, ove-rexpressed mir-138-5p could repressed cell viabili-ty, invasion and migration, and induced cell apop-tosis of RB in vitro. Further researches showed that pyruvate dehydrogenase kinase 1 (PDK1) could be a potential target spot of mir-138-5p.

Materials and Methods

Cell LinesThe Institute of Biochemistry and Cell Biolo-

gy, Chinese Academy of Science (Shanghai, Chi-na) provided us with RB cell line Y79 and retinal pigment epithelial cell line ARPE-19. Culture me-dium was consisted of penicillin, 10% fetal bo-vine serum (FBS, Invitrogen Life Technologies, Carlsbad, CA, USA) and Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scienti-

European Review for Medical and Pharmacological Sciences 2017; 21: 5624-5629

Z. WANG1, Y.-J. YAO1, F. ZHENG1, Z. GUAN1, L. ZHANG1, N. DONG2, W.-J. QIN3

1Department of Ophthalmology, Fuzhou Second Hospital, Fujian, China2Corneal Disease Clinic, Xiamen Eye Center Affiliated Xiamen University, Fujian, China3Department of Radiation Oncology, Zhongshan Hospital Affiliated Xiamen University, Fujian, China

Corresponding Author: Nuo Dong, Ph.D; e-mail: wangzhfz@126.com; Wenjuan Qin, Ph.D; e-mail:proftomato@163.com

Mir-138-5p acts as a tumor suppressor by targeting pyruvate dehydrogenase kinase 1 in human retinoblastoma

Mir-138-5p in retinoblastoma

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fic, Waltham, MA, USA). Besides, cells were cul-tured in humidified incubator, which contained 5% CO2 and was set at 37°C. The miRNA mimics and inhibitor provided by Genepharma Co., Ltd. (Shanghai, China) were used for transfection of RB cells. Negative control (NC) was transfected non-specific siRNA.

Quantitative RT-PCRFirstly, total RNA was separated with TRIzol

reagent (Invitrogen, Carlsbad, CA, USA). Then, RNA was reverse-transcribed to cDNAs via re-verse Transcription Kit (TaKaRa, Dalian, China). Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed on ABI 7500 RT‑PCR detection system (Applied Biosy-stems, Foster City, CA, USA). The thermal cycle was as follows: 30 s at 95°C, 5 s at 95°C for 40 cycles, 35 s at 60°C.

Western BlotReagent radioimmunoprecipitation assay

(RIPA) (Beyotime, Shanghai, China) was utili-zed to extract protein from cells. Bicinchoninic acid (BCA) protein assay kit (TaKaRa, Dalian, China) was chosen for quantifying protein con-centrations. The target proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Next, they were transformed onto the polyvinylidene fluoride (PVDF) membrane and incubated with antibo-dies. Cell Signaling Technology (CST, Beverly, MA, USA) provided us with rabbit anti-PDK1 and rabbit anti-GAPDH, as well as goat anti-rabbit se-condary antibody. Chemiluminescent film was applied for assessment of protein expression with ImageJ software.

Luciferase AssaysThe 3ʹ-UTR of PDK1 was cloned into the

pGL3 vector (Promega, Madison, WI, USA) na-med wild-type (WT) 3’-UTR. Site-directed mu-tagenesis of the miR-138-5p binding site in PDK1 3’-UTR named mutant (MUT) 3ʹ-UTR was con-ducted with quick-change site-directed mutage-nesis kit (Stratagene, Cedar Creek, TX, USA). Y79 RB cells were infected with WT-3’-UTR or MUT-3ʹ-UTR for 48 h and were performed on the dual Luciferase reporter assay system (Promega, Madison, WI, USA).

Cell Counting Kit-8 (CCK8) AssayCell viability of theses treated cells in 96-

well plates was monitored every 24 h by CCK8

assay following the protocol (Dojindo Molecu-lar Technologies, Inc. Kumamoto, Japan). The absorbance was measured at 450 nm on the spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Wound Healing AssayCells were cultured in DMEM medium in

6-well plates overnight. After scratched with a plastic tip, cells were cultured in serum-free DMEM. Wound closure was viewed at different time points. Each assay was independently repe-ated in triplicate.

Matrigel AssayTo detect the invasion of RB cells, 5×104 cells

in 200 µL serum-free DMEM were transformed to the top chamber of an insert (8 μm pore size; Millipore, Billerica, MA, USA) coated with 50 µg Matrigel (BD Biosciences, San Jose, CA, USA). The bottom chamber was added with DMEM me-dium (10% FBS). 48 h later, top surface of cham-bers was wiped by cotton swab, immersed for 10 min with precooling and stained in crystal violet for 30 min. The data for invasion were counted from three fields per membrane.

Statistical AnalysisSPSS 21.0 (SPSS Inc., Armonk, NY, USA) was

used in our study. Student t-text was selected. We present data as mean ± SD. All experiments were repeated three times. p values < 0.05 were consid-ered statistically significant.

Results

Downregulated mir-138-5p in RB CellsFirstly, RT-qPCR was conducted for detecting

mir-138-5p level in RB cell Y79 and retinal pig-ment epithelial cell line ARPE-19. As the result, mir-138-5p was significantly lower in Y79 com-pared with ARPE-19 (Figure 1A).

Mir-138-5p Suppresses Cell Viability and Promoted RB cell Apoptosis

Firstly, Y79 cells were infected with mir-138-5p mimics and inhibitor. Then the mir-138-5p level in treated cells was detected by qRT-PCR (Figure 1B and Figure 1C). The cell viability was examined by CCK8 assay. As the result, mir-138-5p mimics significantly reduced cell viability (Figure 2A) and mir-138-5p inhibitor significantly prompted cell viability (Figure 2B). Additional-

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ly, apoptosis assay was performed to detect cell apoptosis of Y79 cells. We found that mir-138-5p mimics increased apoptosis rate while mir-138-5p inhibitor reduced apoptosis rate in Y79 cells (Fig-ure 2C and 2D).

Mir-138-5p Suppresses Migrated and Invaded Ability of RB Cells

The migration in mir-138-5p mimics cells was decreased (Figure 3A) and the migration in mir-138-5p inhibitor cells was increased (Figure 3B).

Figure 1. Mir-138-5p expression level in RB cells. (A) mir-138-5p expression was significantly decreased in the RB cells (Y79) compared with retinal pigment epithelial cell line (ARPE-19). (B) The mir-138-5p expression level in cells transfected with mir-138-5p mimics and negative control was determined by qRT‑PCR. (C) The mir-138-5p expression level in cells trans-fected with mir-138-5p inhibitor and negative control was determined by qRT‑PCR. *p<0.05; **p<0.01.

Figure 2. Mir-138-5p suppresseD cell viability and promoted apoptosis of RB cell. (A) mir-138-5p mimics significantly re-duced cell viability compared with negative control. (B) mir-138-5p inhibitor significantly prompted cell viability compared with negative control. (C) Apoptosis assay showed that the apoptosis rate of cells transfected with mir-138-5p mimics was increased compared with those transfected with negative control. (D) Apoptosis assay showed that the apoptosis rate of cells transfected with mir-138-5p inhibitor was decreased compared with those transfected with negative control. Matrigel assay demonstrated that numbers of invading cells were reduced in pcDNA-mir-138-5p cells compared with EV cells. The results represent the average of three independent experiments (mean ± standard error of the mean); *p<0.05.

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Besides, matrigel assay showed that numbers of invading cells were reduced in mir-138-5p mimics cells compared with negative control cells (Figure 3C). Numbers of invading cells were more in mir-138-5p mimics cells (Figure 3D).

Correlation Between PDK1 and mir-138-5pTo further understand how mir-138-5p partici-

pated in RB development, we searched the miR-NAs that contained mir-138-5p binding site by Starbase v2.0. The search results demonstrated that PDK1 was one of those candidate targets (Fig-ure 4A). The luciferase assay revealed that Y79 cells transfected with WT-PDK1-3′-UTR had less luciferase activity than other groups (Figure 4B). Western blot assay showed that PDK1 could be suppressed at protein level by mir-138-5p mimics, and PDK1 could be upregulated at protein level by mir-138-5p inhibitor (Figure 4C and Figure 4D). PDK1 was upregulated in RB cells (Figure 4E).

Discussion

RB, as a rare malignant tumor, frequently hap-pens in childhood. Recently, microRNAs partic-

ipate in development and metastasis of RB. For example, mir-497 suppresses proliferation and metastasis of human retinoblastoma cells via tar-geting VEGF10. Moreover, mir-320 inhibits RB progression via repressing specificity protein 111. In addition, microRNA-34a, MicroRNA-183 and some other microRNAs also play important role in the development of RB12,13. Thus, more and more effort for exploring the function of microR-NAs in RB is really urgent.

MicroRNAs, which are less than 20 bp in length, usually take effect by targeting the 3’-UTR section of genes14. Then, the target genes will be degraded and the function will be lost. So, microRNAs could affect diverse function of tu-mors, such as migration, proliferation, invasion, cell cycle, cell apoptosis and so on15. This inspires us to make more efforts to explore the function of miRNAs. In our study, mir-138-5p was lowly ex-pressed in RB cells. Furthermore, the cell viabili-ty, migrated and invaded ability was inhibited and cell apoptosis was suppressed in RB cells after mir-138-5p was overexpressed. Data above sug-gested that mir-138-5p suppresses the progression and tumorigenesis of RB. Accumulating evidence revealed that PDK1 acts as an oncogene and in-

Figure 3. Mir-138-5p suppressed cell migration and invasion of RB cells. (A) The migration in mir-138-5p mimics cells was decreased compared with negative control cells. (B) The migration in mir-138-5p inhibitor cells was increased compared with negative control cells. (C) Matrigel assay demonstrated that numbers of invading cells were reduced in mir-138-5p mimics cells compared with negative control cells. (D) Numbers of invading cells were increased in mir-138-5p inhibitor cells com-pared with negative control cells. *p<0.05.

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duces tumor genesis of cancers. For instance, expression level of PDK1 is correlated to poor prognosis of hepatocellular cancer16. PDK1 down-regulated by mircroRNA-379 suppresses tumor development of osteosarcoma17. Moreover, PDK1, as a critical regulator of PDK1/Akt/NF-kappa B axis, can be repressed by mir-128b and further regulates cell proliferation of gastric cancer18. In ovarian serous cancer, PDK1 is associated with better survival19. Moreover, PDK1 emerges as an oncogene in lung cancer through enhancing War-burg effect20. Besides, animal experiments reveal that PDK1 is associated with aggressiveness of bladder cancer mouse. A recent report21 showed that upregulated PDK1 was discovered in RB and may be a potential therapeutic target. In our study, overexpression of mir-138-5p downregulat-ed PDK1 in RB cells.

Conclusions

Downregulated mir-138-5p was firstly discov-ered in RB cells. Besides, overexpressed mir-138-5p could inhibit development and metastasis of RB through regulating PDK1. These findings implied that mir-138-5p could be a prospective therapeutic target for RB.

AcknowledgementsThis study was supported by National Natural Science Foundation of China (No. 81470600), Xiamen Science and Technology Plan Projects (No. 3502Z20159017), Nat-ural Science Foundation of Fujian Province of China (No. 2017J01331; No. 2015-ZQN-ZD-34 and 2016D013) and Huaxia Translation Medicine funding (No. 2017-A-02).

Conflict of InterestThe Authors declare that they have no conflict of interest.

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Figure 4. Correlation between PDK1 and mir-138-5p in RB cells. (A) PDK1 was a possible target of mir-138-5p. (B) Lucif-erase assay revealed that Y79 cells transfected with WT-PDK1-3′-UTR and mir-138-5p had less luciferase activity than other groups. PDK1 was higher expressed in RB cell lines compared with 16HBE cells. (C) Western blot assay showed that PDK1 was downregulated at protein level after cells were transfected with mir-138-5p mimics. (D) Western blot assay showed that PDK1 was upregulated at protein level after cells were transfected with mir-138-5p inhibitor. (E) PDK1 was significantly high-er expressed in RB cells (Y79) compared with retinal pigment epithelial cell line (ARPE-19). *p<0.05.

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