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

European Review for Medical and Pharmacological Sciences 2017; 21: 5624-5629 Mir-138-5p acts as a tumor suppressor by targeting pyruvate dehydrogenase kinase 1 in human retinoblastoma Z. WANG 1, Y.-J. YAO 1, F. ZHENG 1, Z. GUAN 1, L. ZHANG 1, N. DONG 2, W.-J. QIN 3 1 Department of Ophthalmology, Fuzhou Second Hospital, Fujian, China 2 Corneal Disease Clinic, Xiamen Eye Center Affiliated Xiamen University, Fujian, China 3 Department of Radiation Oncology, Zhongshan Hospital Affiliated Xiamen University, Fujian, China Abstract. OBJECTIVE: MicroRNAs have caught more attention for their role in tumor progression. Retinoblastoma (RB) is one of these ordinary 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 expression in RB cells was monitored by RT-qPCR. Besides, the role of mir-138-5p in RB development was explored through function experiments in vitro. The potential mechanism was further explored by RT-qPCR, luciferase assay, and Western blot assay. RESULTS: In our investigation, mir-138-5p was lower-expressed in RB cells than that in retinal pigment 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 overexpression of mir-138-5p, while PDK1 was upregulated 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 vision for interpreting the mechanism of RB tumorigenesis. 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 world 1. Although effective therapy is available for RB patients, children with RB have greater life-threatening risks 2. Therefore, it is urgent to find more potential regulators and targets for treatment of RB. Recently, aberrant non-coding RNAs expression is identified in most cancers and closely associated with patients prognosis. Moreover, mounting evidence indicates that micrornas contribute to regulating oncogene expression, which further acts on progression of malignant cancers, including RB 3-5. Accumulating evidence demonstrates that mir-138-5p participates in tumor carcinogenesis. Mir-138-5p knockdown promotes cell growth and invaded ability via suppressing survivin in bladder cancer 6. By targeting GPR124 in non-small cell lung cancer, mir-138-5p could reverse resistance to gefitinib 7. Moreover, mir-138-5p can inhibit autophagy via suppressing SIRT1 in pancreatic carcinoma 8. Mir-138-5p contributes to suppressing development and metastasis of colorectal carcinoma via targeting PD-L1 9. However, 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, overexpressed mir-138-5p could repressed cell viability, invasion and migration, and induced cell apoptosis 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 Lines The Institute of Biochemistry and Cell Biology, Chinese Academy of Science (Shanghai, China) provided us with RB cell line Y79 and retinal pigment epithelial cell line ARPE-19. Culture medium was consisted of penicillin, 10% fetal bovine serum (FBS, Invitrogen Life Technologies, Carlsbad, CA, USA) and Dulbecco s Modified Eagle Medium (DMEM, Thermo Fisher Scienti- 5624 Corresponding Author: Nuo Dong, Ph.D; e-mail: wangzhfz@126.com; Wenjuan Qin, Ph.D; e-mail:proftomato@163.com

Mir-138-5p in retinoblastoma fic, Waltham, MA, USA). Besides, cells were cultured in humidified incubator, which contained 5% CO 2 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-PCR Firstly, total RNA was separated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Then, RNA was reverse-transcribed to cdnas via reverse Transcription Kit (TaKaRa, Dalian, China). Quantitative reverse transcriptase-polymerase chain reaction (qrt-pcr) was performed on ABI 7500 RT PCR detection system (Applied Biosystems, 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 Blot Reagent radioimmunoprecipitation assay (RIPA) (Beyotime, Shanghai, China) was utilized to extract protein from cells. Bicinchoninic acid (BCA) protein assay kit (TaKaRa, Dalian, China) was chosen for quantifying protein concentrations. 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 antibodies. Cell Signaling Technology (CST, Beverly, MA, USA) provided us with rabbit anti-pdk1 and rabbit anti-gapdh, as well as goat anti-rabbit secondary antibody. Chemiluminescent film was applied for assessment of protein expression with ImageJ software. Luciferase Assays The 3ʹ-UTR of PDK1 was cloned into the pgl3 vector (Promega, Madison, WI, USA) named wild-type (WT) 3 -UTR. Site-directed mutagenesis of the mir-138-5p binding site in PDK1 3 -UTR named mutant (MUT) 3ʹ-UTR was conducted with quick-change site-directed mutagenesis 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) Assay Cell viability of theses treated cells in 96- well plates was monitored every 24 h by CCK8 assay following the protocol (Dojindo Molecular Technologies, Inc. Kumamoto, Japan). The absorbance was measured at 450 nm on the spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Wound Healing Assay Cells 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 repeated in triplicate. Matrigel Assay To detect the invasion of RB cells, 5 10 4 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 medium (10% FBS). 48 h later, top surface of chambers 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 Analysis SPSS 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 considered statistically significant. Results Downregulated mir-138-5p in RB Cells Firstly, RT-qPCR was conducted for detecting mir-138-5p level in RB cell Y79 and retinal pigment epithelial cell line ARPE-19. As the result, mir-138-5p was significantly lower in Y79 compared 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- 5625

Z. Wang, Y.-J. Yao, F. Zheng, Z. Guan, L. Zhang, N. Dong, W.-J. Qin 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 transfected with mir-138-5p inhibitor and negative control was determined by qrt PCR. * p<0.05; ** p<0.01. 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 (Figure 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 2. Mir-138-5p suppressed cell viability and promoted apoptosis of RB cell. (A) mir-138-5p mimics significantly reduced 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. 5626

Mir-138-5p in retinoblastoma 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 compared with negative control cells. *p<0.05. 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-138-5p To further understand how mir-138-5p participated 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 (Figure 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 happens in childhood. Recently, micrornas participate in development and metastasis of RB. For example, mir-497 suppresses proliferation and metastasis of human retinoblastoma cells via targeting VEGF 10. Moreover, mir-320 inhibits RB progression via repressing specificity protein 1 11. In addition, microrna-34a, MicroRNA-183 and some other micrornas also play important role in the development of RB 12,13. 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 genes 14. Then, the target genes will be degraded and the function will be lost. So, micrornas could affect diverse function of tumors, such as migration, proliferation, invasion, cell cycle, cell apoptosis and so on 15. This inspires us to make more efforts to explore the function of mirnas. In our study, mir-138-5p was lowly expressed in RB cells. Furthermore, the cell viability, migrated and invaded ability was inhibited and cell apoptosis was suppressed in RB cells after mir-138-5p was overexpressed. Data above suggested that mir-138-5p suppresses the progression and tumorigenesis of RB. Accumulating evidence revealed that PDK1 acts as an oncogene and in- 5627

Z. Wang, Y.-J. Yao, F. Zheng, Z. Guan, L. Zhang, N. Dong, W.-J. Qin Figure 4. Correlation between PDK1 and mir-138-5p in RB cells. (A) PDK1 was a possible target of mir-138-5p. (B) Luciferase 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 higher expressed in RB cells (Y79) compared with retinal pigment epithelial cell line (ARPE-19). *p<0.05. duces tumor genesis of cancers. For instance, expression level of PDK1 is correlated to poor prognosis of hepatocellular cancer 16. PDK1 downregulated by mircrorna-379 suppresses tumor development of osteosarcoma 17. 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 cancer 18. In ovarian serous cancer, PDK1 is associated with better survival 19. Moreover, PDK1 emerges as an oncogene in lung cancer through enhancing Warburg effect 20. Besides, animal experiments reveal that PDK1 is associated with aggressiveness of bladder cancer mouse. A recent report 21 showed that upregulated PDK1 was discovered in RB and may be a potential therapeutic target. In our study, overexpression of mir-138-5p downregulated PDK1 in RB cells. Conclusions Downregulated mir-138-5p was firstly discovered 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. Acknowledgements This study was supported by National Natural Science Foundation of China (No. 81470600), Xiamen Science and Technology Plan Projects (No. 3502Z20159017), Natural 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 Interest The Authors declare that they have no conflict of interest. References 1) Zhou D, Liu P, Sun DW, Chen ZJ, Hu J, Peng SM, Liu YL. USP22 down-regulation facilitates human retinoblastoma cell aging and apoptosis via inhibiting TERT/P53 pathway. Eur Rev Med Pharmacol Sci 2017; 21: 2785-2792. 2) Yang Q, Tripathy A, Yu W, Eberhart CG, Asnaghi L. Hypoxia inhibits growth, proliferation, and increases response to chemotherapy in retinoblastoma cells. Exp Eye Res 2017; 162: 48-61. 3) Wei D, Yang L, Lv B, Chen L. Genistein suppresses retinoblastoma cell viability and growth and induces apoptosis by upregulating mir-145 and inhibiting its target ABCE1. Mol Vis 2017; 23: 385-394. 4) Xin ZC, Yang HQ, Wang XW, Zhang Q. Diagnostic value of micrornas in breast cancer: a meta-analysis. Eur Rev Med Pharmacol Sci 2017; 21: 284-291. 5628

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