Mir-222 Inhibited OS Via YWHAG

European Review for Medical and Pharmacological Sciences 2018; 22: 2588-2597 MicroRNA-222 contributed to cell proliferation, invasion and migration via regulating YWHAG in osteosarcoma Y.-W. CHU1, C.-R. WANG1, F.-B. WENG1, Z.-J. YAN1, C. WANG2 1Department of Orthopedic, First People’s Hospital of Wujiang District, Affiliated Hospital of Nantong University Medical School, Suzhou, China 2Department of Orthopedic, The Third People’s Hospital of Yancheng, Affiliated Yancheng Hospital of Southeast University Medical School, Yancheng, China Yawei Chu and Churong Wang contributed equally to this work Abstract. – OBJECTIVE: The aim of this study Introduction was to investigate the role of microRNA-222 (miR-222) in osteosarcoma (OS), and to further Osteosarcoma (OS) is a common malignant explore the potential molecular mechanism. osteogenic tumor, which is predisposed to attack PATIENTS AND METHODS: We measured the the adolescence, and has a poor prognosis with a level of miR-222 in OS tissues and cell lines using high degree of malignancy1. The current 5-year quantitative Real-time polymerase chain reaction. Synthesized miR-222 mimics or inhibitors were survival rate is only about 55% to 68%, with the obtained to up-regulate or down-regulate the ex- vast majority of patients eventually occurring 2 pression of miR-222 in U2OS or Saos2 cells. Cell tumor metastasis . Therefore, looking for new counting kit-8 (CCK8) and colony formation assay and effective prevention and control measures to were employed to detect the ability of cell prolif- improve the quality of life and survival of pa- eration, and transwell assay was used to confirm tients and to prevent or delay the transfer of OS the ability of cell invasion. Furthermore, lucifer- become a high priority. ase assay and Western blot were applied to verify the target of miR-222 in OS. With the continuous innovation of experimen- RESULTS: The level of miR-222 in OS tumor tal techniques in molecular biology and the com- tissue samples was significantly lower than that pletion of the human genome project, research- in normal group. Over-expression of miR-222 de- ers have become more and more understanding creased cell proliferation and invasion in U2OS about the RNA and its functions3. microRNAs cells while knockdown of miR-222 promoted cell are a huge family which relative to the function- growth and metastasis in Saos2 cells. Further- 4 more, YWHAG was found to be a candidate tar- al RNA regulation mechanism . About 50% of the get of miR-222 using several databases. Elevat- known miRNAs in human are found to locate in ed level of miR-222 inhibited YWHAG expression the chromosome region closely related to the tu- while reduced miR-222 promoted YWHAG ex- mor, suggesting that they play important roles in pression. Also, up-regulation of YWHAG re- the development of tumors5. In OS, several studies6 stored the inhibiting effect of miR-222 mimics. CONCLUSIONS: have identified miRNAs participated in the devel- We identified for the first opment and progression of OS. For example, miR- time that the expression level of miR-222 was reduced in OS tissues as well as in OS cell lines. 92b could promote osteosarcoma proliferation, miR-222 could inhibit cell proliferation and inva- invasion, and migration via targeting RECK; miR- sion via down-regulating YWHAG. These data 199a-5p could dual-target PIAS3 and p27 and the could provide a potential target for the biologi- promote tumor growth in human OS; also, miR- cal treatment of OS. 135b and TAZ formed a feedback loop which promoted epithelial-mesenchymal transition (EMT) Key Words: miR-222, Proliferation, Invasion, YWHAG, Osteosar- and tumorigenesis in OS; miR-491 acted as a tu- coma. mor suppressor via inhibiting OS lung metastasis and chemoresistance through αB-crystallin7-10. 2588 Corresponding Author: Chao Wang, MM; e-mail: [email protected] miR-222 inhibited OS via YWHAG Several researches have reported miR-222 Rockville, MD, USA) and maintained at 37°C in functioned as a tumor suppressor in many can- humid air containing 5% CO2. The cells were pas- cers, which belongs to a miRNA cluster miR- saged when the cell confluence reached 90%, and 221/222. Liu et al11 found that in colorectal can- kept in logarithmic growth state. We took cells cer cells, a miR-221/222-mediated feedback loop under logarithmic growth phase to conduct the maintained constitutive activation of NF-κB and experiments. STAT3. Galardi et al12 confirmed that in human prostate carcinoma cell lines, miR-221/222 target- RNA Isolation and Quantitative Real-Time ing p27Kip1, affected the growth potential. Wong Polymerase Chain Reaction (qRT-PCR) et al13 demonstrated in hepatocellular carcinoma, Total RNA of tissue samples and established over-expression of miR-222 conferred cell metas- cell lines were lysed using RIPA reagent (Invitro- tasis through activating protein kinase B (AKT) gen, Carlsbad, CA, USA). ATaqMan microRNA pathway. However, the expression and function in Reverse Transcription kit (Biosystems, Medford, OS have not been mentioned before. MA, USA) was employed to reverse the RNA In present study, we found that miR-222 expres- to complementary Deoxyribose Nucleic Acid sion level was decreased in OS samples compared (cDNA), and the expression level of miR-222 was to normal control using quantitative Real-time detected using a SYBR Green PCR master mix polymerase chain reaction (PCR). Gain-of-func- Kit (TaKaRa, Dalian, China). U6 was applied as tion and loss-of-function experiments were con- an internal control and the relative expression lev- ducted to further confirm the influences of miR- el was measured by 2-∆∆CT method. Each experi- 222 in OS. Furthermore, we verified YWHAG as ment was repeated at least three times. an immediate target of miR-222 in OS. This study might provide a novel target for OS biological di- Cell Transfection agnosis and prognosis prediction. miR-222 mimcs, inhibitors, negative control (NC), inhibitors negative control (INC) and pcD- NA for over-expressing YWHAG (YWHAG) Patients and Methods were synthesized by GenePharma (Shanghai, China). Cells were transfected with these synthet- Tissue Samples of OS and Normal ics using lipofectamine 2000 (Invitrogen, Carls- Control Group bad, CA, USA) in accordance with the manufac- A total of 21 cases of osteosarcoma tissue turer’s instructions. The efficiency of transfection specimens surgically removed from Department was confirmed using qRT-PCR. of Orthopedic in our hospital from 2010 to 2016 were collected. There were 13 males and 8 fe- Cell Counting Kit-8 (CCK8) Assay males, ranged from 6 to 35 years, with an aver- The Cell Counting Kit (Dojindo, Kumamoto, age of 20.42 years. 21 cases of normal bone tis- Japan) was employed to measure the ability of cell sue samples collected from normal bone tissue proliferation. Cells after treatment were seeded in specimens after the hip joint replacement and 96-well plates and cultured in 100 μL normal me- the sex, age, and tumor group, had no significant dium at a density of 3000 cells per well. At 0 h, difference. Specimens were cut and then placed 24 h, 48 h, and 72 h after culture, 10 μLCCK8 in liquid nitrogen at -80°C for preservation. This reagents were added into the well and the absor- study was approved by the Ethics Committee of bance was measured at 450 nm. Each experiment the Third People’s Hospital of Yancheng. Signed was replicated for at least three times. written informed consents were obtained from all participants before the study. Colony Formation Assay A total of 400 cells were planted in each 3.5 cm Cell Lines and Culture plate after transfection, and maintained in normal Human OS-derived cell line Saos2, 143B, RPMI-1640 medium for two weeks. Then, the HOS, MG63, U2OS and normal osteoblast line colony containing more than 50 cells was calcu- NHost cells were purchased from Shanghai Keil- lated. We repeated the assay for three times. ton Biological Co., Ltd. (Shanghai, China). All these cell lines were cultured in Roswell Park Transwell Assay Memorial Institute-1640 (RPMI)-1640 medium To detect the cell invasion and migration ability, containing 10% fetal bovine serum (FBS) (Gibco, we used 8-μm transwell inserts (Millipore, Billeri- 2589 Y.-W. Chu, C.-R. Wang, F.-B. Weng, Z.-J. Yan, C. Wang ca, MA, USA) to realize the experiments. For inva- Statistical Analysis sion, the upper surface of the membrane was cov- Statistical product and service solutions 17.0 ered with Matrigel (BD Science, Franklin Lakes, software (SPSS Inc., Chicago, IL, USA) were ap- NJ, USA). A total of 4x104 treated cells in FBS-free plied to analyze the data. The measurement data medium were seeded into the top chamber of the were shown as mean ± SD. Comparison between insert, and 500 μL RPMI-1640 medium contain- groups was done using One-way ANOVA test ing 10% fetal bovine serum (FBS) were added into followed by Least Significant Difference (LSD). the lower chamber. After 48 h incubation, cells that p-values < 0.05 were considered having signifi- failed to pass through the membrane were removed. cant difference. The membrane containing cells on its lower surface was fixed with pre-cooling methanol and stained with 0.5% crystal violet. Next, the cells stained were Results calculated after pictures taken using a microscope in five random visions. For migration, the upper miR-222 was Reduced in OS Tissue and surface of the membrane was covered with nothing. Cell Lines The other steps were the same as the invasion one. To examine the expression level of miR-222, we measured the expression of miR-222 in 21 OS pa- Dual-Luciferase Assay tients’ tissue samples compared with 21 normal con- Dual-Luciferase assay (Promega, Madison, trol tissue samples.

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