European Review for Medical and Pharmacological Sciences 2018; 22: 6350-6357 MicroRNA-203 inhibits invasion and induces apoptosis of laryngeal cancer cells via targeting LASP1 J. TAN, Y.-Y. JING, L. HAN, H.-W. ZHENG, J.-X. SHEN, L.-H. ZHANG, L.-S. YU Department of Otorhinolaryngology and Head and Neck Surgery, Peking University People’s Hospital, Beijing, China Abstract. – OBJECTIVE: To explore the role Introduction of microRNA-203 in laryngeal cancer and its un- derlying mechanism in regulating cell invasion Head and neck neoplasms rank sixth in the in- and apoptosis. cidence of all cancers that severely endanger hu- PATIENTS AND METHODS: MicroRNA-203 man health. As a kind of common head and neck expression in laryngeal cancer tissues and paracancerous tissues was detected by quanti- neoplasms, laryngeal cancer ranks second in the tative real time-polymerase chain reaction (qRT- incidence of respiratory tract tumors. Based on PCR). The regulatory effects of microRNA-203 the pathological types of laryngeal cancer, squa- on the invasion and apoptosis of laryngeal can- mous cell carcinomas are the most common ones cer cells were detected by transwell assay and in clinical patients1. Although the diagnosis and flow cytometry, respectively. Dual-Luciferase re- treatment for laryngeal cancer have been greatly porter gene assay was performed to access the advanced, the 5-year survival is still unsatisfacto- binding condition of microRNA-203 and LASP1. ry due to the frequent recurrence and lymph node Both mRNA and protein levels of LASP1 in la- 2 ryngeal cancer cells were detected after trans- metastasis . The potential pathogenic factors of fection with microRNA-203 mimic or microR- laryngeal cancer include smoking, alcohol, air NA-203 inhibitor by qRT-PCR and Western blot, pollution, occupational factors, etc.3. It is report- respectively. Rescue experiments were finally ed that some certain oncogenes (such as bcl-2, performed to detect whether microRNA-203 reg- c-myc, etc.) and tumor-suppressor genes (such as ulates laryngeal cancer development via target- p53, Rb, p16, p21, etc.) are involved in the occur- ing LASP1. 4 RESULTS: MicroRNA-203 was lowly expressed rence and development of laryngeal cancer . The in laryngeal cancer tissues and cell lines. Mi- specific mechanism of laryngeal cancer has not croRNA-203 knockdown in Hep-2 cells can pro- been fully elucidated and is required for further mote the invasion and inhibit the apoptosis of la- in-depth investigations5. ryngeal cancer cells. Subsequently, LASP1 was MicroRNA is a type of non-coding RNAs with predicted to be the target gene of microRNA-203, approximately 19-25 nt in length. It degrades which was further verified by the Dual-Lucifer- mRNA level or inhibits the translation of the ase reporter gene assay. LASP1 expression was negatively regulated by microRNA-203. Further- target gene by pairing with the 3’UTR of target 6 more, rescue experiments showed that the reg- gene . MicroRNA exerts diverse functions in ulatory effects of microRNA-203 on the invasion cell proliferation, differentiation, apoptosis and and apoptosis of laryngeal cancer cells were re- migration7,8. Recent studies have suggested that versed by LASP1. microRNAs could be served as oncogenes or CONCLUSIONS: We showed that lowly ex- tumor-suppressor genes, and are involved in on- pressed microRNA-203 could promote the inva- 9 sion and inhibit apoptosis of laryngeal cancer cogenesis, tumor cell invasion and angiogenesis . cells via inhibiting LASP1. Some certain microRNAs have been served as biomarkers and therapeutic targets of laryngeal cancer10,11. For example, microRNA-203 is lowly Key Words: expressed in laryngeal cancer tissues12. The spe- MicroRNA-203, Laryngeal carcinoma, Cell invasion, Apoptosis, LASP1. cific role of microRNA-203 in laryngeal cancer still needs to be comprehensively studied. 6350 Corresponding Author: Lisheng Yu, MD; e-mail: [email protected] MicroRNA-203 inhibits invasion and induces apoptosis of laryngeal cancer cells This study aims at exploring the regulatory ef- in the study were as follows: MicroRNA-203, fect of microRNA-203 on invasion and apoptosis F: 5’-GTGAAATGTTTAGGACCACTAGAA-3’, of laryngeal cancer cells, as well as its underlying R: 5’-GCTGTCAACGATACGCTACGT-3’; U6, mechanism. F: 5’-CGCTTCGGCAGCACATATAC-3’, R: 5’-TTCACGAATTTGCGTGTCAT-3’; GAPDH, F: 5’-GGAATCCACTGGCGTCTTCA-3’, R: Patients and Methods 5’-GGTTCACGCCCATCACAAAC-3’; LASP1, F: 5’-TGCGGCAAGATCGTGTATCC-3’, R: Sample Collection 5’-GCAGTAGGGCTTCTTCTCGTAG-3’. Laryngeal cancer tissues and paracancerous tissues were surgically resected from laryngeal Cell Apoptosis Detection cancer patients treated in our hospital. Tissues Hep-2 cells were washed with phosphate-buff- were immediately preserved in liquid nitrogen ered saline (PBS), digested with trypsin and for the following experiments. Enrolled patients centrifuged at 1000 rpm/min for 5 min. After were pathologically diagnosed as laryngeal can- washing with pre-cooled PBS twice, cells were cer and informed consent has been signed. This resuspended in PBS at a density of 1×105/mL. study was approved by the Ethics Committee of Subsequently, 1 μL of propidium iodide (PI) and Peking University People’s Hospital. 5 μl of Annexin V-fluorescein isothiocyanate (FITC) were added in 100 μL of cell suspension, Cell Culture followed by incubation at room temperature in M2E, TU212, Hep-2, and M4E cells were cul- the dark for 15 min. Finally, cell apoptosis was tured in Roswell Park Memorial Institute-1640 analyzed by flow cytometry (Partec AG, Ar- (RPMI-1640; Gibco, Grand Island, NY, USA, lesheim, Switzerland). USA) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomy- Transwell Assay cin (Hyclone, South Logan, UT, USA). Cells were Transwell chamber was pre-coated with 1 mg/ incubated in a 5% CO2 incubator at 37°C. mL Matrigel at 37°C for 3-5 h. Cells were ad- justed at a density of 2×105/mL. 100 μL of cell Cell Transfection suspension and 600 μL of culture medium con- Cells in logarithmic growth phase were taining 10% FBS was added in the upper and seeded in the 6-well plates and transfected lower chamber, respectively. 24 h later, non-ad- with microRNA-203 mimics, microRNA-203 herent cells were cleaned. Cells were fixed with inhibitor, pcDNA-LASP1 or negative control formaldehyde for 30 min and stained with violet following the instructions of Lipofectamine crystal for 15-30 min. Finally, penetrating cells 2000 (Invitrogen, Carlsbad, CA, USA). Trans- were observed and captured under a microscope. fection plasmids were provided by GenePhar- ma (Shanghai, China). Culture medium was Western Blot replaced 4-6 h later. The RIPA (radioimmunoprecipitation assay) protein lysate (Beyotime, Shanghai, China) was RNA Extraction and Quantitative used to extract the total protein in each group Real Time-Polymerase Chain Reaction of cells and tissues. The BCA (bicinchoninic (qRT-PCR) acid) method was performed to quantify the Total RNA in tissues was extracted using protein concentration. Protein samples were elec- TRIzol method (Invitrogen, Carlsbad, CA, USA) trophoresed on polyacrylamide gels and then for reverse transcription according to the instruc- transferred to polyvinylidene difluoride (PVDF) tions of PrimeScript RT reagent Kit (TaKaRa, membranes (Merck Millipore, Billerica, MA, Otsu, Shiga, Japan). RNA concentration was de- USA). After blocking with 5% skimmed milk, tected using a spectrometer and those samples the membranes were incubated with primary an- with A260/A280 ratio of 1.8-2.0 were selected tibody (LASP1, Cell Signaling Technology, Dan- for the following qRT-PCR reaction. QRT-PCR vers, MA, USA) at 4°C overnight. The membrane was then performed based on the instructions of was incubated with the secondary antibody after SYBR Premix Ex Taq TM (TaKaRa, Otsu, Shiga, rinsing with Tris-buffered saline and Tween 20 Japan) at 94°C for 15 s, 55°C for 30 s, and 72°C (TBST). Chemiluminescence was used to expose for 30 s, for a total of 40 cycles. Primers used the protein bands on the membrane. 6351 J. Tan, Y.-Y. Jing, L. Han, H.-W. Zheng, J.-X. Shen, L.-H. Zhang, L.-S. Yu Dual-Luciferase Reporter Gene Assay Results Wild-type and mutant-type LASP1 were con- structed. Cells were co-transfected with microR- MicroRNA-203 Was Lowly Expressed NA-203 mimics or negative control and wild-type in Laryngeal Cancer or mutant-type LASP1, respectively. Luciferase We detected the expression level of microR- activity was detected according to the instruc- NA-203 in laryngeal cancer tissues and cor- tions of Dual-Luciferase detection kit (Promega, responding paracancerous tissues by qRT-PCR. Madison, WI, USA). The results showed that microRNA-203 was low- ly expressed in laryngeal cancer tissues com- pared with that of paracancerous tissues (Figure Statistical Analysis 1A). Similarly, microRNA-203 expression was Statistical Product and Service Solutions SPSS also downregulated in laryngeal cancer cell lines 20.0 statistical software (IBM, Armonk, NY, (LL212, M2E, Hep-2, and M4E) compared with USA) was used for data analysis. Measurement that of controls (Figure 2B). Hep-2 cells were se- data were expressed as mean ± standard deviation lected for subsequent experiments. To further de- (x– ± s) and compared using the t-test. p < 0.05 was velop the specific mechanism of microRNA-203 considered statistically significant. in regulating the laryngeal cancer development, Figure 1. MicroRNA-203 was lowly expressed in laryngeal cancer. A, MicroRNA-203 was lowly expressed in laryngeal cancer tissues compared with that of paracancerous tissues. B, MicroRNA-203 was downregulated in laryngeal cancer cell lines (LL212, M2E, Hep-2, and M4E) compared with that of controls. C, D, Transfection efficacies of microRNA-203 mimic and microRNA-203 inhibitor in Hep-2 cells were verified by qRT-PCR. 6352 MicroRNA-203 inhibits invasion and induces apoptosis of laryngeal cancer cells microRNA-203 mimic and microRNA-203 in- Hep-2 cells, manifesting as a lower amount of hibitor were constructed.