European Review for Medical and Pharmacological Sciences 2019; 23: 6186-6193 Downregulation of lncRNA SNHG7 inhibits proliferation and invasion of nasopharyngeal carcinoma cells through repressing ROCK1 L. WANG1,2, T. XU3, X. CUI4, M. HAN2, L.-H. ZHOU2, Z.-X. WEI2, Z.-J. XU2, Y. JIANG2 1Department of Otorhinolaryngology, Qilu Hospital of Shandong University, Jinan, China 2Department of Otorhinolaryngology, The Affiliated Hospital of Qingdao University, Qingdao, China 3Department of Orthidontics, The Affiliated Hospital of Qingdao University, Qingdao, China 4Department of Otorhinolaryngology, Qingdao Women and Children’s Hospital Affiliated to Qingdao University, Qingdao, China Lin Wang and Tao Xu contributed equally to this work Key Words: Abstract. – OBJECTIVE: Recent studies have revealed the important role of long noncoding Long noncoding RNA, SNHG7, Nasopharyngeal RNAs (lncRNAs) in the progression of tumori- carcinoma, ROCK1. genesis. This study aimed to identify the bio- logical function of lncRNA small nucleolar RNA host gene 7 (SNHG7) in the progression of naso- Introduction pharyngeal carcinoma (NPC). PATIENTS AND METHODS: LncRNA SNHG7 Nasopharyngeal carcinoma (NPC) is one of expressions in NPC cell lines and 50 paired NPC tissue samples were detected by Real-time the most common head and neck epithelial can- quantitative polymerase chain reaction (RT-qP- cers with high morbidity in Southern China and CR). Transwell assay, wound healing assay and Southeast Asia1. With the advances made in inten- proliferation assay were conducted to evaluate sity-modulated radiotherapy and combined che- the in vitro function of SNHG7 in NPC cells. Xe- moradiotherapy, the prognosis for patients with nograft model was established for determining local and regional NPC has been significantly the in vivo effect of SNHG7 on tumor formation improved. However, metastatic rate remains high and metastasis of NPC. The underlying mech- anism of SNHG7 in mediating the progression in NPC patients, which is the leading cause of tre- of NPC was explored by RT-qPCR and Western atment failure and cancer-related death in NPC, 2,3 blot. with a median survival about 12 months . The- RESULTS: SNHG7 expression was remarkably refore, clarifying the molecular mechanisms un- downregulated in NPC tissues compared with derlying NPC is urgently required to promote the that in adjacent normal samples. Knockdown of development of effective individualized therapy SNHG7 attenuated proliferation, invasion and and improve the clinical outcomes of NPC pa- migration of NPC cells. Moreover, tumor size tients. With the development of high-throughput and the number of metastatic nodules were re- duced in mice administrated with NPC cells sequencing and microarrays, researchers have di- transfected with sh-SNHG7. Knockdown of SN- scovered that more than 90% of mammalian ge- HG7 downregulated ROCK1 at mRNA and pro- nome can be transcribed into noncoding RNAs tein level. Besides, the expression of ROCK1 in (ncRNAs). Long noncoding RNAs (lncRNAs), tumor tissues was positively correlated to SN- one subgroup of ncRNAs in over 200 nt long, are HG7 expression. important clusters of transcripts with little pro- CONCLUSIONS: Knockdown of SNHG7 inhib- tein-coding potential. Recently, evidence proved its migration, invasion and proliferation of NPC cells through downregulating ROCK1, which that lncRNAs are important regulators in many mayRETRACTED offer a new therapeutic intervention for biological behaviors, including carcinogenesis. NPC patients. For example, lncRNA TP73AS1 dramatically 6186 Corresponding Author: Yan Jiang, MD; e-mail: [email protected] LncRNA SNHG7 inhibits the progression of nasopharyngeal carcinoma promotes cell apoptosis and depresses cell proli- SNHG7 was amplified and inserted into pcD- feration in colorectal cancer by functioning as a NA3.1 (Invitrogen, Carlsbad, CA, USA). Cell competing endogenous RNA sponging miR103 to transfection was conducted with Lipofectamine modulate the expression of PTEN (gene of pho- 2000 (Invitrogen, Carlsbad, CA, USA). Tran- sphate and tension homology deleted on chromo- sfection efficacy at 48 h was detected by Real-ti- some ten)5. Long non-coding RNA ZFAS1 promo- me quantitative polymerase chain reaction (RT- tes nasopharyngeal carcinoma through activating qPCR). Wnt/beta-catenin pathway6. In addition, lncRNA CDKN2BAS promotes cell growth and migration RNA Extraction and RT-qPCR in hepatocellular carcinoma through miR-153-5p/ Total RNA from tissues and cells were ex- ARHGAP18 signaling pathway7. However, the tracted using TRIzol reagent (Invitrogen, Car- potential role of lncRNA small nucleolar RNA lsbad, CA, USA). The total RNA was reversely host gene 7 (SNHG7) in the development of NPC transcribed to complementary deoxyribose nu- remains unexplored. In the present study, expres- cleic acids (cDNAs) through reverse Transcrip- sion level of lncRNA SNHG7 was remarkably tion Kit (TaKaRa Biotechnology Co., Ltd., Da- upregulated in NPC samples. Moreover, fun- lian, China). Thermocycling conditions were as ctional experiments revealed that knockdown of follows: 30 s at 95°C, 5 s at 95°C and 35 s at 60°C, SNHG7 depressed cell proliferation, invasion and for 40 cycles. Following were the primers used for migration in NPC. Furthermore, we discovered RT-qPCR: SNHG7, forward: 5′- GTGACTTCGC- that lncRNA SNHG7 exerted its role in the pro- CTGTGATGGA-3′ and reverse: 5′-GGCCTCTA- gression of NPC by upregulating ROCK1. TCTGTACCTTTATTCC-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward: 5′-CCAAAATCAGATGGGGCAATGCTGG-3′ Patients and Methods and reverse: 5′-TGATGGCATGGACTGTG- GTCATTCA-3′. 2-ΔΔCt method was utilized for Tissue Specimens calculating relative expression. Paired NPC tissues and adjacent non-tumor tissues (≥ 5 cm away from the tumor edge) were Western Blot surgically resected from 50 NPC patients under- Total proteins were extracted from cells via ra- going surgery from February 2016 to December dioimmunoprecipitation assay (RIPA) buffer and 2018 in the Affiliated Hospital of Qingdao Uni- quantified by bicinchoninic acid (BCA) protein versity. The Ethics Committee of the Affiliated quantification kit (Beyotime, Shanghai, China). Hospital of Qingdao University approved this stu- The target proteins were separated by sodium do- dy protocol, and all participants provided written decyl sulphate-polyacrylamide gel electrophore- informed consents. sis (SDS-PAGE). Protein samples were loaded on the polyvinylidene difluoride (PVDF) membrane Cell Lines (Millipore, Billerica, MA, USA). Membranes were Human NPC cell lines CNE2, CNE1, 5-8F and blocked in 5% skim milk and incubated with pri- 6-18B and immortalized normal nasopharynge- mary antibodies (rabbit anti-GAPDH and rabbit al epithelial cell line NP69 were offered by the anti-ROCK1) and secondary antibodies. Finally, Chinese Academy of Science (Shanghai, China). the Pierce (Rockford, IL, USA) enhanced chemi- Cells were cultured in Roswell Park Memorial luminescence (ECL) was utilized for visualizing Institute-1640 (RPMI-1640) (HyClone, South Lo- Western blotting substrate immunoreactive bands. gan, UT, USA) containing 10% fetal bovine se- rum (FBS) (Gibco, Rockville, MD, USA) and 1% Cell Proliferation Assay penicillin (epidermal growth factor was applied Cell proliferation was monitored every 24 h by for culturing NP69 cells). Cells were maintained cell counting kit-8 (CCK-8) assay. Spectrophoto- in an incubator with 5% CO2 at 37°C. meter (Thermo Scientific, Waltham, MA, USA) was utilized to measure the absorbance at 450 nm. Cell Transfection Lentivirus expressing short-hairpin RNA Wound Healing Assay (shRNA) directed against SNHG7 was provided Cells seeded into 6-well plates, were cultured by GenePharmaRETRACTED (Shanghai, China). Complemen- in Roswell Park Memorial Institute 1640 (RPMI- tary deoxyribose nucleic acid (cDNA) encoding 1640) medium overnight. After scratched with 6187 L. Wang, T. Xu, X. Cui, M. Han, L.-H. Zhou, Z.-X. Wei, Z.-J. Xu, Y. Jiang a plastic tip, cells were cultured in serum-free the number of metastatic nodules in the lung was (RPMI-1640). Wound closure was viewed at the counted. The animal experiments were approved appointed time points. Each assay was indepen- by the Animal Ethics Committee of Qingdao Uni- dently repeated for three times. versity (Qingdao, China). Matrigel Assay Statistical Analysis 24 h after transfection, 2 × 105 cells suspended Statistical analysis was conducted by Statisti- in 100 µL of serum-free Roswell Park Memorial cal Product and Service Solutions (SPSS) 18.0 Institute-1640 (RPMI-1640) were applied on the (SPSS Inc., Chicago, IL, USA). Chi-square test, top chamber of an 8-μm culture inserts (Corning, Student t-test and Kaplan-Meier method were se- Corning, NY, USA) pre-coated with 50 µg Matri- lected when appropriate. Data were presented by gel (BD Biosciences, Franklin Lakes, NJ, USA). mean ± SD (Standard Deviation). It was conside- Roswell Park Memorial Institute-1640 (RPMI- red statistically significant when p<0.05. 1640) containing 20% fetal bovine serum (FBS) was added in the bottom chamber. 24 h later, the- se inserts were treated by methanol for 30 min Results and stained by hematoxylin for 20 min. An inver- ted microscope (×40) (Nikon, Tokyo, Japan) was Expression Level of SNHG7 in utilized for counting penetrating cells in three NPC Tissues and Cells random fields. First, RT-qPCR was conducted for detecting SNHG7 expression in 50 paired NPC tissues and Xenograft Model 4 NPC cell lines. The result indicated