Lncrna HOTAIR Promotes Nasopharyngeal Carcinoma Cell

European Review for Medical and Pharmacological Sciences 2017; 21: 5143-5152 LncRNA HOTAIR contributes to the tumorigenesis of nasopharyngeal carcinoma via up-regulating FASN

D.-D. MA1, L.-L. YUAN2, L.-Q. LIN3

1Department of Otorhinolaryngology, Affiliated Hospital of Jining Medical College, Jining, China 2Department of Histology and Embryology, Jining Medical College, Jining, China 3Department of Otorhinolaryngology, Linyi People’s Hospital, Linyi, China

Abstract. – OBJECTIVE: The therapeutic op- Key Words: tions for nasopharyngeal carcinoma (NPC) treat- Nasopharyngeal carcinoma, lncRNA HOTAIR, Fatty ment have been restricted mainly to radiothera- acid synthase, Oncogene, Tumorigenesis. py or chemotherapy, and the clinical treatment effect remains unsatisfactory. The primary pur- pose of this study was to investigate the molecular mechanisms of NPC and to find effective Introduction novel therapeutic targets for NPC. PATIENTS AND METHODS: In order to ana- Nasopharyngeal carcinoma (NPC) is an ag- lyze the expression level of lncRNA HOTAIR and gressive squamous cell carcinoma that resides in FASN in human NPC clinical tissues or NPC cells, nasopharynx, which is the most common type of total RNA was extracted with TRIzol and the relative mRNA expression levels were detected by malignancy of the neck and head in Southeast Asia 1 quantitative Real-time PCR. The endogenous ex- and North Africa . There are three major patho- pression of HOTAIR was modulated using lenti- genic factors of NPC, including Epstein-Barr virus virus vectors transfection. The protein levels of infection, genetic susceptibility, and environmen- Fatty acid synthase (FASN), p21 and MMP-9 in tal factors2-4. Due to the anatomical location and NPC cells were determined by Western blot when the inconspicuous early symptoms, NPC is usually HOTAIR was knockdown. A free Fatty acid quan- 5 titation assay was performed to detect the intra- diagnosed at the advanced stage . At present, the cellular free Fatty acid in NPC cells. The CCK-8 main treatments of NPC are mono-radiotherapy and colony formation assays were performed for and concurrent adjuvant chemotherapy. However, cell viability and proliferation determination. The the side effects of the treatments influence the life cell cycle of NPC cells was also determined by of the patients seriously6,7. According to Setton flow cytometric analysis. A matrigel invasion as- et al8, the relapse rate of NPC after the combined say was performed to analyze the invasive abili- radiation and chemotherapy treatment was 82%. ty of NPC cells. RESULTS: In this study, we observed a sig- The therapeutic approaches remain limited and nificant upregulation of lncRNA HOTAIR in NPC the clinical outcome is still unsatisfactory be- cells and clinical NPC specimens. The expres- cause the regulatory mechanisms of NPC remain sion of Fatty acid synthase (FASN) was posi- unclear9. Therefore, researches on the molecular tively correlated to HOTAIR in NPC specimens. mechanisms and genetic alterations involved in the Knockdown of HOTAIR led to downregulation of progression of NPC are needed. FASN in NPC cells, thus suppressing cell proliferation and invasion. Additionally, de novo syn- Long non-coding RNAs (lncRNAs) are widely thesis of cellular free fatty acid in NPC cells was defined as transcripts longer than 200 nucleotides inhibited when HOTAIR was knockdown. Fur- and do not have open reading frames that encode thermore, the protein levels of MMP-9 and p21 proteins10,11. A recent experiment indicates that were downregulated when HOTAIR was knock- there are upwards of 15,000 lncRNAs encoded down. CONCLUSIONS: by the human genome, suggesting lncRNAs are We suggest that HOTAIR is ubiquitous in mammalian genomes12. Although important in the progression and recurrence of NPC, perhaps through upregulating FASN. Tar- lncRNAs could not be translated into proteins, geting HOTAIR may be an effective therapeutic the majority of them play a crucial role in many strategy for NPC. biological processes such as in the regulation

Corresponding Author: Lili Yuan, MD; e-mail: [email protected] 5143 D.-D. Ma, L.-L. Yuan, L.-Q. Lin of gene transcription, post-transcriptional regu- between FASN and HOTAIR expression in NPC lation, and epigenetic regulation. Additionally, specimens. Further studies showed that FASN as some lncRNAs are associated with protein lo- well as the synthesis of cellular free fatty acid calization and fatty acid metabolic process in in NPC cells were inhibited when HOTAIR was numerous human diseases13,14. HOX antisense in- knockdown. The subsequent functional studies tergenic RNA (HOTAIR), an lncRNA that was revealed that HOTAIR knockdown suppressed first identified from a custom-tilling array of the cell proliferation and invasion in vitro. Our study HOXC locus, is encoded from the HOXC locus dissected a novel de novo palmitate synthesis on chromosome 12q13.1315. HOTAIR with 2,158 function of HOTAIR in NPC and it might serve nucleotides plays an important role in gene regu- as a promising diagnostic and therapeutic target lation by modifying chromatin structure15-17. As a for NPC patients. potential oncogene, HOTAIR was initially identified in breast cancer. It was reported to have a role in tumorigenesis and metastasis in a variety Patients and Methods of carcinomas, such as colorectal cancer, cervical cancer, bladder cancer, hepatocellular carcinoma, Cell Culture and Clinical Specimens gastrointestinal stromal tumors, and pancreat- The normal human nasopharyngeal NP460 and ic cancer18. Its expression positively correlates the human NPC cell lines CNE1, CNE2, 6-10B, with poor prognosis, tumor progression and re- 5-8F and HONE1, were obtained from the South- currence in these cancers. Although HOTAIR ern Medical University (Guangzhou, China). All has been reported as an independent prognostic the NPC cells were maintained in Roswell Park marker for NPC progression and survival, as Memorial Institute-1640 (RPMI-1640) (Gibco, well as mediated angiogenesis19,20, the underlying Grand Island, NY, USA) supplemented with 10% mechanism still needs to be further explored. fetal bovine serum (FBS) BI (Salt Lake City, UT, Fatty acid synthase (FASN) is a multi-func- USA) and 1% penicillin-streptomycin (Gibco, tional enzyme that catalyzes the biosynthesis of Grand Island, NY, USA). The normal human palmitate in a NADPH dependent manner21. It has nasopharyngeal NP460 was maintained in a 1:1 been reported FASN ubiquitously expressed in mixture of refined KSFM medium (Invitrogen, normal cells in adult tissues, but the levels were Carlsbad, CA, USA) and Epilife (Sigma-Aldrich, from low to moderate. However, most of normal St. Louis, MO, USA). The cells were cultured in cells which primarily import lipids from the a humidified 5% CO2 atmosphere at 37°C. extracellular milieu, do not have a strict require- A total of 23 clinical NPC specimens were ob- ment for FASN activity22,23. In contrast, an in- tained from the affiliated Hospital of Jining Med- creased requirement for lipids in functions, such ical College (Jining, Shandong, China). The study as membrane biosynthesis, protein modification, was approved by the Ethics Committee of our and signaling molecules, were observed in tumor hospital (JN2015-015, approval date, 2015.3.1), cells. Therefore, tumor cells are more dependent and all the recruited patients signed informed on de novo palmitate synthesis catalyzed by consent before participating in this study. Spec- FASN than normal cells24,25. Accordingly, FASN imens were obtained immediately after surgical is overexpressed in many solid and hematopoietic resection and stored at -80°C for further analysis. tumors, such as breast, ovarian, prostate, colon, Among them, 14 specimens were from male and lung, and pancreatic26-30. Moreover, it was also 9 from female patients. found FASN inhibition has anti-tumor activities in biologically diverse preclinical tumor models RNA Extraction and qRT-PCR and provides mechanistic and pharmacologic ev- Total RNA was extracted from NPC tissues idence that FASN inhibition presents a promis- or NPC cells using TRIzol reagent (Invitrogen, ing therapeutic strategy for treating a variety of Carlsbad, CA, USA) according to manufacturer’s cancers31. All these reports suggested that FASN instructions. Complementary DNA (cDNA) was could be used as diagnostic biomarker and thera- synthesized with Reverse Transcription M-MLV peutic target in treatment of NPC. (TaKaRa Biotechnology, Dalian, Liaoning, Chi- In the present study, our results confirmed na). Primers used for HOTAIR amplification the expressions of HOTAIR and FASN were were: forward 5’-GGTAGAAAAAGCAACCAC- markedly upregulated in NPC cells and speci- GAAGC-3’ and reverse 5’-ACATAAACCTCT- mens, and also revealed a positive correlation GTCTGTGAGTGCC-3’. Primers used for FASN

5144 LncRNA HOTAIR promotes nasopharyngeal carcinoma cell proliferation and invasion amplification were: forward 5’-TGCTAGCT- mL medium per well one day before the experi- GATCGATCGATCGTCG-3’ and reverse 5’-CG- ment. The cell viability was examined by CCK-8 TAGCTGATCGATGCTAGCTAGC-3’. GAPDH kit (Keygentec, Nanjing, China) according to the (forward 5’-TGCTAGGCTAGGACGCTAGC- manufacturer’s instruction. For colony formation TAC-3’ and reverse 5’-CTGGGCTAGATCGAC- assay, cells were seeded into 6-well plate at a GAGAGCTC-3’) was used as an internal control. density of 5 × 102 cells per well and cultured for The relative expression levels of HOTAIR and another fortnight. The colonies were stained with FASN were quantified by Applied Biosystems Coomassie brilliant blue (Beyotime, Shanghai, 7500 Real-Time PCR System (Applied Biosyste- China) and counted under a fluorescent micro- ms, Foster City, CA, USA) with Power SYBR1 scope (IX70, Olympus, Tokyo, Japan). All the Green PCR Master Mix (Applied Biosystems, experiments were performed in triplicate. Foster City, CA, USA) according to the supplier’s protocol. The relative mRNA expression levels Flow Cytometric Analysis were analyzed and expressed relative to threshold The cell cycle of NPC cells was also deter- cycle values (ΔCt), then converted to fold changes mined by flow cytometric analysis. HOTAIR using the 2-ΔΔCt method32,33. knockdown stable cells or the shNC control cells (2 × 104/well) were seeded in 12-well plate. After Construction of HOTAIR siRNA Lentiviral 72 h, cells for cell cycle analysis were harvest- Expression Vector ed by trypsinization and fixed in 70% ethanol The RNA interference sequences for human overnight. The fixed cells were rehydrated in HOTAIR were obtained from previous arti- phosphate-buffered saline (PBS) and digested cles20,34. The sequences of HOTAIR siRNA and with RNase A and labeled with propidium iodide negative control were 5’-GAACGGGAGUACA- (PI), then analyzed by Flow cytometry. Data were GAGAGA-3’(siHOTAIR-1), 5’-CCACAUGAAC- analyzed using Flow Jo analysis software (Tree GCCCAGAGAUU-3’(siHOTAIR-2), and 5’-CG- Star, Ashland, OR, USA). GAUCAGCUCGCGCUAUCAUCGCA-3’ (siNC). The oligonucleotides encoding short hairpin RNA Western Blotting (shRNA) were then constructed and synthesized For Western blotting analysis, total protein by Shanghai GenePharma Co., Ltd. (Shanghai, was extracted and quantified using the Bradford China) and annealed into double strands by An- method. About 60 g protein of each sample were nealing Buffer for RNA Oligos (Beyotime Insti- separated by 12% sodium dodecyl sulphate-poly- tute of Biotechnology, Haimen, Jiangsu, China). acrylamide gel electrophoresis (SDS-PAGE) and The double stranded DNA molecules were in- then transferred to polyvinylidene difluoride serted into PLKO.1 lentiviral vector. All of the (PVDF) membranes (Millipore, Billerica, MA, constructed plasmids were confirmed by DNA USA). After the membranes were blocked with sequencing. The plasmids carrying shHOTAIR 5% milk in phosphate buffered saline (PBS)- or the negative control shNC and packaging vec- 0.05% Tween, they were incubated with prima- tors were co-transfected into HEK293T to pro- ry antibody at 4°C overnight. Subsequently, the duce lentiviruses designated as Lv-ShHOTAIR-1, membranes were incubated with horse radish Lv-ShHOTAIR-2 and Lv-ShNC. The lentiviruses peroxidase (HRP)-conjugated secondary anti- particles were further used for infection of NPC bodies and finally detected with ECL substrate cells. For stable cell line, infected NPC cells were kit (Tanon, Shanghai, China). The HRP-conju- selected by puromycin (Sigma-Aldrich, St. Lou- gated secondary antibodies and primary antibody is, MO, USA) in the concentration of 2 mg/mL. against β-actin were purchased from Santa Cruz After antibiotics selection for about 7 days, the Biotechnology (Santa Cruz, CA, USA). FASN cultured cells were collected and the knockdown was purchased from Abcam (Cambridge, MA, of HOTAIR was confirmed by qRT-PCR. USA), MMP-9 and p21 were purchased from Cell Signaling Technology (Beverly, MA, USA). CCK-8 Assay and Colony Formation Assays Invasion Assay The CCK-8 and colony formation assays were Transwell invasion assay was performed using performed for cell viability and proliferation de- Boyden’s chambers. Cells were planted in the termination. For CCK-8 assay, cells were plated upper chamber consisting of 8-m membrane filter in 96-well plates at a density of 5000 cells in 100 inserts coated with Matrigel (BD Biosciences,

5145 D.-D. Ma, L.-L. Yuan, L.-Q. Lin

Franklin Lakes, NY, USA). The chemoattractant Results in the lower chamber was supplemented with medium containing 10% fetal bovine serum (FBS). LncRNA HOTAIR and FASN Were Cells on the upper surface were removed by a Up-Regulated in Human NPC Clinical wet cotton swab after 24 h, and those attached on Tissues and NPC Cell Lines the lower side of the membrane were fixed and In order to analyze the expression level of stained with crystal violet before counting under lncRNA HOTAIR and FASN in human NPC a microscope in five randomly selected fields. clinical tissues, 23 NPC tissues and their adjacent normal ones were collected. Next, the total RNA Intracellular Free Fatty Acid Assay of each specimen was extracted and the gene ex- Intracellular free fatty acid in NPC cells was pression levels were detected by Real-time PCR. determined by a free fatty acid quantitation kit Our results revealed that both HOTAIR and FASN according to manufacturer’s instructions (Sig- were considerably upregulated in NPC tissues ma-Aldrich, St. Louis, MO, USA). compared to adjacent normal ones (Figure 1A-B). Moreover, as shown in Figure 1C, FASN mRNA Statistical Analysis levels were positively related to the elevation of All quantitative data were expressed as mean ± HOTAIR in NPC tissues (r = 0.4520, p = 0.0304). standard deviation. Comparison between groups Furthermore, we evaluated the expression levels was done using One-way ANOVA test followed of lncRNA HOTAIR and FASN in six NPC cell by Least Significant Difference (LDS). p values lines (CNE1, CNE2, 6-10B, 5-8F and HONE1) by < 0.05 were considered statistically significant. Real-time PCR. The results showed HOTAIR and

Figure 1. Relative lncRNA HOTAIR and FASN expression in NPC tissues and NPC cell lines assessed by Real-time PCR. A-B, lncRNA HOTAIR and FASN were upregulated in 23 NPC tissues compared with 23 normal nasopharyngeal epithelial samples. C, qRT-PCR assays examined the expression of both HOTAIR and FASN mRNA in each of 23 NPC tissues, and the relevance is listed in each blot (r = 0.4520, p = 0.0304). D-E, lncRNA HOTAIR and FASN were upregulated in a panel of NPC cells compared with normal nasopharyngeal epithelial NP460 cell line. The data were analyzed using 2-ΔΔCt method and presented as the mean ± standard deviation. n = 3, *p < 0.05, **p < 0.01.

5146 LncRNA HOTAIR promotes nasopharyngeal carcinoma cell proliferation and invasion

FASN were also significantly increased in NPC The relative expression level of HOTAIR was cells compared with the immortalized nasopha- detected by RT-qPCR at 48 h after transfection. ryngeal cell line NP460 (Figure 1D-E). Among Figure 2A shows that the relative level of HO- the five cell lines evaluated, CNE2 and 5-8F with TAIR in NPC cells was significantly decreased by higher HOTAIR and FASN levels, were more Lv-shHOTAIR-1 or Lv-shHOTAIR-2, indicating aggressive cell lines when compared with CNE1 that HOTAIR can be knocked down by Lv-shHO- and 6-10B, respectively. Therefore, high level of TAIR-1 or Lv-shHOTAIR-2 successfully. The HOTAIR frequently occurs in NPC consistent relative expression level of HOTAIR transfected with previous studies, suggesting HOTAIR may with Lv-shHOTAIR-1 was reduced by 66.15% in mediate NPC development and progression. CNE2 cells and 69.28% in 5-8F cells; the reduced proportion was each with 53.23% and 73.48% Knockdown of lncRNA HOTAIR Inhibits when transfected with Lv-shHOTAIR-2. The rel- FASN in NPC Cells ative expression level of FASN was also detected To explore the relationship between HOTAIR by RT-qPCR, the level of FASN in NPC cells was and FASN in vitro, two NPC tumor cell lines in- significantly decreased when HOTAIR was knock- cluding CNE2 and 5-8F were selected for the high- down (Figure 2B). Next, Western blot assays were est upregulation of HOTAIR among the five NPC carried out to detect the protein levels of FASN in tumor cell lines. SiRNAs and lentiviral vectors CNE2 and 5-8F when HOTAIR was silenced. Our were used to limit HOTAIR expression in CNE2 results showed that the FASN protein levels were and 5-8F cells, investigating whether knockdown significantly reduced in Lv-shHOTAIR groups, of HOTAIR could inhibit FASN in NPC cells. compared with the control groups (Figure 2C).

Figure 2. HOTAIR was relevant to FASN expression in vitro. A, HOTAIR expression was suppressed by Lv-shHOTAIR in NPC cells. B, FASN expression was suppressed when HOTAIR knockdown. C, Protein levels of FASN were downregulated in the Lv-shHOTAIR infected CNE1 and 5-8F cells. Actin served as the internal control. D, HOTAIR knockdown inhibits the production of cellular free fatty acid in CNE1 and 5-8F cells. The data were presented as the mean ± standard deviation. *p < 0.05, **p < 0.01, n = 6.

5147 D.-D. Ma, L.-L. Yuan, L.-Q. Lin

Furthermore, the fatty acid synthase abilities of vival. As our results indicated that the expression tumor cells were also detected by measuring cel- level of FASN positively correlated with HOTAIR lular free fatty acid. HOTAIR knockdown signifi- expression, HOTAIR knockdown inhibits FASN cantly decreased the cellular free fatty acid from in NPC cells. In this study, the effect of HOTAIR 25.3 mM to 14.2 mM or 17.9 mM in CNE2 when on the proliferation of NPC cells was investigated. transfected with Lv-shHOTAIR-1 and Lv-shHO- After CNE1 and 5-8F cells were infected with TAIR-2, respectively. In 5-8F, cellular free fatty Lv-shHOTAIR-1 or Lv-shHOTAIR-2 or Lv-shNC, acid was decreased from 39.8 mM to 26.5 mM CCK–8 assays were used to examine the effect or 24.7 mM when transfected with shHOTAIR-1 of lncRNA HOTAIR on the proliferation of NPC and Lv-shHOTAIR-2, respectively (Figure 2D). cells. Although the cell proliferative ability among In summary, our results showed knockdown HO- shHOTAIR-1, Lv-shHOTAIR-2 and Lv-shNC in- TAIR inhibits FASN in NPC cells, which makes fected cells without significantly changes within HOTAIR a possible therapy target in treatment of 24 h, the cell proliferative ability of shHOTAIR-1 NPC. and Lv-shHOTAIR-2 groups markedly inhibited compared to Lv-shNC groups from 48 h (Figure Downregulation of lncRNA HOTAIR 3A). Furthermore, the cell cycle of shHOTAIR-1, Inhibited the Proliferation of NPC Cells Lv-shHOTAIR-2 and Lv-shNC groups was al- FASN is a vital enzyme in tumor cell biolo- so analyzed by flow cytometry when the cells gy, and inhibition of FASN prevents tumor cell were infected for 72 h. The results showed that growth, blocks tumor cell proliferation and sur- the percentage of G1 phase cells was increased

Figure 3. HOTAIR mediated cell growth in NPC cells. A, Growth of CNE2 and 6‑10B cells were detected by Cell Counting Kit‑8 assay every 24 h from 0 h to 72 h when HOTAIR expression was suppressed by Lv-shHOTAIR. B, The cell cycles of CNE2 and 6-10B cells were analyzed by Flow cytometry when the cells were infected for 72 h. C-D, Downregulation of lncRNA HOTAIR inhibited colony formation of CNE2 and 6-10B cells. n = 3, *p < 0.05, **p < 0.01.

5148 LncRNA HOTAIR promotes nasopharyngeal carcinoma cell proliferation and invasion about 15% (p < 0.01) after HOTAIR knockdown cells. We subsequently investigated the inva- by shHOTAIR-1 and about 12% (p < 0.05) when sion of CNE2 and 5-8F cells after infected with knockdown by Lv-shHOTAIR-2 in CNE1 cells LV-shHOTAIR. Matrigel invasion assays were compared with Lv-shNC groups, and the percent- performed to investigate whether lncRNA HO- age of G1 phase increased in 5-8F cells was about TAIR was involved in the invasion of NPC cells. 11% and 14%, respectively. The results showed the The results showed that lncRNA HOTAIR knock- cell cycle of NPC cells was arrested in G1 phage down cells exhibited impeded invasion abilities after downregulation of HOTAIR. There were not in both CNE2 and 5-8F cell lines (Figure 4A). significant changes among the remains phages of Statistical analysis indicated that the difference cell cycle (Figure 3B). Moreover, Lv-ShHOTAIR between LV-shHOTAIR-1 or LV-shHOTAIR-2 infected NPC cells formed much fewer colonies group compared with LV-shNC group was sig- compared with those obtained with Lv-ShNC in- nificantly (p < 0.01); the cells that passed through fected cells (Figure 3C-D). All these data sug- the filters were reduced more than 60% when gested that downregulation of HOTAIR inhibits infected with LV-shHOTAIR (Figure 4B). Our proliferation of NPC cells. results demonstrated that downregulation of HO- TAIR inhibits invasiveness of NPC cells in vitro. Knockdown of HOTAIR Suppresses Cell We further investigated the mechanism of HO- Invasion of NPC TAIR in the progression of cell metastasis in NPC Our studies indicated that silence of HOTAIR cells. Western blot showed that the expression inhibited the proliferation of CNE2 and 5-8F of matrix metalloproteinase (MMP)-9 and p21

Figure 4. Silencing of HOTAIR suppresses the invasion capacity of CNE2 and 5-8F cells. A, Representative images of invading cells (×100). B, Average number of invading cells from three independent experiments. C, Western blot examined the expression levels of MMP-9 and p21 in CNE2 and 5-8F cells after HOTAIR knockdown. n = 5, ** p < 0.01.

5149 D.-D. Ma, L.-L. Yuan, L.-Q. Lin were downregulated when HOTAIR knockdown that HOTAIR promotes invasion and metastasis (Figure 4C). It has been reported that MMP-9 in a group of tumors. In the present work, the and p21 positively correlated with migration and expression of lncRNA HOTAIR was examined invasiveness of tumor cells. When MMP-9 and in 23 primary NPC tissues and 23 normal naso- p21 were silenced, the invasion of tumor cells pharyngeal epithelial tissues, and in five NPC and was suppressed. Although HOTAIR in regulating one normal nasopharyngeal epithelial cell line. the protein levels of MMP-9 and p21 directly or The results revealed that HOTAIR expression through FASN remains to be investigated, our was upregulated in NPC specimens compared findings showed that silencing HOTAIR could in- with the normal tissues, and the extent of the inhibit cells invasion by downregulation of MMP-9 crease in five NPC cells ranged from about 5- to and p21 in NPC cells. 20-fold compared with the normal nasopharyngeal epithelial cell line. HOTAIR level was much higher in two aggressive cell lines: CNE2 and Discussion 5-8F. To explore the biological function of HO- TAIR in NPC progression, a lentivirus-mediated As one of the most common tumors in Southern shRNA expression system capable of interfering China and Southeast Asia, NPC is a specific re- with HOTAIR expression in CNE2 and 5-8F gional genetic disease and its development might cells was developed in the present study. The be associated with multiple factors20. Among all data demonstrated that knockdown of HOTAIR of these factors, the activation of oncogenes or significantly suppressed the proliferation and in- the inactivation of tumor suppressor genes that vasion of CNE2 and 5-8F cells in vitro. disrupt the homeostasis of cellular gene expres- Previous reports31,42,43 suggested that inhibi- sion results in the occurrence of NPC as multiple tion of de novo palmitate synthesis via FASN other human cancers35-37. Although encouraging inhibition provides an interesting approach to developments in the mechanism of NPC have cancer therapy with a strong biological rationale. been achieved, the prognosis and treatment for It has been found that lncRNAs are functionally patients with advanced NPC remain unfavor- associated with essential biological processes, able38. Hence, the molecular-targeted therapy will including fatty acid metabolic process44. The provide a more specific treatment for NPC and present investigation examined the expression of might provide new insight into its pathogenesis. FASN in NPC tissues and in NPC cell lines. Our LncRNA is considered as an important factor that results revealed that the mRNA level of FASN influences the development of human tumors12. positively correlated with HOTAIR expression in With the development of technological methods, NPC specimens. Furthermore, the protein levels such as lncRNA microarray and RNA sequenc- of FASN were downregulated when HOTAIR ing, more and more lncRNAs have been found was silenced in NPC cell lines. In all, knockdown to be dysregulated in tumors, which function as of HOTAIR inhibits fatty acid synthase in NPC oncogenes or tumor suppressors39. Previous stud- cells. Furthermore, the progression promotion in ies12,18 have demonstrated that abnormal expres- NPC by HOTAIR was partially activated by the sion of lncRNAs was able to change the biologi- FASN, MMP-9 and p21. However, whether the cal functions of tumor cells by affecting various effects of HOTAIR in regulating the protein lev- cellular processes. A recent research40 revealed els of MMP-9 and p21 were directly via targeting that LncRNA SLERT regulates the transcription FASN, need to be further investigated. Based of RNA polymerase I. Dysregulated rRNA syn- on this, we suggested that HOTAIR promotes thesis by RNA polymerase I is associated with nasopharyngeal carcinoma cell proliferation and uncontrolled cell proliferation which may afford invasion via targeting FASN. However, further the development of tumor. These data suggest the research is necessary to clarify the exact relation- important roles of lncRNAs in the pathogenesis ship between HOTAIR and FASN. and treatment of tumors. However, the specific functions and mechanisms of lncRNA in the occurrence of tumors remain to be elucidated. Conclusions As demonstrated in several previous research- es18,20, the expression of lncRNA HOTAIR was We identified the role of HOTAIR in mediat- upregulated in many primary tumor tissues in- ing tumorigenesis via upregulating FASN. These cluding NPC. Subsequent studies41 documented data demonstrated that HOTAIR might serve as

5150 LncRNA HOTAIR promotes nasopharyngeal carcinoma cell proliferation and invasion

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