LncRNA MNX1-AS1 drives aggressive laryngeal squamous cell carcinoma progression and serves as a ceRNA to target FoxM1 by sponging microRNA-370

Long non-coding RNA (LncRNA) MNX1 antisense RNA 1(MNX1-AS1) is associated with the pathology of numerous cancers. But, the role and underlying pathways of MNX1-AS1 in the regulation of laryngeal squamous cell carcinoma (LSCC) is not known. We demonstrated remarkably elevated levels of MNX1-AS1 in the LSCC tissues, which was correlated with poor disease prognosis. Moreover, MNX1-AS1-silencing strongly suppressed LSCC cell proliferation, migration, and invasion. We also demonstrated that MNX1-AS1 sequesters that activity of miR-370, thereby releasing Forkhead Box ml (FoxM1) from the inhibitory actions of MNX1-AS1. Furthermore, the positive correlation of MNX1-AS1 and FoxM1 as well as the converse correlation between miR-370 and MNX1-AS1 (or FoxM1) were revealed in LSCC tissues using experiments. Based on rescue assays, FoxM1 overexpression or miR-370 downregulation partially recovered the inhibitory effect of MNX1-AS1 silencing on LSCC cells. Moreover, knockdown of MNX1-AS1 retarded tumor growth in nude mice model. In summary, these findings verified that MNX1-AS1 modulated LSCC progression by competitively binding with miR-370 to regulate FoxM1.


INTRODUCTION
Laryngeal squamous cell carcinoma (LSCC) is a highly prevalent form of laryngeal cancers that has been rising worldwide in recent years [1]. Although much advancements have been made in multimodal therapy like surgical resection, chemotherapy, radiotherapy, and combined therapies, the 5-year overall survival (OS) rate of LSCC patients have not altered significantly due to late diagnosis and high propensity for metastasis [2,3]. As such, it is urgent and crucial to explore novel prognosis markers and therapeutic targets for LSCC.
Long non-coding RNAs (lncRNAs) are >200 nucleotides (nt) long RNAs that do not code for proteins and are associated with numerous forms of cancers via its regulation of chromatin remodeling, transcriptional modulation, and post-transcriptional control [4,5]. Multiple studies report involvement of lncRNAs in LSCC progression and function as tumor suppressors or oncogenes [6,7]. For instance, LncRNA DLX6-AS1 contributes to the LSCC growth via the regulation of miR-376c [8]. Similarly, lncRNA XIST modulates the miR-144/IRS1 axis to accelerate LSCC progression [9]. Furthermore, LncRNA CDKN2B-AS1 serves as an oncogene in the pathogenesis of LSCC and work via sponging of miR-497 to upregulate CDK6 [10]. Alternately, LncRNA GAS5 functions as a tumor suppressor that inhibits LSCC progression by sponging miR-21 [11]. These studies implied that lncRNAs might act as novel targets for diagnosis and treatment of LSCC.
AGING LncRNA MNX1-AS1 was first implicated in the progression of ovarian cancer [12]. Accumulating evidence demonstrated that MNX1-AS1 played an oncogenic role in multiple malignancies including gastric cancer [13], esophageal squamous cell carcinoma [14], osteosarcoma [15], non-small cell lung cancer [16], hepatocellular carcinoma [17], prostate cancer [18], breast cancer [19] and cervical cancer [20] and so on. Although one study reported increased MNX1-AS1 expression in LSCC tissues based on the Cancer Genome Atlas Database (TCGA) analysis [21], little is known about its role and regulation in the pathogenesis of LSCC.
In this study, we demonstrated elevated MNX1-AS1 levels in LSCC tissues, which closely related with advanced UICC stage, lymph node metastasis, and poor prognosis. Additionally, using both in vitro and in vivo experimentation, we revealed MNX1-AS1 silencing reduced LSCC growth and metastasis by targeting the miR-370/FoxM1 pathway. Hence, this study established a new regulatory pathway of MNX1-AS1/miR-370/FoxM1 axis that modulates LSCC progression.

LSCC tissues had elevated MNX1-AS1 expression, which closely correlated with poor prognosis
To establish MNX1-AS1 levels in LSCC, we examined MNX1-AS1 levels in LSCC tissues and adjoining healthy tissue (ANT) samples. As illustrated in Figure  1A, MNX1-AS1 was augmented in LSCC tissues, relative to ANT. Next, using the average MNX1-AS1 level in LSCC tissues as a cut off, 40 LSCC patients were categorized as either high expressing or low expressing for subsequent Kaplan-Meier analysis. We showed that patients with high expression of MNX1-AS1 had advanced UICC stage, lymph node metastasis, and poor overall survival (OS) ratio, relative to patients with low expression of MNX1-AS1 (Table 1, Figure 1B).

MNX1-AS1 knockdown inhibits LSCC growth in cell culture and nude mice
MNX1-AS1 levels were assessed in a LSCC cell line TU212 and in normal bronchial epithelial cell line (16HBE). As depicted in Figure 2A, MNX1-AS1 was strongly expressed in TU212 cells, as opposed to 16HBE cells. To elucidate the role of MNX1-AS1 in LSCC, we silenced MNX1-AS1 levels in TU212 cells, using sh-MNX1-AS1 ( Figure 2B). CCK8 assay showed that MNX1-AS1 silencing severely inhibited LSCC cell proliferation in TU212 cells, as opposed to sh-NCs ( Figure 2C). Moreover, colony forming assays confirmed that reduced number of colonies in the MNX1-AS1 silenced cells, relative to sh-NCs ( Figure  2D). To ascertain the effect of MNX1-AS1 silencing on LSCC tumor growth in mice, we administered MNX1-AS1 silenced cells to nude mice and demonstrated significantly smaller tumor volume and weight in the TU212-sh-MMNX1-AS1 mice, as compared to the sh-NCs ( Figure 2E-2F). IHC assay showed that the positive cells of Ki-67 were greatly reduced in sh-MNX1-AS1 group verses the sh-NC group ( Figure 2G). Based on these data, MNX1-AS1 knockdown significantly decreased tumor growth in cell culture and in nude mice.

Knockdown of MNX1-AS1 inhibits LSCC cell migration and invasion
Using wound healing and transwell invasion assays, we measured the impact of MNX1-AS1 depletion on LSCC migration and invasion abilities. We observed that MNX1-AS1 depletion suppressed TU212 cell migration and invasion ( Figure 3A and 3B).

DISCUSSION
Multiple reports confirmed that lncRNAs played a vital role in LSCC cell physiology and pathogenesis, and might act as diagnosis markers and therapy agents [6,7]. Here, we aimed to clarify the precise role and uncover potential pathway of MNX1-AS1 regulation in LSCC. Here, we discovered that lncRNA MNX1-AS1 accelerated LSCC progression via miR-370/FoxM1 axis, indicating that MNX1-AS1 may be a potential therapy target for LSCC. AGING MNX1-AS1 was reported to serve as an oncogenic lncRNA in numerous forms of cancers [12][13][14][15][16][17][18][19][20]. For instance, MNX1-AS1 overexpression promotes gastric cancer progression via the EZH2/BTG2 and miR-6785-5p/BCL2 axis [13]. MNX1-AS1 contributed to bladder cancer initiation and progression via the modulation of AGING miR-218-5p/RAB1A pathway [25]. MNX1-AS1 drove lung cancer growth and metastasis using the miR-527/BRF2 pathway [26]. Here, we discovered MNX1-AS1 levels to be up-regulated in LSCC tissues, which further confirmed the previous result from TCGA [21]. In addition, increased MNX1-AS1 was closely correlated with advanced UICC stage, lymph node metastasis and short survival of patients with LSCC. Subsequent loss-of-function assays revealed that MNX1-AS1 depletion reduced LSCC growth and metastasis in vitro, as well as inhibited tumorigenesis of LSCC in vivo. These data imply that MNX1-AS1 serves as an oncogene in LSCC.
Here, we select miRNAs that can bind with MNX1-AS1 using LncBook online software. Among miRNAs, we chose to study miR-370 due to its essential role in cancer progression. miR-370 was shown to be downregulated and play a suppressive role in multiple cancers [29][30][31]. For LSCC, miR-370 expression was downregulated and function as a tumor suppressor in LSCC [24]. Through luciferase and RNA-Pull down assays, we further confirmed that miR-370 could bind with MNX1-AS1 in LSCC cells. Moreover, our results revealed that miR-370 levels were markedly elevated in MNX1-AS1depleted TU212 cells. We also found that miR-370 levels were downregulated in LSCC tissues and cells. MNX1-AS1 levels were inversely associated with miR-370 levels in LSCC tissues. Moreover, miR-370 silencing partly rescued the inhibitory effects mediated by MNX1-AS1 depletion in LSCC cells. Based on these data, MNX1-AS1 modulated LSCC progression by acting as a ceRNA by sponging miR-370.
Forkhead box M1 (FoxM1) is a well established downstream target of miR-370 in LSCC [24]. FoxM1, belonging to the Fox transcription factor family, was related to tumor formation in multiple cancers [32,33]. In LSCC, FoxM1 was shown to be elevated and served as a tumor promoting gene [34,35]. Thus, we hypothesized that MNX1-AS1 modulates the miR-370/FoxM1 pathway in LSCC. Here, we discovered that FoxM1 expression was elevated in LSCC tissues, similar to what we demonstrated previously [34,35]. In addition, the positive correlation of MNX1-AS1 and FoxM1 as well as the converse correlation between miR-370 and MNX1-AS1 (or FoxM1) were revealed in LSCC tissues. Of note, downregulation of miR-370 or upregulation of FOXM1 partially reversed the MNX1-AS1 silencing-mediated suppressive effects in TU212 cells. These findings indicate that MMX1-AS1 serves as an oncogene in LSCC by modulating miR-370/FoxM1 pathway.
In summary, we showed that MNX1-AS1 depletion retarded LSCC progression via miR-370/FoxM1 axis. Thus, MNX1-AS1 may be a promising new therapeutic target for LSCC. However, further studies are required to provide a deep understanding of the clinical translation of the MNX1-AS1/miR-370/FoxM1 axis in LSCC.

Clinical samples
40 LSCC tissue samples and adjoining non-cancerous matched tissue samples were retrieved from patients undergoing partial or total laryngectomy between March 2014 and Mach 2015 at the First Hospital of Jilin University, under approval from Jilin University. All informed consents were signed by all patients. All patients did not receive any tumor treatment before admission. All tissues were flash-frozen in liquid nitrogen within 10 min of extraction and stored at −80°C until further examination.

RNA purification and real-time quantitative PCR
Total RNA was extracted from cells or tissues with TRIzol reagent (Invitrogen, Carlsbad, CA) following manufacturer's guidelines. The RNA was employed for the synthesis of cDNAs with Primescript RT reagent kit (Takara, Dalian, China). cDNAs were amplified and quantified by with SYBR Green mix (Takara) in Applied Biosystems 7500 instrument. Primer sequences used in this study were described previously [13,24,28]. Relative gene expression was measured by −2 ΔΔCt method. Internal controls were GAPDH and U6 for MNX1-AS1/FOXM1 and miR-370, respectively.

Cell culture
Human LSCC cell line TU-212 and healthy bronchial epithelial cell line (16HBE) were bought from Shanghai Huiying Biological Technology (Shanghai, China), and maintained in Dulbecco's modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 0.1 mg/mL streptomycin in a humid environment at 37°C with 5% CO2.

Cell proliferation and colony formation assays
The impact of MNX1-AS1 on proliferation assay was evaluated using the Cell Counting Assay kit (CCK8, Sigma), based on manufacturer's guidelines. Transfected cells were seeded in 96-well plates (5x10 3 cells/well) for 24, 48 or 72 h. Next, CCK8 solution was introduced followed by a 4-h incubation. Absorbance was examined at 450 nm with a Microplate Reader (Bio-Rad, Hercules, CA, USA).
For colony formation, 1,000 plasmid incorporated cells were grown in 6-well plates over 2 weeks. Then cells were PBS-washed Twice, fixed in methanol for 15 min, and dyed with 0.1% crystal violet for 15 min at room temperature. The clones were quantified using Image J.

Cell migration and invasion assays
Wound healing and transwell chamber assays were carried out to assess migration and invasion properties, as we previously described (9). For wound healing assay, 2 × 10 5 transfected cells were plated in 12-well plates and allowed to grow till confluency. Next, a sterile pipette tip was employed to introduce a wound on the cell surface, before incubation in serum-free medium over 24 h before imaging at 0 and 24 h with an inverted microscope (Leica Microsystems, Inc., Buffalo Grove, IL. USA). For transwell invasion assay, 24-well Transwell chambers with polycarbonate filters (8-μm pores; Corning Inc.) were applied. Plasmid incorporated cells (1×10 4 cells per well) in zero serum medium were introduced to the top chamber coated with Matrigel, and medium with 10% FBS was included in the bottom chamber for chemoattraction. After 24-h, the migrated cells were fixed with 4% formaldehyde for 20 min and then stained with 0.1% crystal violet for 5 min. Cell invasion was quantified with an inverted microscope (Leica Microsystems, Inc.) by choosing 5 random vision field per treatment.

Subcellular fractionation assay
The NE-PER Nuclear and Cytoplasmic Extraction Reagents (Cat no: 78833; Thermo Fisher Scientific, Waltham, MA, USA) were used to isolate the cytoplasmic and nuclear extracts from TU212 cells. The distribution of MNX1-AS1 in cytoplasm or nucleus was examined using qRT-PCR. GAPDH and U6 served as controls for the cytoplasm and nucleus, respectively.

Luciferase reporter assay
A wild-type (WT) or mutant (MT) MNX1-AS1 fragment containing the miR-370 binding site were introduced into the pGL3-basic vector (Promega, Madison, WI, USA).For luciferase assay, TU212 cells were simultaneously incorporated with the miR-370 mimics or corresponding NC. 48 h later, luciferase reporter assay system (Promega) was applied to examine the luciferase activity.

Tumor evaluation in nude mice
All animal protocols in this study were agreed upon by the Animal Research Ethics Committee of Jilin University (Changchun, China). Ten male athymic nude BALB/c mice (5-6 weeks, 18-25g) from the Laboratory Animal center of Jilin University were housed in this center. All mice received standard mouse irradiated food and tap water ad libitum. TU212 cells stably incorporated with sh-MNX1-AS1 or sh-NC were subcutaneously administered into flank of nude mice (Five mice in each group). Tumor volumes were quantified weekly formulas follows: tumor volume (V) = width 2 × length × 0.5. After 28 days, all mice were sacrificed, tumors were harvested, and weighted. The tumor was paraffin-embedded to examine the cell proliferation marker Ki-67 expression with immunohistochemical (IHC) using an anti-Ki-67 antibody (Abcam, Cambridge, UK) as described previously [13]. The residual tumor tissues were sorted at −80°C until RNA extraction.

Statistical analysis
Each experiment was repeated 3 times and analyzed with SPSS software, version 17.0 (IBM SPSS, Armonk, NY, USA). The data presented is average ± SD (standard deviation). Continuous variables were AGING analyzed with Student's two-tailed t-test (2 groups data) or one-way analysis of variance (>2 groups data). A χ2 test was used for comparison of dichotomous variables. The differences in overall survival rate was assessed using Kaplan-Meier method and analyzed with log-rank test. Pearson's correlation analysis was employed for relationship investigations. A P value < 0.05 was considered as significant difference.

AUTHOR CONTRIBUTIONS
XW conceived and designed the study and drafted the manuscript. XC, YH and YT performed the experiments and interpreted the results. XD analyzed the data. All authors have read and approved the final version of this manuscript.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interests.

FUNDING
This study was not funded by any commercial or notfor-profit agencies.