SNHG17 enhances the malignant characteristics of tongue squamous cell carcinoma by acting as a competing endogenous RNA on microRNA-876 and thereby increasing specificity protein 1 expression

ABSTRACT A long noncoding RNA called SNHG17 (small nucleolar RNA host gene 17) is aberrantly expressed and plays essential roles in multiple human cancer types. Nevertheless, its expression pattern and specific functions in tongue squamous cell carcinoma (TSCC) have not been well studied until now. Hence, in this study, we aimed to measure SNHG17 expression in TSCC and to examine the actions of SNHG17 on the malignant characteristics of TSCC cells. The regulatory mechanism that mediates the oncogenic effects of SNHG17 on TSCC cells was investigated too. In this study, SNHG17 was found to be upregulated in TSCC, and this overexpression closely correlated with adverse clinical parameters and shorter overall survival among the patients with TSCC. The SNHG17 knockdown significantly decreased TSCC cell proliferation, migration, and invasion in vitro and tumor growth in vivo. Mechanism investigation revealed that SNHG17 acts as a competing endogenous RNA on microRNA-876 (miR-876) in TSCC cells. In addition, specificity protein 1 (SP1) was validated as a direct target gene of miR-876 in TSCC cells. SP1 expression restoration in TSCC cells reversed miR-876 overexpression–induced anticancer effects. MiR-876 downregulation strongly attenuated the actions of the SNHG17 knockdown in TSCC cells. SNHG17 plays an oncogenic part in TSCC cells both in vitro and in vivo via sponging of miR-876 and thereby upregulating SP1, which could be regarded as a promising target for TSCC therapy.


Introduction
Tongue squamous cell carcinoma (TSCC) is the most prevalent subtype of oral cancer and is characterized by high malignancy, a high rate of tumor metastasis, and frequent recurrence, causing malfunctions of mastication and speech as well as dysphagia [1,2]. The morbidity rate of TSCC in China is 4.81%, and the mortality rate of the patients is 2.21% [3]. The therapeutic methods for TSCC, including surgery, chemotherapy, and radiotherapy, advanced substantially in recent years; the TSCCrelated mortality rate is still increasing, and the 5-year survival rate of the patients is only approximately 50% [4]. Over half of TSCC cases are usually detected and diagnosed at an advanced stage of this cancer; this situation is another reason for the poor clinical outcomes [5]. Thus, a better understanding of the molecular mechanisms behind the genesis and progression of TSCC may facilitate the diagnosis and treatment of this aggressive malignant tumor.
Long noncoding RNAs (lncRNAs) are a group of noncoding regulatory RNA transcripts longer than 200 nucleotides [6]. LncRNAs have no proteincoding ability but can interact with RNA, protein, and DNA [7]. In recent years, increasing evidence shows that lncRNAs play important roles in all the characteristics of physiological and pathological processes, including inflammation, differentiation, metabolism, carcinogenesis, and cancer progression [8][9][10]. In the field of TSCC research, a number of lncRNAs are reported to be strongly involved, eg, lncRNAs THOR [11], MALAT1 [12], and LINC00961 [13]. Aberrantly expressed lncRNAs can affect the aggressiveness of TSCC by acting as oncogenic factors or tumor suppressors [14][15][16]. Accordingly, investigating the expression and functions of lncRNAs in TSCC as well as their mechanisms of action may clarify the link between lncRNAs and TSCC, thus providing novel insights into TSCC management via targeting of lncRNAs.
MicroRNAs (miRNAs) are a family of endogenous, highly conserved, short noncoding RNA molecules containing 18 to 24 nucleotides [17]. They downregulate gene expression through a direct interaction with the 3′-untranslated region (3′-UTR) of complementary messenger RNAs (mRNAs, ie, target transcripts) by promoting mRNA degradation and/or inhibiting translation [18]. To date, over 2,600 miRNA genes have been confirmed in the human genome, and the encoded miRNAs are predicted to regulate the expression of~60% of human protein-coding genes [19]. Nowadays, a growing number of studies suggest that miRNAs exert oncogenic or tumorsuppressive effects on the formation and progression of TSCC and participate in the regulation of various biological activities [20][21][22]. Therefore, exploration of TSCC-associated miRNAs may be useful for the identification of novel therapeutic targets in this disease.
SNHG17 is aberrantly expressed and performs important functions in multiple human cancer types [23][24][25][26]. Inspired by the above research, we tested the hypothesis that SNHG17 is involved in the progression of TSCC. To this end, we determined the expression of SNHG17 in TSCC and studied the regulatory actions of SNHG17 on the malignant phenotype of TSCC cells in vitro and in vivo. Meanwhile, the mechanisms underlying the influence of SNHG17 on TSCC progression were characterized in detail.

Patients and tissue samples
A total of 56 pairs of TSCC tissue samples and adjacent non-tumor tissue samples were collected from patients at the University of the Chinese Academy of Sciences, Shenzhen Hospital between February 2013 to March 2014. All these patients had a diagnosis of primary TSCC and were treated with surgical resection. All the patients were followed-up for 5 years. Patients who had received chemotherapy and/or radiotherapy were excluded from this study. Following the surgical resection, all tissue samples were immediately immersed in liquid nitrogen and then stored at -80°C. This study was conducted with the approval of the Ethics Committee of the University of the Chinese Academy of Sciences, Shenzhen Hospital. All the analyzes of human subjects and their biological samples were carried out in accordance with the principles of the Helsinki Declaration. Written informed consent was provided by all the participating patients before the tissue collection.

Cell lines
Three human TSCC cell lines, Tca8113, SCC-15, and CAL-27, and normal gingival epithelial cells were bought from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained at 37°C in an incubator supplied with 5% of CO 2 . The TSCC cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% of fetal bovine serum (FBS) and 1% of a penicillin/streptomycin solution (all from Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The normal gingival epithelial cells were grown in the Minimum Essential Medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% of FBS and 1% of the penicillin/streptomycin solution.

Oligonucleotides, plasmids, and transient transfection
The small interfering RNA (siRNA) targeting SNHG17 (si-SNHG17) and its negative control siRNA (si-NC) were chemically synthesized by GenePharma Co., Ltd. (Shanghai, China). The full-length sequence of SP1 lacking the 3′-UTR was amplified by GenePharma Co., Ltd., and then inserted into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.), resulting in a plasmid called pcDNA3.1-SP1 (pc-SP1). The miR-876 mimics, NC mimics (miR-NC), miR-876 inhibitor, and NC inhibitor were acquired from RiboBio Co., Ltd. (Guangzhou, China). To obtain different expression levels of the genes under study in cell models, one of the aforementioned oligonucleotides and one of the plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Reverse-transcription quantitative polymerase chain reaction (RT-qPCR)
Extraction of total RNA from tissues or cells was conducted by means of the TRIzol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.). To assess SP1 mRNA and SNHG17 expression, the PrimeScript RT Reagent Kit (Takara Biotechnology Co., Ltd., Shiga, Japan) was applied for conversion of the total RNA into cDNA products. The resultant cDNA was then subjected to qPCR with SYBR Premix Ex Taq™ (Takara Biotechnology Co., Ltd.). GAPDH served as the internal reference control for the SP1 mRNA and SNHG17 analyses. Measurement of miR-876 expression was performed via the miScript Reverse Transcription Kit (Qiagen GmbH, Hilden, Germany) and miScript SYBR Green PCR Kit, respectively, for reverse transcription and qPCR. U6 small nuclear RNA served as a control template for miR-876. The 2 −ΔΔCt method was utilized to analyze relative expression of the genes under study [27].

Cell counting Kit-8 (CCK-8) assay
Transfected cells were collected after 24 h of incubation, were used to prepare a cell suspension, and were seeded in 96-well plates at an initial density of 2 × 10 3 cells per well. The cells were then maintained at 37°C and 5% CO 2 for 0, 24, 48, or 72 h, and the CCK-8 assay was carried out at the indicated time points. Another 1 h incubation was carried out with the additional 10 µl of the CCK-8 solution (Yeasen Biotechnology Co., Ltd., Shanghai, China). Absorbance was detected on a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The growth curves were plotted with time points on the X-axis and the absorbance values on the Y-axis.

Transwell assays
The migratory status of cells was evaluated as the ability to migrate through 24-well Transwell filters (8 ◽m pore size; BD Biosciences, San Jose, CA, USA), whereas Matrigel (BD Biosciences)precoated Transwell filters were employed to determine the invasiveness of cells. Following 48 h transfection, the cells were detached, washed twice with phosphate-buffered saline, and resuspended in FBS-free DMEM. A suspension containing 5 × 10 4 cells was inoculated into the upper compartments, whereas the lower compartments were covered with 500 µl of DMEM supplemented with 20% of FBS. All the inserts were incubated for 24 h at 37°C and 5% CO 2 . Cells remaining on the upper side of the membrane were gently removed with a cotton swab, and the migratory and invasive cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Images were captured by means of an inverted microscope (x200 magnification; Olympus Corporation, Tokyo, Japan). Finally, five visual fields were randomly selected, and the migratory and invasive cells were counted.

Tumor xenograft experiment
The animal experiment was conducted with the approval of the Ethics Committee of the University of the Chinese Academy of Sciences, Shenzhen Hospital. The animal experiment was carried out according to the Animal Protection Law of the People's Republic of China-2009 for experimental animals. Male BALB/c nude mice (4 to 6 weeks old) were bought from Guangdong Medical Laboratory Animal Center (Guangzhou, China) and housed in a specific-pathogen-free grade barrier environment. Cells transfected with either si-SNHG17 or si-NC were treated with 0.25% trypsin for detachment and were resuspended in phosphate-buffered saline. The cell suspension was then inoculated subcutaneously into a flank of mice. The volume of tumor xenografts was monitored and calculated via the following formula: tumor volume = ½ × length × tumor width [2]. All the mice were euthanized at 4 weeks after inoculation, and their tumor xenografts were resected, imaged, and weighed.

Nuclear/cytoplasmic fractionation
Extraction of the cytoplasmic and nuclear fractions was conducted with the PARIS Kit (Invitrogen; Thermo Fisher Scientific, Inc.).

RNA immunoprecipitation (RIP) assay
The Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore Inc., Billerica, MA, USA) was employed to assess the binding of miR-876 to SNHG17. In particular, a cell lysate was prepared via RIPA lysis buffer and subjected to incubation with the magnetic beads conjugated with either an anti-AGO2 antibody or control immunoglobulin G (IgG; Millipore Inc.). At the end of the experiments, the immunoprecipitated RNAs were isolated and analyzed by RT-qPCR to determine SNHG17 and miR-876 expression.

Luciferase reporter assay
The SP1 3′-UTR fragments containing the predicted wild-type (wt) or mutant (mut) miR-876binding site were synthesized by GenePharma Co., Ltd., and inserted into the pmirGLO luciferase reporter plasmid (Promega, Madison, WI, USA) to generate reporter plasmids SP1-wt and SP1mut, respectively. The SNHG17-wt and SNHG17mut plasmids were constructed in a similar way. For the reporter assay, either the miR-876 mimics or miR-NC and either the wt or mut reporter plasmid were cotransfected into cells with Lipofectamine 2000. After 48 h of incubation, luciferase activity was determined with a Dual-Luciferase Reporter Assay System (Promega). Renilla luciferase activity was normalized to that of firefly luciferase.

Western blot analysis
Total protein was isolated using RIPA buffer, and protein concentration was measured with the Bicinchoninic Acid Assay Kit (both from Beyotime Institute of Biotechnology, Haimen, China). Equal amounts of protein were loaded and resolved by SDS-PAGE on a 10% gel and then transferred onto polyvinylidene difluoride (PVDF) membranes. After blockage with 5% fatfree milk at room temperature for 2 h, the membranes were probed with primary antibodies against SP1 (cat. No. ab124804; dilution 1:1,000; Abcam, Cambridge, UK) or GAPDH (cat. No. ab181602; dilution 1:1,000; Abcam), followed by incubation with a goat anti-rabbit IgG antibody conjugated with horseradish peroxidase secondary antibody (cat. No. ab205718; dilution 1:5,000; Abcam). The protein signals were developed with the Pierce ECL Western Blotting Substrate (Pierce; Thermo Fisher Scientific, Inc.). GAPDH served as the loading control.

Statistical analysis
Each experiment was performed for at least three times. All results are presented as the mean ± standard deviation and were analyzed using the SPSS software (version 19.0; SPSS Inc.). The correlation between SNHG17 expression and the clinical parameters among the 56 patients with TSCC was assessed by the χ 2 test. Differences between two groups were evaluated by Student's t test; oneway analysis of variance in combination with Tukey's post hoc test was conducted to compare the data among multiple groups. The association between SNHG17 expression and overall survival of TSCC patients was analyzed via the Kaplan-Meier curve and logrank test. Data with P < 0.05 were considered statistically significant.

High expression of SNHG17 correlates with poor clinical outcomes of patients with TSCC
First, RT-qPCR was carried out to measure SNHG17 expression in the 56 pairs of TSCC tumors and adjacent non-tumor tissue samples. The results indicated that the expression of SNHG17 was higher in TSCC tissue samples than in the adjacent non-tumor tissue samples (Figure 1(a), p < 0.05). Consistently with this result, SNHG17 expression was higher in all three TSCC cell lines (Tca8113, SCC-15, and CAL-27) than in the normal gingival epithelial cells (Figure 1(b), p < 0.05).
We next investigated the clinical value of aberrant SNHG17 expression in patients with TSCC. The median value of SNHG17 among the TSCC tissue samples was defined as the cutoff value (2.74), and according to the cutoff, all the patients with TSCC were subdivided into either a high SNHG17 expression group (n = 28) or low SNHG17 expression group (n = 28). The χ 2 test indicated that higher SNHG17 expression notably correlated with tumor size (P = 0.015), TNM stage (P = 0.029) and lymph node metastasis (P = 0.031) among the 56 patients with TSCC (Table 1). In addition, patients with TSCC featuring high TSCC expression showed shorter overall survival compared with that in the group with low SNHG17 expression ( Figure 1(c), p = 0.030). These findings suggested that SNHG17 expression is high in TSCC, and this overexpression may be associated with TSCC progression.
The knockdown of SNHG17 inhibits the proliferation, migration, and invasiveness of Tca8113 and CAL-27 cells The expression of SNHG17 was higher in Tca8113 and CAL-27 cells among the three TSCC cell lines; accordingly, these two cell lines were chosen for subsequent functional assays. SNHG17 expression was silenced in Tca8113 and CAL-27 cells using the siRNA called si-SNHG17. RT-qPCR confirmed that SNHG17 was remarkably downregulated in the Tca8113 and CAL-27 cells that were transfected with si-SNHG17 (Figure 2(a), p < 0.05). The CCK-8 assay revealed that the transfection of si-SNHG17 obviously decreased the proliferative ability of Tca8113 and CAL-27 cells (Figure 2(b), p < 0.05). We then tested whether the reduction in SNHG17 expression affects the migration and invasiveness of TSCC cells. The results of Transwell assays meant that the migratory (Figure 2(c), p < 0.05) and invasive (Figure 2(d), p < 0.05) abilities were notably hindered in the Tca8113 and CAL-27 cells with the SNHG17 knockdown. The above results showed that SNHG17 may play a tumor-suppressive part in the malignancy of TSCC.

SNHG17 acts as a sponge on miR-876 in TSCC cells
Currently, the most widely accepted theory is that the cytoplasmic lncRNAs work as a competing endogenous RNA (ceRNA) by sponging certain miRNAs and thereby enhancing the expression of these miRNAs' targets [28]. To reveal the molecular events underlying the oncogenic influence of SNHG17 on TSCC progression, we first determined the subcellular localization of SNHG17 in Tca8113 and CAL-27 cells. As depicted in Figure 3(a), SNHG17 was mostly present in the cytoplasm of both Tca8113 and CAL-27 cells. Bioinformatics software starBase 3.0 was applied to search for the potential miRNAs that may be sponged by SNHG17. The bioinformatics prediction uncovered complementary sequences between SNHG17 and miR-876 (Figure 3(b)). MiR-876 was selected for subsequent validation because this miRNA has been demonstrated to exert important actions on tumorigenesis and tumor progression [29][30][31][32][33][34]. The luciferase reporter assay was performed to investigate whether miR-876 can bind to SNHG17. The miR-876 mimics-mediated overexpression of miR-876 (Figure 3(c), p < 0.05) significantly decreased the luciferase activity of SNHG17-wt in Tca8113 and CAL-27 cells (P < 0.05), but upregulation of miR-876 had no influence on the luciferase activity of SNHG17-mut (Figure 3(d)).
The RIP assay was conducted to gain more insight into the interaction between SNHG17 and miR-876 in TSCC cells. The results showed that SNHG17 and miR-876 were preferentially enriched on AGO2binding beads after immunoprecipitation in the lysates of Tca8113 and CAL-27 cells, suggesting that miR-876 is a target of SNHG17 in TSCC cells (Figure 3(e), p < 0.05). Furthermore, the expression of miR-876 was measured in the 56 pairs of TSCC tissue samples and adjacent non-tumor tissues, and expression correlation with SNHG17 was evaluated. The data revealed that miR-876 was underexpressed in TSCC tumors (Figure 3(f), p < 0.05), manifesting an inverse correlation with SNHG17 amounts (Figure 3(g); r = −0.5811, P < 0.0001). Moreover, miR-876 turned out to be significantly upregulated in SNHG17 knockdown Tca8113 and CAL-27 cells, as evidenced by RT-qPCR analysis (Figure 3(h), p < 0.05). Consequently, SNHG17 functioned as a molecular sponge of miR-876 thereby inhibiting miR-876 function in TSCC.

MiR-876 overexpression exerts inhibitory effects on the proliferation, migration, and invasiveness of Tca8113 and CAL-27 cells
To investigate whether miR-876 is associated with TSCC progression, a series of experiments was conducted on miR-876-overexpressing Tca8113 and CAL-27 cells. The CCK-8 assay showed that miR-876 mimics-transfected Tca8113 and CAL-27 cells manifested slower proliferation compared with that of the cells transfected with miR-NC (Figure 4(a), p < 0.05). Furthermore, the Transwell assay results revealed that ectopic miR-876 expression obviously restricted the migration (Figure 4

MiR-876 directly targets SP1 mRNA in TSCC cells
To decipher the molecular mechanisms via which miR-876 reduces the malignancy of TSCC cells, potential targets of miR-876 were predicted by means of starBase 3.0, TargetScan, and miRDB. Two highly conserved possible miR-876-binding sites were found in the 3′-UTR of SP1 mRNA ( Figure 5(a)). The luciferase reporter assay was conducted to test whether miR-876 can directly bind to the 3′-UTR of SP1 mRNA. The luciferase activity was noticeably lower in SP1-wt-transfected (1 and 2) Tca8113 and CAL-27 cells that were cotransfected with the miR-876 mimics (P < 0.05); however, no suppression of the luciferase activity was observed among the cells that were transfected with SP1-mut instead (both 1 and 2; Figure 5(b)).
Besides, RT-qPCR was performed to determine SP1 mRNA expression in the 56 pairs of TSCC tissue samples and adjacent non-tumor tissues. The mRNA of SP1 was found to be overexpressed in the TSCC tumors in comparison with that in the adjacent non-tumor tissues

Restoration of SP1 expression abrogates the tumor-suppressive effects of miR-876 on TSCC cells
On the basis of the above observations, it was hypothesized that miR-876 upregulation attenuated the growth and metastasis of TSCC cells by decreasing SP1 expression. To test this hypothesis, rescue experiments were performed on miR-876-overexpressing Tca8113 and CAL-27 cells via cotransfection with the SP1-overexpressing plasmid (pc-SP1). Western blotting confirmed that the SP1 protein was significantly downregulated in miR-876-overexpressing Tca8113 and CAL-27 cells; meanwhile, cotransfection with pc-SP1 almost abrogated the SP1 protein downregulation that was caused by miR-876 overexpression ( Figure  6(a), p < 0.05). Furthermore, a series of functional assays indicated that restoration of SP1 expression abrogated the inhibitory effects of miR-876 overexpression on the proliferation (Figure 6  These data suggested that the decrease in SP1 expression is essential for the inhibitory actions of miR-876 on the malignancy of TSCC cells. The miR-876-SP1 axis is involved in SNHG17driven stimulation of TSCC cell proliferation, migration, and invasion On the basis of the above results, it was hypothesized that the SNHG17 knockdown inhibits the proliferation, migration, and invasiveness of TSCC cells via downregulation of the miR-876-SP1 axis output. Hence, rescue experiments were conducted to validate this notion. First, the transfection efficiency was evaluated by RT-qPCR. It was noted that miR-876 expression was markedly silenced in Tca8113 and CAL-27 cells by miR-876 inhibitor transfection (Figure 7(a), p < 0.05). Then, si-SNHG17 and either the miR-876 inhibitor or NC inhibitor were introduced into Tca8113 and CAL-27 cells. The downregulation of SNHG17 increased miR-876 expression (Figure 7(b), p < 0.05) and decreased SP1 protein amounts (Figure 7 (c), p < 0.05) in Tca8113 and CAL-27 cells, whereas miR-  (Figure 7(f), p < 0.05) of Tca8113 and CAL-27 cells were notably suppressed by the SNHG17 knockdown, and conversely, these effects were reversed after cotransfection with the miR-876 inhibitor. In short, all these findings led to a conclusion that the SNHG17 knockdown restricted TSCC progression in vitro by reducing the sponging of miR-876, with a consequent decrease in SP1 expression.

The SNHG17 knockdown impairs tumor growth of TSCC cells in vivo
The tumor xenograft experiment was carried out to corroborate the oncogenic effect of SNHG17 in TSCC more comprehensively. Tca8113 cells transfected with either si-SNHG17 or si-NC were harvested and then injected subcutaneously into the flank of mice. The volume of tumor xenografts was monitored, and these data were used for plotting the tumor growth curve. It was observed that the growth rate of tumor xenografts was markedly slower in the si-SNHG17 group than in the si-NC group (Figure 8(a), p < 0.05). At 4 weeks after the inoculation, all the mice were euthanized. The tumor xenografts from the si-SNHG17 and si-NC groups are illustrated in Figure 8(b). In addition, the tumor xenografts originating from the Tca8113 cells transfected with si-SNHG17 manifested a lower weight compared with that in the si-NC group (Figure 8(c), p < 0.05).
Furthermore, we determined the expression of SNHG17, miR-876, and SP1 in the tumor xenografts. RT-qPCR revealed that SNHG17 expression was lower (Figure 8 SNHG17-transfected Tca8113 cells. Furthermore, western blotting results suggested that the protein amount of SP1 was lower in the si-SNHG17 group than in the si-NC group (Figure 8(f)). Therefore, these results meant that a reduction in SNHG17 expression restrained tumor growth in vivo by regulating the miR-876-SP1 axis.

Discussion
A variety of lncRNAs are aberrantly expressed in TSCC, and their anomalous expression has been extensively demonstrated to play crucial roles in TSCC progression [35][36][37]. LncRNAs have tumorsuppressive or oncogenic functions and are implicated in the regulation of diverse cancer-related abnormal cellular behaviors [38][39][40]. Hence, in-depth research into the functional involvement of lncRNAs in TSCC may help to identify diagnostic or prognostic biomarkers of (and therapeutic targets in) TSCC. In this work, we first measured the expression of SNHG17 in TSCC and evaluated its clinical significance among the 56 patients with TSCC. Additionally, we investigated the interactions of SNHG17 with the miRNA-mRNA axis in relation to the regulation of aggressive characteristics of TSCC cells. This study illustrates a novel lncRNA-miRNA-mRNA pathway that contributes to the malignant phenotype of TSCC. SNHG17 is overexpressed in gastric cancer, and its upregulation is closely associated with invasion  , c) The expression of the miR-876 and SP1 protein in Tca8113 and CAL-27 cellsthat were cotransfected with si-SNHG17 and either the miR-876 inhibitor or NC inhibitorwas respectively determined by RT-qPCR and western blotting. *P < 0.05 vs. group si-NC. # P < 0.05 vs. group si-SNHG17+ NC inhibitor. (d-f) CCK-8 and Transwell assays were performed to examine the proliferation, migration, and invasiveness of Tca8113 and CAL-27 cells that were treated as described above. *P < 0.05 vs. the si-NC group. # P < 0.05 vs. group si-SNHG17+ NC inhibitor.
depth, TNM stage, lymph node metastasis, and distant metastasis [23,24]. Patients with gastric cancer harboring SNHG17 overexpression exhibit worse overall survival and progression-free survival than do the patients with low SNHG17 expression [23]. Of note, SNHG17 expression has been identified as an independent biomarker predicting the overall survival and progression-free survival of patients with gastric cancer [23]. SNHG17 expression is also excessive in colorectal cancer [25] and non-small cell lung cancer [26]. Nonetheless, the expression status of SNHG17 in TSCC has been unclear. Herein, a total of 56 pairs of TSCC tissue samples and adjacent non-tumor tissue samples were collected and assayed by RT-qPCR to quantitate SNHG17 expression. The results showed that SNHG17 is overexpressed in TSCC, and this overexpression correlated with tumor size, TNM stage and lymph node metastasis. Patients with TSCC featuring high SNHG17 expression showed shorter overall survival in comparison with the patients with low SNHG17 expression. These results suggest that SNHG17 might be a diagnostic and/or prognostic biomarker of TSCC.
Previously, SNHG17 has been validated as an oncogenic lncRNA in carcinogenesis and cancer progression. For instance, silencing of SNHG17 expression suppresses colorectal-cancer cell proliferation in vitro, promotes apoptosis and cell cycle arrest in vitro, and slows tumor growth of colorectal-cancer cells in vivo [25]. In non-small cell lung cancer, SNHG17 downregulation restricts tumor cell proliferation and migration and induces apoptosis in vitro [26].However, the specific participation of SNHG17 in TSCC has not yet been elucidated. In this study, a series of functional experiments revealed that SNHG17 downregulation causes evident suppression of TSCC cell proliferation, migration, and invasion in vitro and their tumor growth in vivo. These observations suggest that SNHG17 may be an effective target for managing TSCC.
Our identification of the mechanism of SNHG17 action should advance the understanding of its involvement in cancer initiation and progression. Previously, the oncogenic roles of SNHG17 have been convincingly attributed to an epigenetic knockdown of P57 in colorectal cancer [25] and to regulation of FOXA1, XAF1, and BIK expression in non-small cell lung cancer [26]. In the present study, we demonstrated that SNHG17 works as a ceRNA of miR-876 in TSCC and thereby relieves the repressive action of miR-876 on its direct target gene, SP1. MiR-876 is known to be downregulated in hepatocellular carcinoma [29], breast cancer [30,31], gastric cancer [32], osteosarcoma [33], and lung cancer [34]. MiR-876 plays tumorsuppressive roles and participates in the regulation of a variety of cancer-associated processes in human malignant tumors [29][30][31][32][33][34]. To the best of our knowledge, the present study is the first to show that miR-876 is sponged by SNHG17 and may therefore inhibit the malignancy of TSCC cells.
SP1, a sequence-specific DNA-binding protein, is encoded by a gene in chromosomal region 12q13.1 [41]. SP1 can directly interact with GC/ GT-rich promoter elements via its C(2)H(2)-type zinc fingers at C-terminal domains and subsequently enhances or attenuates the activity of gene promoters [42]. SP1 expression is high in a variety of human malignant tumors, including prostate cancer [43], breast cancer [44], gastric cancer [45], and thyroid cancer [46]. In TSCC, SP1 is overexpressed too and significantly promotes the genesis and progression of TSCC by regulating numerous cellular behaviors [47][48][49] in agreement with our findings. In this study, our data uncovered a novel upstream molecular mechanism controlling the expression of SP1 in TSCC. Namely, SNHG17 acts as a ceRNA on miR-876 and thus upregulates SP1 in TSCC cells. Consequently, the SNHG17-miR-876-SP1 pathway facilitates TSCC progression in vitro and in vivo, highlighting promising targets for TSCC therapy.

Conclusion
We demonstrated that SNHG17 serves as a sponge of miR-876 in TSCC thereby weakening the inhibitory action of miR-876 on SP1 expression and driving aggressive TSCC progression. The components of this regulatory pathway, including SNHG17, miR-876, and SP1, are potential targets for the development of novel therapies for TSCC.

Disclosure statement
No potential conflict of interest was reported by the authors.