LncRNA SNHG16 Contributes to Tumor Progression via miR-302b-3p/SLC2A4 in Pancreatic Cancer

Background: It is reported that lncRNA SNHG16 is signicantly highly expressed in pancreatic cancer (PC). However, the functions and mechanisms of SNHG16 are not clear. The aim of this study was to explore the effects of SNHG16 in PC. Methods: qRT-PCR analyze was applied to detect the expression levels of SNHG16, miR-302b-3p and SLC2A4 in PC tissues and cells. CCK8 and EdU assays were used to determine the proliferation of PC cells. Transwell assay were used to measure the capacities of PC cells migration and invasion. Apoptosis were evaluated by ow cytometry, and the expression of apoptosis related proteins (including Bax, Bcl-2, cleaved caspase-3 and cleaved caspase-9), which were tested by western blot. The interactions between miR-302b-3p and SNHG16 or miR-302b-3p and SLC2A4 mRNA 3’UTR were claried by Dual luciferase reporter assay and RNA immunoprecipitation. Results: SNHG16 was signicantly elevated in PC tissues and cell lines, which was associated with poor prognosis of PC patients. Knockdown of SNHG16 reduced PC cells proliferation, migration and invasion. SNHG16 acted as a sponge to regulate miR-302b-3p expression in PC cells. And miR-302b-3p targeted SLC2A4 directly. Conclusions: SNHG16 promoted the progression of PC via miR-302b-3p/SLC2A4 axis and was expected to be a potential


Introduction
Pancreatic cancer (PC) is one of the most severe gastrointestinal malignancies, which is the fourth most common cause of cancer-related deaths [1] . PC is characterized by the rapid, atypical and advanced disease progression [2] . As a result of a biological process involving multiple steps, there is no clinically sensitive early diagnosis indicator or effective treatment point [3] . Therefore, the research for diagnostic markers and targeted therapies to gradually inhibit the progression of PC has become the focus of attention in the treatment of PC.
It has been proved that only 2% of the genome sequence is capable of coding proteins, whereas more than 95% transcripts are identi ed as non-coding RNA [4] . Long non-coding RNAs (lncRNAs) is a class of non-coding RNA which is featured with no protein coding ability and longer than 200 nt. Emerging evidences demonstrate that lncRNA involved in multifarious malignant cancers, including PC [5][6][7] . For example, lncRNA HOTTIP contributes to PC to enhance the Wnt/β-catenin pathway via binding to WDR5 [8] . However, it was reported that LINC01197 was down-regulated in PC tissues, which inactivated Wnt/β-catenin pathway through interfering with β-catenin binding to TCF4 in PC cells [9] .
Recently, there has gradually been discovered in the study of small nucleolar RNA host gene 16 (SNGH16) in tumorigenesis of many cancer types [10,11] . And SNHG16 was also widely regarded as an essential oncogene [12] . In hepatocellular carcinoma, SNHG16 was high expressed in HCC-resistant tissues and promoted HCC cells viability and autophagy, suppressed apoptosis via regulating miR-23b-3p/EGR1 pathway [13] . However, the exact roles of SNHG16 in PC have not been clari ed yet.
In this study, we purposed to explore the roles of lncRNA SNHG16 functionally and mechanically impacting on PC. By detecting SNHG16 expression in paired PC tissues and cell lines, we found lncRNA SNHG1 was signi cantly overexpressed in PC. In addition, elevated SNHG1 exhibited an oncogenic role in PC cell proliferation, migration, invasion and apoptosis. Mechanistic investigations revealed that SNHG16 served as a sponge of miR-302b-3p, which reversed SNHG16-induced proliferation by targeting SLC2A4 in PC.

Materials And Methods
Tissue collection PC samples and corresponding adjacent non-cancerous tissues are collected from PC patients who received a surgical operation at the First Hospital of Lanzhou University. The study was approved by the ethics committee of the First Hospital of Lanzhou University, and all included patients signed informed consent.

Cell culture
The human normal pancreatic duct epithelial cell line (HPY-Y5) and pancreatic cancer cell lines (including BxPC3, Panc-1, MIA Paca-2 and SW1990) are all bought in the Chinese Academy of Sciences, Shanghai, China. All PC cells are cultured in high-glucose Dulbecco's modi ed Eagle's medium (DMEM) containing with 10% fetal bovine serum (Gibco, USA) in a sterile incubator of 5% CO 2 , 37 °C. RNA isolation, reverse transcription and qRT-PCR assay Total RNA was extracted from tumor tissues and cells by TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. cDNAs are produced by the reverse transcript kit from Takara (Dalian, China). SNHG16 and SLC2A4 expression was detected by using SYBR Green real-time PCR Kit (Qiagen, Germany). miR-302b-3p expression was analyzed by Hairpin-it TM miRNAs qPCR kit (GenePharma, China). The data of mRNA and miRNA are presented as the fold change in mRNA/miRNA abundance normalized against the β-actin or U6 snRNA. The primer sequences used in this study are presented in Table 1. Table 1 Primer sequences for qRT-PCR

Genes
Primer sequences Plasmids construction and cell transfection The miR-302b-3p mimics, inhibitors, small hairpin RNA of SLC2A4 (sh-SLC2A4) and corresponding negative control (NC mimics or sh-NC) are purchased from RiboBio, Guangzhou, China. The inhibitors and shRNAs were transfected into BxPC3 and Panc-1 cells using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's directions. The transfection e ciencies were detected at 48 hours post transfection by qRT-PCR. Transfected cells were converged for the subsequent experiments.

Construction of stably transfected cells
The lentiviral particles of LV-shSNHG16 and LV-sh-NC were purchased from GenePharma, China. The lentivirus was used directly to infect BxPC3 and Panc-1 cells with the addition of 8 μg/ml polybrene (Sigma-Aldrich) to construct the stably transfected cells. Puromycin (Invitrogen, USA) was added to the media 48 h after infection and maintained for 2 weeks to select stably transfected cells (BxPC3/Panc-1sh-SNHG16 or BxPC3/Panc-1-sh-NC).

EdU assay
The regents used in the EdU assay was purchased from Ribobio (Guangzhou, China). PC cells were sowed in confocal dishes at a density of 3 × 10 5 . The 4% paraformaldehyde (Beyotime, China) was used to x the cells for 10 min. The 1% triton (Beyotime, China) was used to transparent the cells for 5 min after washing three times with PBS. Subsequently, the cells were incubated with dyeing agent for 30 min in the dark, following stained with DAPI (Olympus, Tokyo, Japan) and incubated for 5 min at 37 °C. The images were trapped using a microscopy with a magni cation of 400×.
Wound healing assay PC cells were seeded in 6-well plates and grown to 70-80% con uence. The wound was scraped by 100 μl pipette tips. The remaining cells were cultured in serum-free DMEM medium for 48 h. Images of migration were trapped at 0 and 48 h after scraping.

Migration and invasion assays
Cells were suspended in serum-free DMEM medium. 100 µl of the cell suspension was seeded on the upper part of a 24-well culture plate with an 8 µm transwell chamber (Corning, USA) for 48 h, and 500 µl of DMEM medium supplemented with 10% FBS was added to the lower chamber. The chambers uniformly covered with Matrigel (BD Biosciences) are used to invasion assay. The transwell chambers were xed in 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 30 min at room temperature. Then, cells above the chambers were wiped off by a cotton swab. And stained cells were washed with PBS and observed with a microscope. The capacities of cell migration and invasion were assessed with average stained cells in 5 areas.
Luciferase reporter assay The full-length of SNHG16 (WT or Mut) is synthesized and cloned into the pGL3-promoter vectors (Promega, USA). Dual luciferase reporter activities are conducted in BxPC3 and PANC-1 cells cotransfected with pGL3-promoter-SNHG16 (WT or Mut) and NC mimics or miR-302b-3p mimics. The luciferase activity was analyzed by the dual Luciferase reporter assay system (Promega) according to the manufacturer's instruction.

Western Blot analysis
Total proteins were extracted from cells with RIPA (Beyotime, China). The concentration of total proteins was texted by BCA protein assay kit (Beyotime, China). 30 μg of total proteins was split by 6%, 8% or 10% SDS-PAGE gels and then transferred to PVDF membrane (Millipore, USA). After blocked with 5% skim milk for 2 h, the membrane was incubated overnight with a speci c primary antibody at 4 ℃. The speci c primary antibodies (including Bax, Bcl-2, Cleaved caspase-3, Cleaved caspase-9) were purchased from Proteintech Group and the concentration of these antibodies applied in this study was 1:500. The speci c primary antibodies (including ICAM-1, VCAM-1 and MMP9) were purchased from Abcam, the concentration of these antibodies applied in this study was 1:1000. Next, the membrane was incubated with corresponding secondary antibodies for 2 h at room temperature. The signals at protein level are visualized with the ECL western blotting substrate (Tanon, Shanghai, China).

Statistical analysis
All statistical analyses were accomplished with SPSS 22.0 software (Chicago, USA) and Prism version 7.0 software (California, USA). Data are presented as the mean ± SEM. And each experiment was repeated at least three times independently. An unpaired t-test was performed to compare the mean of two groups. The one-way ANOVA and Bonferroni multiple comparison test were applied to analyze the differences between two or more groups. Spearman correlation analysis was performed to detect the statistical dependence between SNHG16, miR-302b-3p and SLC2A4 levels. Signi cant differences were de ned as P 0.05.

Results
LncRNA SNHG16 is elevated in PC tissues and cell lines In this study, the expression level of SNHG16 in human PC tissues was rstly evaluated by qRT-PCR. Compared with the pair-matched adjacent normal samples, SNHG16 expression was signi cantly increased in PC tissues (Fig. 1A). Moreover, the expression level of SNHG16 was up-regulated in four PC cell lines BxPC3, Panc-1, MIA Paca-2 and SW1990, compared with HPY-Y5 (Fig. 1B). In addition, Kaplan-Meier analysis indicated that PC patients with increased SNHG16 shown a shorter over-all survival than those with decreased SNHG16 (Fig. 1C). The above results indicate that elevated SNHG16 might be a critical role in the progression of PC.

SNHG16 affects PC cells proliferation and apoptosis
In order to explore the roles of SNHG16 in PC process, we established BxPC3 and Panc-1 cells that stably silenced SNHG16, which were named sh-SNHG16 ( Fig. 2A). Next, CCK8 and EdU assays were employed to determinate the proliferation of PC cells, and it was revealed that knockdown of SNHG16 inhibited BxPC3 and Panc-1 cells proliferation (Fig. 2B and 2C). Consistently, the protein levels of cell proliferation markers, PCNA and Ki-67, were decreased as SNHG16-silencing (Fig. 2D). Then, ow cytometry cell apoptosis analysis demonstrated that SNHG16 knockdown increased the proportion of apoptotic PC cells (Fig. 2E), and western blot analysis revealed that SNHG16 knockdown promoted the expression of apoptosis-related proteins Bax, cleaved-caspase-3 and cleaved-caspase-9, while inhibited the expression of Bcl-2 (Fig. 2F). Above these, SNHG16 knockdown suppressed PC cells proliferation and promoted apoptosis.

Knockdown of SNHG16 suppresses PC cells migration and invasion
Subsequently, we examined the roles of SNHG16 in migration and invasion. Wound healing assay indicated that the motility of PC cells that stably silenced SNHG16 was signi cantly decreased (Fig. 3A). Transwell assay was implemented to evaluate the abilities of PC cells migration and invasion, and it is observed the reduced migration and invasion abilities of PC cells that stably silenced SNHG16 (Fig. 3B).
The results of western blot demonstrated that reduced SNHG16 inhibited the expression of ICAM-1, VCAM-1 and MMP-9 (Fig. 3C). In general, SNHG16 knockdown suppressed PC cells migration and invasion.
SNHG16 acts as a sponge to regulate miR-302b-3p expression in PC cells It is well-known that the commonly molecular mechanism of lncRNAs is acted as molecular sponges of miRNAs. Previous researches con rmed that SNHG16 regulated miRNA expression [14] . Therefore, we hypothesized that SNHG16 might exert functions via interacting with miRNAs in PC. The online software ENCORI was used for predicting the miRNAs which might be regulated by SNHG16, and qRT-PCR was performed to determine the expression levels of these predicted miRNAs in PC cells. It was found that only miR-302b-3p was downregulated in PC cells (Fig. 4A), and the biding site of miR-302b-3p on SNHG16 were displayed in Fig. 4B. The expression of miR-302b-3p was increased in SNHG16-knockdown PC cells (Fig. 4C). Dual luciferase reporter assays and RIP were performed to verify whether miR-302b-3p binds to SNHG16 directly. The luciferase activity in PC cells co-transfected with SNHG16-WT and miR-302b-3p mimics were weaker than these in cells co-transfected with SNHG16-WT and NC mimics, or cotransfected with SNHG16-Mut and miR-302b-3p (Fig. 4D). The results of RIP revealed that SNHG16 and miR-302b-3p were highly enriched in anti-Ago2 beads compared with IgG beads (Fig. 4E). In addition, miR-302b-3p was reduced both in PC tissues and cell lines ( Fig. 4F and 4G). Taken together, above results proved that SNHG16 acts as a molecular sponge to regulate miR-302b-3p expression in PC cells.

Overexpression of miR-302b-3p inhibits PC cells proliferation, migration and invasion, and promotes apoptosis
The effects of miR-302b-3p on biological phenotypes of PC cells were further explored in PC cells transfected with miR-302b-3p mimics or negative controls. The transfection e ciency of miR-302b-3p was detected by qRT-PCR (Fig. 5A). The results of CCK8 and EdU assays indicated that overexpressed miR-302b-3p inhibited the proliferation of PC cells ( Fig. 5B and 5C). Apoptosis experiment revealed that miR-302b-3p overexpression induces PC cells apoptosis (Fig. 5D). And miR-302b-3p overexpression attenuated the capacities of PC cells migration and invasion (Fig. 5E).

Discussion
PC is a highly aggressive solid tumor that frequently causes local invasion and early metastasis, which induces more than 300,000 deaths each year. The prognosis of PC patients is very poor, and the overall 5- year survival rate is less than 5% [15] . Therefore, it is important to identify the promising diagnostic markers or targeted therapies to gradually inhibit the progression of PC. With the development of sequencing technology, more and more non-coding RNA have been discovered. Among them, lncRNAs have been getting more and more attention due to their wide range of functions.
Small nucleolar RNA host gene 16 (SNHG16) has been reported as an oncogenic lncRNA in multiple cancers such as colorectal cancer [16] , non-small cell lung cancer [17] , breast cancer [18] , clear cell renal cell carcinoma [19] . A further research indicated that SNHG16 served as a novel prognostic marker which promote tumor formation and metastasis in vivo and vitro by sponging miR-146a further inducing MUC5AC in NSCLC [17] . Here, we investigated novel biological effects of SNHG16 in PC.
The pivotal conclusion of this study is that SNHG16 plays a critical role in PC. The results demonstrated that SNHG16 was signi cantly upregulated in human PC tissues and PC cells, and that increasing SNHG16 was correlated with poor prognosis of PC patients. Functional experiments indicated the inhibition effects of silenced SNHG16 on PC cell proliferation, migration and invasion, whereas promotion of apoptosis.
Mechanistically, the target gene, miR-302b-3p, was predicted and identi ed by luciferase reporter assay and RIP. MiR-302b-3p was highly expressed both in PC tissues and cells. Following functional experiments con rmed that miR-302b-3p promoted the proliferation, migration and invasion of PC cells, which was opposite with SNHG16. These data implied that SNHG16 acts as a sponge to negatively regulate miR-302b-3p expression in PC cells. Moreover, we investigated that miR-302b-3p can directly target 3'UTR of SLC2A4 and inhibited SLC2A4 expression in PC cells.
SLC2A4 is a gene codi ed the insulin-sensitive glucose transporter GLUT4, which is an insulin-sensitive glucose transporter that plays a key role in glucose homeostasis [20] . SLC2A4 is an e cient glucose transporter, which is located in the cytoplasmic vesicles and can be transferred to the plasma membrane to take up glucose when stimulated by insulin [21] . It has been shown that SLC2A4 was elevated in cancer progression [22,23] , which is in conformity with our results. SLC2A4 inhibition abolished the biological effects of PC cells in proliferation, migration and invasion induced by miR-302b-3p downregulation. Thus, we suggested that SNHG16 was an oncogene in PC progression via targeting miR-302b-3p/SLC2A4 axis and expected to be a potential target for early diagnosis and treatment of PC.

Consent for publication
The authors agrees to publication in the Journal.
Authors' contributions Wence Zhou conceived and designed the study. Chen Bo and Xin Miao performed the literature search. Xin Li and Haofei performed data extraction. Hao Xu drafted the manuscript. All authors read and approved the nal manuscript.

Availability of data and material
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no con icts of interest.