LncRNA GAS8-AS1 suppresses papillary thyroid carcinoma cell growth through the miR-135b-5p/CCND2 axis

The aim of the present study was to investigate the potential role of GAS8 antisense RNA 1 (GAS8-AS1) in papillary thyroid carcinoma (PTC). PcDNA3.1-GAS8-AS1 and si-GAS8-AS1, miR-135b-5p mimic and si-CCND2 were transfected into PTC cells. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8). QRT-PCR was used to determine expressions of GAS8-AS1, miR-135b-5p, and CCND2, and Western blot were detected protein level of CCND2. The miRNA target gene prediction site TargetScan was used to predict potential targets of GAS8-AS1 and miR-135b-5p. Cell cycle progression was analyzed by flow cytometry. We found that GAS8-AS1 was down-regulated in PTC cell lines and inhibited proliferation and cycle of PTC cell. GAS8-AS1 directly targets miR-135b-5p, and GAS8-AS1 could regulate a downstream target of miR-135b-5p, Cyclin G2 (CCNG2), in an miR-135b-5p-mediated manner. In addition, we also proved that overexpressed GAS8-AS1 inhibited tumor formation in vivo. GAS8-AS1 suppresses PTC cell growth through the miR-135b-5p/CCND2 axis.


Introduction
Thyroid cancer is the most frequent malignancy of the endocrine system, representing 1-2% of total cancer cases, and the morbidity of thyroid cancer has been substantially increasing over the past few decades all over the world [1,2]. Papillary thyroid cancer (PTC) is the prevalent type of thyroid cancer, and comprises approximately 80% of all thyroid cancers [3]. Regarding the epidemiology of thyroid cancer, more recent data are available that generally support the hypothesis that the increasing incidence of thyroid cancer diagnosis is primarily due to indolent PTC with a relatively stable rise mortality [4]. Most of PTCs could be managed successfully with a combination of adjuvant radioactive iodine therapy and levothyroxine suppression treatment after complete surgical intervention, nevertheless a fraction of patients with PTCs remain irresponsive to treatment and result in comorbidity and mortality. Therefore, further studies on the molecular mechanisms underlying the development and progression of PTC are still urgently needed.
Long non-coding RNAs (lncRNAs) are a group of RNAs, greater than 200 nts in length, which are important for the regulation of gene function and various cellular processes [5]. LncRNAs regulate a variety of processes including cell proliferation, metastasis, invasion migration, apoptosis, development, and differentiation [6]. The growing data have indicated that many lncRNAs are differentially expressed between PTC tissues and adjacent non-cancerous tissues, and are involved in progress of PTC [7][8][9][10][11], but only a small subset have been determined to be biologically significant [12]. GAS8 antisense RNA 1 (GAS8-AS1), which is located in the second intron of GAS8 and transcribes a 994-nt lncRNA in the opposite orientation of GAS8, has been reported to be a novel tumor suppressor that can affect tumor cell proliferation in PTC [13], but the mechanism of how GAS8-AS1 regulates tumor cell function remains unclear. miRNA, as a class of small non-coding RNA 19-25 nts in length, takes part in post-transcriptional regulation of target genes [14,15]. It has been reported that miRNAs are crucial regulatory factors in numerous cancers [16,17] and numerous cellular processes [18,19]. Mounting evidence have demonstrated that aberrantly expressed miRNAs are involved in regulating development of PTC [20][21][22]. Previous studies have suggested miR-135b-5p acts as a tumor suppressor, involved in various cancers [23][24][25]. One study also demonstrated that miR-135b-5p is significantly up-regulated PTC [26]; however, report about the role of miR-135b-5p in the PTC development is in its infancy, and there is no evidence of a relationship between GAS8-AS1 and miR-135b-5p in PTC.

Cell culture and transfection
The human PTC cell line, BCPAP, the German Collection of Micro-organisms and Cell Cultures (Braunschweig, Germany). The human PTC cell line, TPC1 and normal thyroid cell line Nthy-ori-3-1were obtained from Chinese Science Institute (Shanghai, China), and the human PTC cell line, K1, were purchased from the European Collection of Authenticated Cell Cultures (ECACC, U.K.). The human PTC cell line, IHH4, was obtained from Health Science Research Resources Bank (Osaka, Japan). Cell culture medium was prepared according to the suppliers' instructions. The BCPAP and Nthy-ori-3-1 were cells were cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, U.S.A.) supplemented with 10% FBS (Gibco, Carlsbad, CA, U.S.A.). The TPC1 and K1 were maintained in DMEM high glucose (Gibco, Carlsbad, CA, U.S.A.) supplemented with 10% FBS. IHH4 was maintained in a mixture (1:1) of RPMI 1640 and DMEM high glucose medium supplemented with 10% FBS. All cells were cultured at 37 • C, in a humidified atmosphere with 5% CO 2 .

Cell proliferation
The proliferation rates of TPC1 and BCPAP were measured using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). Approximately 2 × 10 3 cells were seeded in each well of a 96-well plate, and 10 μl of CCK-8 was added to 90 μl of culture medium at the indicated time, and were then transfected with pcDNA3.1-GAS8-AS1, si-GAS8-AS1, miR-135b-5p mimic, and si-CCNG. Proliferation was evaluated at 0, 24, 48, and 72 h after gene transfection. The UV absorbance at 450 nm was measured using a BioTek synergy H1 hybrid reader.

Cell cycle analysis
TPC-1 and BCPAP cells were transfected with pcDNA3.1-GAS8-AS1, si-GAS8-AS1, miR-135b-5p mimic, and si-CCNG, and analyzed by flow cytometry after 48 h. Briefly, cells were harvested in PBS containing 2 mmol/l EDTA, washed once with PBS, and fixed for 2 h in cold ethanol (70%). Fixed cells were washed once in PBS and treated with 40 mg/ml RNase A in PBS for 30 min. They were then washed once in PBS and stained with 50 mg/ml propidium iodide (Roche). Images of the cell cycle were obtained by the FACS Calibur system (Becton-Dickinson, Franklin Lakes, NJ, U.S.A.).

Luciferase reporter assay
Cells (

Western blotting
Proteins were extracted with RIPA buffer. The protein concentration was determined using a protein assay kit (Bio-Rad). Approximately 30 μg of protein from each sample was separated on a SDS/polyacrylamide gel (10% gel) and transferred on to PVDFs. Membranes were blocked with 5% skim milk and incubated with anti-CCNG2 antibody (Abcam, Cambridge, U.K.) overnight at 4 • C followed by incubation with the corresponding secondary antibodies for 1 h at room temperature. Proteins were detected on membranes, after washing in TBST, using Super ECL Plus Detection Reagent (Thermo Fisher Scientific, Carlsbad, CA, U.S.A.).

Tumor formation in nude mice
Twenty-four male BALB/c nude mice (8-week-old, 210-250 g) were randomly divided into three groups, eight mice in each group. BCPAP cells (1 × 10 7 cells in 200 μl serum-free medium) treated with lentiviral vector (LV)-GAS8-AS1 were subcutaneously injected into eight nude mice. All mice were housed in a temperature-controlled room (22-24 • C), with a 12-12 h light/dark cycle. Animals were given free access to chow and water. All procedures in the present study were approved by the Animal Care Committee of The First Affiliated Hospital of Zhengzhou University, and all the methods were carried out in accordance with the approved guidelines. At specific times, the mice were killed, and the weight and volume of the tumors as well as expressions of GAS8-AS1, miR-135b-5p, and CCNG2 were determined.

Statistics
The data are presented as mean + − S.D. Differences between means were determined by Student's t test with in two groups. Differences between more than two groups were analyzed using one-way ANOVA followed by Dunnett's test (Figures 1-3 and 5) and least significant difference (LSD) test (Figure 4). P<0.05 was considered statistically significant. The experiments were repeated three times.

Relative GAS8-AS1 expression was reduced in PTC cell lines
We initially examined the relative expression of GAS8-AS1 in four PTC cell lines, TPC1, BCPAP, K1, IHH4, and one normal thyroid epithelial cell line, Nthy-ori-3-1 by qRT-PCR. The mRNA expression of GAS8-AS1 was significantly decreased in all PTC cell lines compared with Nthy-ori-3-1 (Figure 1). The TPC1 and BPCAP (Supplementary Figure  S1) were selected for further evaluation because GAS8-AS1 expression in which was decreased by more than 2.0-folds.  si-GAS8-AS1 group was four-folds less than in the non-silencing control (scramble) group in TPC1 and BCPAP cells ( Figure 2B). Cell proliferation was detected by CCK-8 assay, si-GAS8-AS1 significantly promoted proliferation compared with the scramble group, and GAS8-AS1 overexpression prominently repressed proliferation compared with the control group in TPC1 ( Figure 2C) and BCPAP cells ( Figure 2D). To demonstrate the growth-inhibiting effect of GAS8-AS1 on PTC cells, cell cycle progression was analyzed by flow cytometry. As shown in Figure 2E,F, GAS8-AS1 overexpression induced significant increase in the percentage of TPC1 and BCPAP cells at pre-G 1 relative to control and significant decrease in the percentage of TPC1 and BCPAP cells at S and G 2 /M phases as compared with control. On the contrary, si-GAS8-AS1 induced marked increase in the percentage of TPC1 and BCPAP at S and G 2 /M phases. Conclusively, GAS8-AS1 inhibited PTC cell proliferation and resulted in an interference with the normal cell cycle distribution of PTC cells.

GAS8-AS1 suppresses PTC cell growth through miR-135b-5p/CCNG2 axis
In our study, we demonstrated the relationships amongst GAS8-AS1, miR-135b-5p, and CCNG2 and their effects on the proliferation and cycle of PTC cell. First, BCPAP cells were transfected with miR-135b-5p mimic and/or GAS8-AS1, si-CCNG2 and/or GAS8-AS1. CCK-8 assay results indicated that GAS8-AS1 repressed the cell proliferation, and this proliferation could be reversed by miR-135b-5p mimic and si-CCNG2 (P<0.05) ( Figure 4A,B). Furthermore, cell cycle arrest induced by GAS8-AS1, miR-135b-5p mimic and si-CCNG2 in BCPAP cells was evaluated detected by flow cytometry. As shown in Figure 4C-F, compared with control, miR-135b-5p mimic and si-CCNG2 induced significant reduction in the percentage of BCPAP cells at G 0 /G 1 compared with control and significant increase in the percentage and BCPAP cells at S and G 2 /M phases. Interesting, GAS8-AS1+miR-135b-5p mimic induced significant increase in the percentage of BCPAP cells at G 0 /G 1 and significant reduction at S and G 2 /M phases compared with control+miR-135b-5p mimic group, while GAS8-AS1+si-CCNG induced similar variation pattern in the comparision with control+si-CCNG group. Therefore, GAS8-AS1 suppresses PTC cell growth through miR-135b-5p/CCNG2 axis.

GAS8-AS1inhibits PTC cell growth through miR-135b-5p/CCNG2 axis in vivo
We further explored the effect of GAS8-AS1 on PTC cell growth in vivo. BCPAP cells stably transfected with LV TU/ml of pcDNA3.1-GAS8-AS1 or control were subcutaneously injected into nude mice, and the tumor was excised at 28 days. Tumor volume was smaller in the LV-GAS8-AS1 group than in the control group or LV-ctrl group (P<0.05) ( Figure 5A). The tumor weight followed the same pattern and was smaller in the LV-GAS8-AS1 group than in the control group and LV-ctrl group (P<0.05) ( Figure 5B). Our results showed that the expressions of GAS8-AS1 and CCNG2 were markedly increased by 3.5-folds in tissues from mice transfected with LV-GAS8-AS1 compared with control and LV-ctrl group whereas miR-135b-5p expression was 3.5-folds higher than control or LV-ctrl group (Figure 5C,D). To conclude, these observations indicate that GAS8-AS1 repressed the development and progression of PTC through inhibition of miR-135b-5p and activation of CCNG2.

Discussion
LncRNAs play crucial roles in the development of various types of cancers [27][28][29], wherein they modulate key cellular processes such as tumor cell proliferation, invasion, and metastasis [30]. GAS8-AS1 is a recently discovered tumor suppressor gene in PTC. GAS8-AS1 expression in plasma was down-regulated in patients with PTC in comparison with those in nodular goiters [31]. A significant depletion of endogenous lncRNA GAS8-AS1 expression was observed in PTC tissue specimens compared with normal tissues of patients [13]. In our study, the expression of GAS8-AS1 was reduced in PTC cell lines compared with a normal thyroid cell line. Overexpressed GAS8-AS1 markedly inhibited proliferation while underexpressed GAS8-AS1 promoted the cell proliferation, and interfered with cell cycle of PTC cells. These findings will provide signifcant contributions to the treatment of PTC. Recently, miR-135b-5p, as an oncogene or tumor suppressor, has been repeatedly reported in cancer-related studies. In these researches, abnormal expression of miR-135b-5p involves in promotion or repression of tumorigenesis and tumor progression by modulating tumor cells proliferation, apoptosis, migration, and so on [25,32]. To determine the mechanism of cell growth induced by GAS8-AS1, we examined the relationship between GAS8-AS1 and miR-135b-5p using miRNA target gene prediction site TargetScan, and found GAS8-AS1 targets miR-135b-5p. In a rescue experiment, we identified the critical role of miR-135b-5p in GAS8-AS1 induced cell growth. Recently, competing endogenous RNA (ceRNA) has been regarded as a new mechanism of post-transcriptional regulation through miRNA competition [33]. CeRNA targets and miRNA can combine to form a complex ceRNA cross-talk, which has pivotal impact on the physiology and development of diseases such as cancer [34]. In this study, the GAS8-AS1 negatively regulated miR-135b-5p in regulating PTC cell growth. However, the ceRNA network is so complex that the GAS8-AS1/miR-135b-5p regulation network in PTC needs intensively study.
CCNG2 is a novel cyclin negatively regulating the cell cycle progression. Several studies indicate that CCNG2 may have an inhibitory role in the progression of cancer as lower expression of CCNG2 is often found in more aggressive cancers [35,36], including thyroid [37]. Therefore, CCNG2 is often proposed to be a tumor suppressor gene through its regulation of cell proliferation. On the other hand, we found that miR-135b-5p directly targets CCNG2 expression by binding to its 3 -UTR. Our results showed that GAS8-AS1/miR-135b-5p influenced cell proliferation and cycle by regulating the expression of CCNG2, and revealed that CCNG2 could be another therapeutic target in PTC.
In summary, the si-GAS8-AS1 promotes cell growth through overexpression of miR-135b-5p and inhibition of CCNG2. Therefore, our study implies the GAS8-AS1/miR-135b-5p-CCNG2 axis may provide a potential target for PTC treatment.