STAT3‐induced upregulation of lncRNA ABHD11‐AS1 promotes tumour progression in papillary thyroid carcinoma by regulating miR‐1301‐3p/STAT3 axis and PI3K/AKT signalling pathway

Abstract Objectives Emerging evidences indicated the importance of long non‐coding RNAs (lncRNAs) in the tumorigenesis and deterioration of malignant tumours. To our knowledge, the study about lncRNAs in papillary thyroid carcinoma (PTC) is still inadequate. ABHD11‐AS1 was highly expressed in the PTC samples of The Cancer Genome Atlas database. This study focused on the biological function and mechanism of lncRNA ABHD11‐AS1 in PTC. Materials and methods qRT‐PCR analysis was used to examine the expression of ABHD11‐AS1 in PTC tissues and cell lines. The prognostic significance of ABHD11‐AS1 for the patients with PTC was analysed with Kaplan‐Meier analysis. The effects of ABHD11‐AS1 knockdown on the cell proliferation and metastasis were evaluated by in vitro functional assays and in vivo experiments. The molecular mechanism which contributed to the oncogenic role of ABHD11‐AS1 in PTC was explored by conducting mechanism experiments. Rescue assays were carried out for final demonstration. Results High expression of ABHD11‐AS1 predicted poor prognosis for patients with PTC and promoted cell proliferation and metastasis in vitro and in vivo. ABHD11‐AS1 was activated by the transcription factor STAT3. ABHD11‐AS1 positively regulated PI3K/AKT signalling pathway. ABHD11‐AS1 acted as a competitive endogenous (ce) RNA to upregulate STAT3 by sponging miR‐1301‐3p. Conclusions STAT3‐induced lncRNA ABHD11‐AS1 promoted PTC progression by regulating PI3K/AKT signalling pathway and miR‐1301‐3p/STAT3 axis.


| INTRODUC TI ON
Thyroid cancer is a kind of commonest endocrine system malignant tumours. The mortality of patients with thyroid cancer is rapidly increasing over the past decades. 1 Papillary thyroid carcinoma (PTC) is the commonest subtype of thyroid cancer. 2 There are some risk factors for PTC, such as ionizing radiation exposure, thyroid nodular disease and the family history. 3 However, the specific mechanism underlying PTC is widely elusive. Therefore, investigating the potential molecular mechanism involved in PTC progression is very essential to find novel biomarkers or therapeutic targets for PTC.
As a subtype of non-coding RNAs (ncRNAs), long non-coding RNAs (lncRNAs) are originally defined as transcriptional noise. 4,5 Importantly, the dysregulation of lncRNAs can affect the progression of various human diseases, including cancers. The most significant function of abnormally expressed lncRNAs is to regulate cellular processes in human cancers and determine the outcomes for cancer patients. 6,7 Epigenetic regulation is a major reason for aberrant expression of lncRNAs. For instance, H3K27 acetylation-mediated activation induces high expression of lncRNA CCAT1 in oesophageal squamous cell carcinoma, 8 whereas the downregulation of p53-induced lncRNA TP53TG1 is caused by the high methylation of its gene promoter. 9 Additionally, upregulation of lncRNAs induced by its upstream transcription factor can promote tumour progression in various human cancers. [10][11][12][13] The dysregulation of lncRNAs has been identified as an oncogenic factor for the tumour progression. [14][15][16][17] Therefore, it is crucial to investigate the mechanism of lncRNAs in human cancers.
Based on the data of The Cancer Genome Atlas (TCGA) database, ABHD11-AS1 was significantly highly expressed in PTC samples.
qRT-PCR exanimation further detects the expression of ABHD11-AS1 in 82 PTC tissues. Kaplan-Meier method was to identify the effect of ABHD11-AS1 expression on the prognosis of PTC patients.
The oncogenic role of ABHD11-AS1 in PTC was identified by performing loss-of-function assays. Therefore, we further detected the molecular mechanism of ABHD11-AS1 in PTC progression. As a transcription activator of lncRNAs, STAT3 has been reported in various human cancers. 18,19 In this study, STAT3 was demonstrated to be able to activate ABHD11-AS1 transcription. Furthermore, we applied microarray analysis and KEGG analysis to find the molecular mechanism involved in ABHD11-AS1-mediated biological behaviours of PTC cells. Previous reports showed that STAT3 can bind to miRNAs by competing with lncRNAs. 20-23 Therefore, we F I G U R E 1 Upregulation of lncRNA ABHD11-AS1 predicted unfavourable prognosis for patients with PTC. A, The expression of lncRNA ABHD11-AS1 in the thyroid cancer tissues of TCGA database was detected. B, The expression of ABHD11-AS1 was measured in 82 pairs of PTC tissues and adjacent non-tumour tissues. C,D, The conspicuous fold change of ABHD11-AS1 in paired tumour tissues and non-malignant tissues was further analysed and illustrated. E, The importance of ABHD11-AS1 expression for the prognosis of PTC patients was analysed by Kaplan-Meier analysis. *P < 0.05, **P < 0.01. PTC: papillary thyroid carcinoma hypothesized that ABHD11-AS1 might positively regulate STAT3 by competitively binding to a miRNA. Bioinformatics analysis and mechanism experiments were conducted to demonstrate our hypothesis.

| Tissue samples
Paired tissues (tumour and non-tumour) were obtained from 82 PTC patients who received surgery at Navy General Hospital. None of these patients received chemotherapy or radiotherapy before this study.
Patients with other tumours or tumour history were excluded from this study. Total samples were dissected immediately, placed on ice, snapfrozen with liquid nitrogen and stored at −80°C for later use. This study had been approved by the ethics committee of Navy General Hospital.
All patients have signed the informed consents before the study.

| Detection of mycoplasma contamination
The mycoplasma contamination of cells was detected with Myco-PCR-Mix mycophenolate mycoplasma detection kit (Qiao Xinzhou Bioengineering Co. Ltd., Shanghai, China). The experimental results are shown in Figure S1.

| Colony formation assay
Following transfection for 72 hours, total cells were trypsinized (Solarbio, Beijing, China), plated into six-well plates at 200 cells each well and cultured for 21 days using RPMI 1640 (Invitrogen) containing 10% foetal bovine serum (Gibco) under routine conditions.
Medium was replaced when needed. After that, colonies were fixed by methanol and stained by 0.5% crystal violet (Sigma). Visible colonies were counted manually.

| Caspase-3 activity assay
The apoptosis condition was examined by using caspase-3 activity kit (Beyotime Institute of Biotechnology, Nantong, China). In short, after cell transfection, 2 × 10 6 cells seeded in six-well plates were lysed in lysis buffer (as provided in the kit) for 15 minutes at 4°C,

| Cell migration assay
Cell migration was examined with transwell assays. In short, indi- and quantified through counting 10 random fields.

| Tumour xenograft model
BCPAP cells stably transfected with sh-NC and sh-ABHD11-AS1 were subcutaneously injected into 5-week-old male BALB/c nude mice at a density of 4 × 10 6 cells per mouse. The nude mice were divided into two groups in accordance with the transfection: sh-NC F I G U R E 5 LncRNA ABHD11-AS1 was upregulated in PTC cells by the transcription factor STAT3. A, The putative binding motif of transcription factor STAT3 was obtained from JASPAR. B,C, Top two binding sites between STAT3 and ABHD11-AS1 promoter were chosen for further analysis. D, ChIP assay analysed the affinity of STAT3 to the part 2 (P2) of ABHD11-AS1 promoter region. E, Luciferase activity assay further demonstrated the combination between STAT3 to the binding sequence of P2 region. F, The expression of STAT3 was detected in PTC tissues and cell lines. G, The correlation between ABHD11-AS1 expression and STAT3 expression was then analysed in PTC tissues by Spearman's correlation analysis. H, The mRNA level of ABHD11-AS1 was examined in PTC cells transfected with STAT3 expression vector. *P < 0.05, **P < 0.01, ***P < 0.001; N.S: no significance. LncRNA: long noncoding RNA; PTC: papillary thyroid carcinoma and sh-ABHD11-AS1 (n = 3 each group). The nude mice were obtained from the Animal Experimental Committee of Navy General Hospital. The nude mice used in this study are healthy. All these mice are with same age and similar weight. Four weeks later, the nude mice were sacrificed. Tumour volume and weight were measured and calculated every 7 days by using a formula (0.5 × length × width 2 ). This study was approved by the Committee Ethics of Animal Center of the Qilu Hospital of Shandong University.

| In vivo tumour metastasis assay
All animal studies had acquired the approval of the Animal Experimental Committee of Qilu Hospital of Shandong University.
The BALB/c nude mice (male, 4-5 weeks old) were obtained from the Experimental Animal Center of Navy General Hospital. The nude mice used in this study are healthy. All these mice are with same age and similar weight. To conduct in vivo metastasis assays, 1 × 10 7 cells were implanted subcutaneously into the right hind flank regions of the two different groups (n = 3 each group). Tumour growth was recorded with the digital callipers every 7 days. Sixty days later, all mice were sacrificed so as to observe the lung and liver metastasis.
Finally, the metastatic tissues were photographed and detected by H&E staining under a microscope.

| Immunofluorescence
Cells were grown on glass coverslips and fixed with 4% formaldehyde for 10 minutes followed with the permeabilization in 0.5% Triton X-100 at room temperature for 15 minutes.

| Fluorescence in situ hybridization (FISH)
Fluorescence in situ hybridization Kit (GenePharma, Shanghai, China) was used to perform FISH assay. ABHD11-AS1 was labelled with the FAM probe, and nucleus was stained with DAPI. U6 was used as the control of nuclear fraction, while 18S rRNA was used as the control of cytoplasmic fraction.

| Luciferase reporter assay
The different fragment sequences predicted by JASPAR were synthe-

| ChIP
ChIP was conducted by the use of the ChIP assay kit following the manufacturer's instruction (17-

| RIP assay
The MagnaRIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) was applied to perform RIP assay. The cell lysates were incubated in a RIP buffer containing the magnetic beads which had F I G U R E 7 ABHD11-AS1 acted as a miR-1301-3p sponge. A,B, The localization of ABHD11-AS1 in cytoplasm or nucleus of PTC cells was identified with subcellular fractionation and FISH assay. C, The potential binding between five candidate miRNAs and ABHD11-AS1 was assessed by luciferase reporter assay. D, RIP assay was conducted to determine the interaction between ABHD11-AS1 and miR-1301-3p. E,F, The expression levels of miR-1301-3p in PTC tissues and cell lines. G, The expression association between ABHD11-AS1 and miR-1301-3p in PTC tissues. *P < 0.05, **P < 0.01. FISH: Fluorescence in situ hybridization; PTC: papillary thyroid carcinoma been coated with Ago2 antibodies. Input and normal IgG were taken as controls. Proteins were digested with proteinase K. Then, the immunoprecipitated RNA was isolated. At last, qRT-PCR was used to examine the purified RNAs.

Immobilon Western Chemiluminescent HRP Substrate (200 μL;
Millipore) was used to cover the surface of membrane. Finally, the signals were captured, and the concentration of the bands was quantified using Image Lab™ software (Bio-Rad Laboratories, Hercules, CA, USA).

| Statistical analysis
Data of all experiments were presented as mean ± standard deviation (SD). All experimental procedures were repeated at least three times. Statistical analysis was conducted using GraphPad Prism 6.0 statistical software (GraphPad Software Inc, La Jolla, CA, USA). Student's t test for two groups or Bonferroni test for more than two groups was used to analyse the differences.
Kaplan-Meier method was used to analyse the overall survival of PTC patients with high or low expression of ABHD11-AS1.
Expression association was analysed with Spearman's correlation analysis. P value <0.05 implied a statistically significant result.

| Upregulation of lncRNA ABHD11-AS1 predicted unfavourable prognosis for patients with PTC
Based on the data of TCGA database, we found that lncRNA ABHD11-AS1 was significantly upregulated in PTC samples ( Figure 1A). Next, we examined the level of ABHD11-AS1 in 82 pairs of PTC tissues and non-malignant tissues. Unsurprisingly, ABHD11-AS1 was expressed higher in PCT tissues ( Figure 1B). The conspicuous fold change of lncRNA ABHD11-AS1 in paired tumour tissues and non-malignant tissues was further analysed and illustrated ( Figure 1C,D). The expression level of ABHD11 presented no significant expression difference between PTC tissue samples and normal tissue samples ( Figure S2A,B). Moreover, the expression of ABHD11 was significantly changed by ABHD11-AS1 knockdown ( Figure S2C). Moreover, we found three transcripts of ABHD11 with specific sequence ( Figure S2D). Then, the expression of these three transcripts was decreased by ABHD11-AS1 knockdown ( Figure S2E). Furthermore, the activity of ABHD11-AS1 promoter was decreased in cells transfected with sh-ABHD11-AS1 ( Figure   S2F). To classify 82 PTC samples, the mean value of ABHD11-AS1 expression was used as threshold. We found that the high expression of ABHD11-AS1 was closely correlated with advanced TNM stage, lymph node metastasis and tumour infiltration (Table 1).
Subsequently, the importance of ABHD11-AS1 expression for the prognosis of PTC patients was analysed by Kaplan-Meier analysis.
PTC patients with higher expression of ABHD11-AS1 had poorer overall survival than patients with lower expression of ABHD11-AS1 ( Figure 1E).

| ABHD11-AS1 knockdown suppressed cell proliferation and induced cell apoptosis in PTC
To investigate the role of ABHD11-AS1 in the tumour progression of PTC, functional assays were conducted in PTC cells. At first, the expression level of ABHD11-AS1 was examined in one normal cell line (Nthy-ori 3-1) and four PTC cell lines (BHP5-16, BCPAP, K1 and BHP2-7). As expected, the higher level of ABHD11-AS1 was detected in PTC cell lines, especially in BCPAP and K1 cells ( Figure 2A). Therefore, shRNA specifically targeted to ABHD11-AS1 (sh-ABHD11-AS1) and negative control shRNA (sh-NC) were transfected into BCPAP and K1 cells to knockdown ABHD11-AS1 effectively ( Figure 2B). Subsequently, functional assays were conducted in BCPAP and K1 cells transfected with sh-ABHD11-AS1. Cell proliferation was found to be drastically suppressed by sh-ABHD11-AS1 ( Figure 2C

| Knockdown of ABHD11-AS1 inhibited the migration and EMT progress of PTC cells
Here, we further detected the effect of differentially expressed ABHD11-AS1 on the migration and EMT progress of PTC cell. The migratory capability of PTC cells transfected with sh-ABHD11-AS1 was tested with transwell assay. The result obviously showed that cell migration was noticeably suppressed by ABHD11-AS1 knockdown ( Figure 3A). Epithelial-mesenchymal transition is known as a biological process which is closely associated with cell migration.
Therefore, we further investigated the influence of sh-ABHD11-AS1 on EMT progress by detecting the protein levels of EMT markers.
Increased level of epithelial markers (E-cadherin, β-catenin) and decreased level of mesenchymal markers (N-cadherin, vimentin) indicated the reversal effect of sh-ABHD11-AS1 on EMT progress in PTC cells ( Figure 3B). Immunofluorescence further demonstrated the reversal effect of sh-ABHD11-AS1 on EMT progress ( Figure 3C).

| Knockdown of ABHD11-AS1 suppressed tumour growth and metastasis in vivo
To further demonstrate the effect of ABHD11-AS1 expression on PTC tumour progression, in vivo experiments were carried out.
As shown in Figure 4A, tumour growth was drastically inhibited by silenced ABHD11-AS1. Similarly, tumour volume and tumour weight in sh-ABHD11-AS1 group were smaller than sh-NC group ( Figure 4B,C). Moreover, in vivo metastasis experiment revealed that inhibition of ABHD11-AS1 expression suppressed the liver metastasis and lung metastasis ( Figure 4D,E). Then, we examined the protein levels of STAT3, E-cadherin and N-cadherin. The results revealed that knockdown of STAT3 led to the downregulation of STAT3, N-cadherin, while led to the upregulation of E-cadherin ( Figure 4F). These data further indicated the oncogenic role of ABHD11-AS1 in PTC.

| LncRNA ABHD11-AS1 was upregulated in PTC cells by the transcription factor STAT3
All findings above suggested that upregulation of ABHD11-AS1 is an oncogenic factor for the tumour progression of PTC. To find the reason why ABHD11-AS1 was upregulated in PTC tissues or cell lines, mechanism investigation was performed. STAT3 was found to be an upstream transcription factor of ABHD11-AS1. The putative binding motif of STAT3 to the ABHD11-AS1 promoter was obtained from JASPAR (http://jaspar.genereg.net/) ( Figure 5A). Top two binding sites between STAT3 and ABHD11-AS1 promoter were chosen for further analysis ( Figure 5B,C). ChIP assay validated the strong affinity of STAT3 to the part 2 (P2) of ABHD11-AS1 promoter region ( Figure 5D). Luciferase activity assay further demonstrated the combination between STAT3 to the binding sequence of P2 region ( Figure 5E). All these findings indicated the binding of STAT3 to ABHD11-AS1 promoter. To demonstrate the regulatory relationship between STAT3 and ABHD11-AS1, the expression of STAT3 was detected in PTC tissues and cell lines. Unsurprisingly, STAT3 was definitely upregulated in PTC tissues and cell lines ( Figure 5F). The positive correlation between ABHD11-AS1 expression and STAT3 expression was then analysed in PTC tissues by Spearman's correlation analysis ( Figure 5G). Furthermore, upregulation of STAT3 led to the increased expression level of ABHD11-AS1 ( Figure 5H), demonstrating the positive regulatory effect of STAT3 on the ABHD11-AS1 expression.

| ABHD11-AS1 exerted oncogenic function in PTC by activating PI3K/AKT signalling pathway
To explore the downstream molecular mechanism of ABHD11-AS1 in PTC progression, microarray analysis was applied to detect the expression change of 500 mRNAs in ABHD11-AS1-downregulated BCPAP cell. A total of 194 mRNAs were found to be significantly upregulated, and 203 mRNAs were obviously downregulated ( Figure 6A). Subsequently, KEGG analysis indicated the PI3K/AKT signalling pathway might involve in ABHD11-AS1-mediated functions ( Figure 6B). It has been reported that PI3K/AKT signalling pathway involved in lncRNAs-mediated tumorigenesis and tumour progression. Thus, we speculated that ABHD11-AS1 might exert its function by activating PI3K/AKT signalling pathway. Western blot analysis revealed that silenced ABHD11-AS1 inhibited the levels of phosphorylated PI3K, AKT and mTOR ( Figure 6C).
Furthermore, RIP assay revealed the binding of ABHD11-AS1 to miR-1301-3p ( Figure 7D). Next, we examined the lower expression of miR-1301-3p in PTC tissues and cell lines ( Figure 7E,F). The negative correlation between the expression of ABHD11-AS1 and that of miR-1301-3p in PTC tissues was analysed ( Figure 7G).
The results indicated that the luciferase activity of STAT3-WT was efficiently decreased by miR-1301-3p mimics ( Figure 8B).
The effect of miR-1301-3p mimics on the luciferase activity of STAT3-WT was partially attenuated by pcDNA-ABHD11-AS1, whereas there was no significant change on the luciferase activity of STAT3-MUT. Furthermore, pull-down assay revealed the interaction between miR-1301-3p and STAT3 ( Figure 8C). The negative expression correlation between miR-1301-3p and STAT3 in PTC tissues was analysed ( Figure 8D).
Furthermore, the mRNA level and protein level of STAT3 were detected in indicated PTC cells. The expression of STAT3 decreased by miR-1301-3p mimics was reversed by ABHD11-AS1 ( Figure 8E,F).

| ABHD11-AS1-miR-1301-3p-STAT3 feedback loop promoted PTC progression
To demonstrate the effect of ABHD11-AS1-miR-1301-3p-STAT3 feedback loop on PTC progression, we designed and performed a series of rescue assays. According to the experimental results of cell proliferation assays, we found that the inhibitory effect of sh-ABHD11-AS1 on cell proliferation was reversed by miR-1301-3p inhibitors or STAT3 overexpression ( Figure 9A,B). Moreover, cell migration and EMT progress suppressed by sh-ABHD11-AS1 were recovered by miR-1301-3p inhibitors or STAT3 overexpression ( Figure 9C,D).

| D ISCUSS I ON
Over the several years, gene mutation has been identified to be crucial risk factor for PTC progression. 27 However, the molecular mechanism underlying the progression of thyroid cancer is still largely unknown. Some studies have provided the evidences to show the crucial role of lncRNAs in the initiation and development of human malignant tumours, 28-32 including PTC. [33][34][35] This study aims to investigate the potential role of a certain lncRNA in PTC. At first, lncRNA ABHD11-AS1 was chosen from TCGA database due to its ectopic expression in thyroid cancer samples.
Subsequently, lncRNA ABHD11-AS1 was subjected to qRT-PCR analysis in 82 pairs of PTC tissues and corresponding non-tumour tissues. Consistently, lncRNA ABHD11-AS1 was conspicuously upregulated in PTC tissues. It is well known that dysregulation of lncRNAs is correlated with the prognosis of cancer patients. [36][37][38][39][40] Here, we applied Kaplan-Meier method to analyse the prognos- To our knowledge, lncRNAs exert function in tumour progression by regulating gene expression or participate in some molecular mechanism. Here, we explored the molecular mechanism involved in ABHD11-AS1-mediated PTC progression. As we all know, transcription factors can modulate the transcription activity of lncRNAs. [44][45][46] Therefore, we hypothesized that the upregulation of ABHD11-AS1 in PTC might be caused by a certain transcription factor. Based on the search result of UCSC, STAT3 was uncovered to be the upstream transcription factor. It has been reported that

ACK N OWLED G EM ENT
The authors appreciate all members contributed to this work.