Exosomal lncRNA DOCK9-AS2 derived from cancer stem cell-like cells activated Wnt/β-catenin pathway to aggravate stemness, proliferation, migration, and invasion in papillary thyroid carcinoma

Exosomal long non-coding RNAs (lncRNAs) are crucial factors that mediate the extracellular communication in tumor microenvironment. DOCK9 antisense RNA2 (DOCK9-AS2) is an exosomal lncRNA which has not been investigated in papillary thyroid carcinoma (PTC). Based on the result of differentially expressed lncRNAs in PTC via bioinformatics databases, we discovered that DOCK9-AS2 was upregulated in PTC, and presented elevation in plasma exosomes of PTC patients. Functionally, DOCK9-AS2 knockdown reduced proliferation, migration, invasion, epithelial-to-mesenchymal (EMT) and stemness in PTC cells. PTC-CSCs transmitted exosomal DOCK9-AS2 to improve stemness of PTC cells. Mechanistically, DOCK9-AS2 interacted with SP1 to induce catenin beta 1 (CTNNB1) transcription and sponged microRNA-1972 (miR-1972) to upregulate CTNNB1, thereby activating Wnt/β-catenin pathway in PTC cells. In conclusion, PTC-CSCs-derived exosomal lncRNA DOCK9-AS2 activated Wnt/β-catenin pathway to aggravate PTC progression, indicating that DOCK9-AS2 was a potential target for therapies in PTC.


Introduction
Papillary thyroid cancer (PTC) takes up around 80% of thyroid cancer (TC) cases 1 . Treatment outcome of PTC is generally satisfactory, and with appropriate treatment, over 95% of PTC patients can survive longer than 5 years 2 . However, there are still approximately 15% of PTC cases presenting aggressive behavior and unsatisfactory prognosis 3 .
Therefore, more efforts are required for the improvement of targeted therapy and diagnosis in PTC.
Long non-coding RNAs (lncRNAs) are known as transcripts without protein-coding ability and consist over 200 nucleotides 4 . LncRNAs can modulte gene expression at diverse levels, such as transcriptional level 5,6 , and posttranscriptional level 7 . Also, lncRNAs have been linked to cancer-related behaviors such as proliferation 8 , stemness 9,10 , and metastasis 11,12 . Mounting lncRNAs have been illustrated to participate in PTC, such as PTCSC2 and n384546 13,14 . DOCK9 antisense RNA2 (DOCK9-AS2) is identified as an important lncRNA related to atherosclerosis 15 . Through GEPIA and circlncRNAnet, DOCK9-AS2 is upregulated in thyroid carcinoma (THCA) specimens, indicating that DOCK9-AS2 participates in PTC development. However, no report has demonstrated the function and modulatory mechanism of DOCK9-AS2 in PTC yet.
Cancer stem-like cells (CSCs) are a small population of tumor cells that contribute to tumor initiation, metastasis and therapy-resistance 16,17 . The equilibrium state between CSCs and non-CSCs is highly dynamic 18,19 , which means that under certain circumstances, non-CSCs could differentiate into CSCs and CSCs into non-CSCs 20 . The involvement of lncRNAs in this process are increasingly documented, even in PTC. For instance, LINC00311 strengthens PTC cell stemness via miR-330-5p/TLR4 pathway 21 . However, the association of DOCK9-AS2 with PTC-CSCs has not been established.
Exosomes are small (30-150 nm) membranous vesicles originated from the multi-vesicular endosomes 22 ,which can transfer some intracellular cargoes between cells 23,24 . Also, tumor cells release exosomes to regulate the tumor microenvironment and impact various target cells 25,26 . Studies have revealed that the molecular cross-talk between CSCs and non-CSCs is important for the CSCs-non-CSCs dynamic equilibrium 20 . Moreover, numerous exosomal lncRNAs are reported to transmit signals and phenotypes between cancer cells, so are in PTC cells 27 . However, whether DOCK9-AS2 functions as an exosomal lncRNA related to PTC-CSCs in PTC microenvironment is unclear.
Therefore, this study proposed to explore whether and how DOCK9-AS2 mediated the crosstalk between CSCs and naïve PTC cells via exosome transmission in PTC.

Patient and tissues collection
Fifty four pairs of PTC and adjacent normal tissue samples of patients with PTC were collected at Nantong Tumor Hospital, with the written informed consents from all participants and the approval from the Ethics Committee of Nantong Tumor Hospital. Patients treated with radiotherapy or chemotherapy before surgery were excluded. After surgical resection, all tissue samples were instantly frozen in liquid nitrogen and then stored at −80°C.

Real-time quantitative PCR (RT-qPCR)
Using Trizol reagent (Invitrogen, Grand Island, NY, USA), the extracted RNA samples were and processed with the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara Bio, Otsu, Japan) to generate the first-strand cDNA. RT-qPCR was run with ABI 7900 system (Applied biosystems, Foster City, CA, USA) and SYBR Green assays (TaKaRa). With GAPDH or U6 as the endogenous control, gene expression was determined via 2 −ΔΔCt method.

Exosome isolation
Exosomes were isolated from plasma or culture medium as previously suggested 28 . In short, samples after centrifugation were subjected to Exoquick exosome precipitation solution (System Biosciences) to obtain exosomes.

Fluorescence in situ hybridization (FISH)
TPC1 and BCPAP cells were 80% confluent at the time of fixation and were then pre-hybridized with 1× PBS/0.5% Triton X-100. Following hybridization with Cy5-labeled probes specific to DOCK9-AS2 all night, the nuclei were counterstained with DAPI solution from Beyotime (Guangzhou, China). Images were finally taken by confocal microscope (Leica Microsystems, Mannheim, Germany).

Subcellular fractionation
RNAs from nucleus and cytoplasm of PTC cells were isolated with PARIS™ Kit (Ambion, Austin, TX, USA) obeying supplier's recommendation. The reaped PTC cells underwent re-suspension in the cell fraction buffer. Thereafter, cells underwent 10-min culturing on ice. The upper solution was then removed by centrifugation, the upper solution was removed and the nuclear pellet was obtained and kept for RNA isolation applying cell disruption buffer. Finally, the isolated RNAs underwent RT-qPCR detection, normalizing to cytoplasm (GAPDH) and nuclear (U6) controls.

Colony formation
Eight hundred PTC cells were plated per well in 6-well plates with complete culture medium for 14 days. The fixed cells in 4% of paraformaldehyde (Sigma-Aldrich) were treated with crystal violet staining (Sigma-Aldrich) for counting.

Invasion assay
Invasive ability of PTC cells were analyzed by Matrigelcoated transwell chamber (Corning Incorporated, Corning, NY, USA). 10,000 cells in serum-free medium were placed to the upper chamber. Complete medium was added into the lower chamber. Invading cells were subjected to fixing in paraformaldehyde and stained in crystal violet solution, followed by counting with microscope (magnification, ×100).

Wound-healing assay
PTC cells were cultivated in 6-well plates all night, cell layer was scratched using sterile plastic pipette tips for creating wounds. After 24 h of incubation with medium, images were obtained by microscopy (Olympus, Tokyo, Japan) at ×100 magnification.

Immunofluorescence staining (IF)
PTC cells on cover slips were fixed with cold acetone for 20 min and treated with primary antibodies against Ecadherin or N-cadherin all night, followed by culturing with secondary antibody for 1 h. DAPI was used to dye cell nuclei. After fluorescent staining, cells were visualized with fluorescence microscope (Olympus).

Sphere-forming assay
PTC cells were initially placed into 96-well ultralow attachment plates (Corning) with 10 cells per well in sphere medium for 7 days. The cell cluster with the diameter more than 50 mm was defined as sphere cells and counted. Sphere formation efficiency equaled to number of spheres/number of seeded cells, and control group was set as 1.

Flow cytometry analysis and cell sorting
The blocking solution containing 2 mM of EDTA, 1% bovine serum albumin (BSA) and PBS was utilized for preparing the single-cell suspension. Then, samples were hybridized with fluorescence conjugated primary antibodies including anti-CD44-APC (BioLegend, San Diego, CA, USA) and anti-CD133-PE (Miltenyi Biotech, Bergish-Gladbach, Germany) Alexa Fluor-488 (Invitrogen). Cell debris was removed by scatter signals. After the samples were stained with the indicated fluorescence-labeled antibodies, the fluorescence compensation was adjusted. Cells under more than 80% purity upon sorting were collected for followed analysis. Data acquisition was performed by FACS Calibur flow cytometer (Becton-Dickinson, Sparks, MD, USA) and cell sorting was performed with FACSAria system (Becton-Dickinson), followed by CellQuest (Becton-Dickinson) and FlowJo software analysis.

Side-population (SP) cell detection and sorting
For detecting SP cells, cells underwent incubation with transport blockers verapamil (100 μM; Sigma-Aldrich, MO, USA), and 20 min later, cells were subjected to Hoechst 33342 incubation. Then, propidium iodide (PI, 2 μg/ml; Sigma-Aldrich, MO, USA) was applied to exclude the dead cells. Cells were analyzed by the FAC-SAriaII (BD Biosciences, CA, USA), and SP cells were visualized following the UV excitation based on blue emission with a 424/44 filter and red emission with a 630/ 22 filter (Omega Optical, Brattleboro, VT). The side and main population (MP) was separately sorted and collected from living cells (PI negative cells).

Exosome labeling
Exosomes isolated from 1.5 × 10 6 PTC-CSCs were cultured with 1 ml of mixed PKH67 (Sigma-Aldrich) in 100 μl of PBS for 5 min at room temperature. Exosome labeling was terminated by adding 2 ml of 0.5% BSA. Stained exosomes were obtained by Exoquick exosome precipitation solution. Exosomes in 9.6 ml of basal medium were incubated with cell sub-confluent layer for 3 h at 37°C, and then washed and fixed. After DAPI staining for nuclei, fluorescence microscope (Zeiss Meditec AG, Jena, Germany) was applied for observation.

Pull-down assay
The experiment was conducted in line with the protocol of Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein extracts from PTC cells were treated with biotin-labeled RNA probes and streptavidin agarose magnetic beads from Thermo Fisher Scientific. The retrieved RNA and protein levels were measured by RT-qPCR and western blotting.

Chromatin immunoprecipitation (ChIP)
According to the instruction of Magna ChIP Kit (Millipore), ChIP assay was performed. After cross-linking, chromatin was broken to 200-500-bp fragments and immunoprecipitated with anti-SP1 or anti-IgG antibody as control (Millipore) at 4°C overnight. Following the addition of magnetic beads, the precipitated chromatin was purified for RT-qPCR.

RNA immunoprecipitation (RIP)
In accordance with the user guide of Magna RIP™ Kit (Millipore), RIP assay was carried out. 1 × 10 7 PTC cells were reaped for culturing in RIP lysis buffer. Thereafter, the lysates were immunoprecipitated with anti-Ago2 or anti-IgG antibody (Millipore). The level of recovered RNA was acquired via RT-qPCR.

Tumor xenograft formation in nude mice assay
Twenty male BALB/c nude mice of 6-weeks were purchased from the National Laboratory Animal Center (Beijing, China) and preserved in the SPF-grade animal laboratory, with the approval of the Animal Research Ethics Committee of Nantong Tumor Hospital. The exosomes isolated from TPC1-CSCs transfected with sh-NC or sh-DOCK9-AS2#1 were incubated with TPC1 cells, with PBS as control. Nude mice were randomly divided into 4 groups (5 mice each group) and injected subcutaneously with the cultured TPC1 cells for 28 days, with tumor volume recorded every 4 days. After sacrificing mice, tumors were carefully excised and weighed. To construct in vivo metastasis model, mice were injected intravenously with TPC1 cells with indicated treatment from the tail vein. Six weeks later, lungs of all mice were collected, with metastatic nodules secondary to lung counted manually and observed by hematoxylin and eosin (HE) staining.

Immunohistochemistry analysis (IHC)
Xenograft tumor tissue samples were fixed by formalin and dehydrated, the 4-mm-thick sections were cut from paraffin-embedded samples. Consecutive sections were analyzed by IHC using antibodies against Ki67, PCNA, E-cadherin, N-cadherin (Abcam). After treatment with secondary antibody, sections were subjected to microscope (Olympus).

Hematoxylin and eosin (HE) staining
To assess tumor metastasis, paraffin-embedded sections were stained by hematoxylin for 5 min. After washing in flowing water and dehydration, sections were stained with eosin and observed.

Statistical analyses
Data of all experiments with independent triplication were analyzed by Prism 6 software (GraphPad, La Jolla, CA, USA) and expressed as mean ± Standard Deviation (S.D.). Correlation analysis was assayed by Pearson's χ 2 analysis. Group comparisons were analyzed with Student's t-test and one-way ANOVA, and the difference was significant with a value of p < 0.05.
Evidence supported that lncRNAs are deeply involved in the communication of tumor microenvironment 28,29 . Therefore, we wondered whether DOCK9-AS2 existed in exosomes. We extracted the exosomes from plasma of 54 PTC patients and 44 healthy controls. Besides, we revealed that these exosomes all had a cup-shaped morphology and their size distributed from 50 to 130 nm, with the existence of several exosomal markers (including CD81, CD63, TSG-101, and Alix) but without Calnexin (an endoplasmic reticulum specific protein) (Fig. S1B). RT-qPCR analysis revealed that DOCK9-AS2 was upregulated in the plasma exosomes from PTC cases versus healthy control (Fig. 1e). In addition, DOCK9-AS2 level was more than 4 fold higher in PTC cell lines (TPC1 and BAPAB) than in Nthy-ori3-1 cell line (Fig. 1f). Also, exosomes derived from TPC1, BAPCB, and Nthy-ori3-1 cells were confirmed according to the size distribution (50-120 nm), the cup-shaped characteristics, and the presence of exosomal markers (Fig. S1C). Interestingly, we found that DOCK9-AS2 was 3-4 fold higher in TPC1 and BCPAP-derived exosomes than in TPC1 and BCPAP cells, but DOCK9-AS2 level presented no significant difference between normal Nthy-ori3-1 cells-derived exosomes and Nthy-ori3-1 cells (Fig. S1D). Consistently, we demonstrated that DOCK9-AS2 level in exosomes from TPC1 and BCPAP was about 10 fold change compared to that in exosomes from Nthy-ori3-1 cells (Fig. 1g). Thus, we guessed that exosomal DOCK9-AS2 might be important in PTC.

DOCK9-AS2 silence abrogated proliferation, migration, invasion, EMT, and stemness of PTC cells
Since we suggested the potent correlation of DOCK9-AS2 with tumor growth and metastasis in PTC, we detected whether DOCK9-AS2 affected growth and metastasis-related behaviors in PTC cells via performing loss-of-function assays. As a result, we uncovered that silencing DOCK9-AS2 led to reduced viability and proliferation in TPC1 and BCPAP cells (Fig. 2a, b and S2A-C). Likewise, the invasion and migration of these two cells were also hindered after DOCK9-AS2 suppression (Fig. 2c-e and S2D-F). Also, PTC cells with DOCK9-AS2 knockdown presented restrained tumor sphere forming efficiency and lowered levels of stemness-related proteins including CD133, Nanog, OCT4, SOX2, EpCAM, and ALDH1A1 (Fig. 2f, g and S2G). Besides, ratio of CD44 + CD133 + PTC cells, and that of mature ones with side population (SP) phenotype (SP + PTC), were both lessened upon DOCK9-AS2 knockdown (Fig. 2h-i and S2H-I). Jointly, DOCK9-AS2 knockdown impaired malignant phenotypes of PTC cells.

Exosomal DOCK9-AS2 derived from PTC-CSCs improved malignant behaviors in PTC
Later, we explored whether exosomal DOCK9-AS2 functioned in the tumor microenvironment. Since DOCK9-AS2 level was higher in the spheres generated by TPC1 and BCPAP cells than the parental cells (Fig. 3a), we then focused on CSCs which have been reported to transmit RNAs to accelerate tumorigenesis and development 30 . In addition, transmission of lncRNAs through exosomes is important for tumor microenvironment communication 28 . Hence, we wondered whether exosomes from PTC-CSCs could transmit DOCK9-AS2 to influence PTC cellular processes. As we observed, DOCK9-AS2 level was not affected under RNase treatment alone, but overtly decreased by co-treatment of RNase and Triton X-100 (Fig. S3A), suggesting that DOCK9-AS2 was packaged by membranes rather than (see figure on previous page) Fig. 2 DOCK9-AS2 silence repressed proliferation, migration, invasion, EMT, and stemness of PTC cells. a Viable PTC cells were detected by CCK-8 under DOCK9-AS2 knockdown. b Images of colonies from PTC cells after transfection with sh-DOCK9-AS2#1/2 was taken and colony number was counted manually. c Images of invasive PTC cells under DOCK9-AS2 deficiency in transwell assay was captured and cell number per field was evaluated. Scale bar: 100 μm. d Pictures of scratch wound of PTC cells at 0 and 24 h under DOCK9-AS2 knockdown was taken and wound width was assessed. e Western blots of E-cadherin, N-cadherin, MMP2, and MMP7 in PTC cells with DOCK9-AS2 knockdown. f Images of 3rd generation spheres formed by PTC cells under DOCK9-AS2. Scale bar: 100 μm. Sphere formation efficiency equaled to sphere number/number of seeded cells, and control group was set as 1. g The levels of stem specific genes including CD133, Nanog, OCT4, SOX2, EpCAM, and ALDH1A1 were analyzed by western blotting. h Flow cytometry plot of CD44 + CD133 + TPC1 cells and quantification of CD44 + CD133 + ratio. i The SP cells were sorted from TPC1 cells with DOCK9-AS2 knockdown by flow cytometry. ** P < 0.01. Error bar denotes Mean ± S.D. being released. Then, we dissociated PTC-CSCs from tumor spheres derived from TPC1 and BCPAP cells, and recognized these PTC-CSCs with higher CD133 + CD44 + and higher expression of stemness specific proteins than parental cells (Fig. 3b and S3B). Thereafter, we isolated exosomes of PTC-CSCs from the medium, and confirmed the morphology, size and exosomal markers (Fig. S3C). RT-qPCR validated that DOCK9-AS2 level was over 4 fold higher in exosomes of PTC-CSCs than PTC-CSCs (Fig. S3D). Also, DOCK9-AS2 was enriched by approximately 4-5-fold higher in exosomes derived from PTC-CSCs than exosomes from naïve PTC cells (Fig. 3c).
Next, we assessed the communication between PTC-CSCs and PTC cells. Interestingly, we observed the localization of PKH67 in the cytoplasm of TPC1 and BCPAP cells incubated with exosomes from PTC-CSCs ( Fig. 3d and S6), which made the recipient TPC1 and BCPAP cells present higher level of DOCK9-AS2 compared to control groups ( Fig. 3e and S3E). Thus, we demonstrated that PTC-CSCs transmitted DOCK9-AS2 to recipient PTC cells via exosomes. Furthermore, we validated that exosomes from PTC-CSCs facilitated proliferation, migration, invasion, EMT and stemness of recipient cells (Fig. 3f-m and S3f-k). Collectively, PTC-CSCs delivered exosomal DOCK9-AS2aggravated malignant activities in PTC cells.
Then, JASPAR identified 2 probable SP1 sites on CTNNB1 promoter (Fig. 4g). ChIP analysis confirmed that both CTNNB1 promoter sections with site 1 or 2 were precipitated by SP1 antibody (Fig. 4h and S4D). More importantly, mutation of either site 1 or 2 partly reduced while mutation of both them completely hindered the binding of SP1 to CTNNB1 promoter (Fig. 4i). Besides, SP1 silence reduced the luciferase activity of WT CTNNB1 promoter, and such reduction was partially reversed when mutating site 1 or site 2, whereas that of CTNNB1 promoter with co-mutation of site 1 and 2 remained unchanged ( Fig. 4j and S4e). These data indicated that SP1 bound to CTNNB1 promoter at both site 1 and 2.
(see figure on previous page) Fig. 4 DOCK9-AS2 activated Wnt/β-catenin signaling through transcriptionally inducing CTNNB1. a The luciferase activity of Wnt/β-catenin signaling was detected using TOP-flash assay with FOP-flash as negative control. b Western blot of p-GSK-3β, GSK-3β, β-catenin, c-Myc in PTC cells under DOCK9-AS2 depletion. c The nuclear and cytoplasm levels of β-catenin were examined by western blotting. d Luciferase activity of CTNNB1 promoter reporter was tested in PTC cells responding to DOCK9-AS2 depletion. e Venn diagram showed SP1 and IRF1 in the intersection of prediction results from Human TFDB, LADAGNA and JASPAR as the potential CTNNB1-targeting TFs. f Western blot of the enrichment of SP1 and IRF1 in the pulldown of DOCK9-AS2 biotin group and DOCK9-AS2 non-biotin group. g 2 SP1 sites on CTNNB1 promoter was obtained from JASPAR. h RT-qPCR tested the enrichment of CTNNB1 promoter fractions with site 1 or site 2 in the ChIP products of SP1. i DNA pulldown assay showed the enrichment of SP1 in the pulldown of WT or Mut CTNNB1 promoter. j The luciferase activity of CTNNB1 promoter was quantified under SP1 knockdown. k RT-qPCR data of the enrichment of CTNNB1 promoter in ChIP products of SP1 in PTC cells with DOCK9-AS2 depletion. l RT-qPCR detected the enrichment of CTNNB1 in the pulldown of DOCK9-AS2 biotin group after silencing SP1. m RT-qPCR of levels of SP1 mRNA and DOCK9-AS2 and western blot of SP1 protein in PTC cells under SP1 knockdown. n RT-qPCR data of CTNNB1 expression in PTC cells transfected with sh-NC, sh-DOCK9-AS1#2, sh-DOCK9-AS2#1+pcDNA3.1 or sh-DOCK9-AS2#1+pcDNA3.1/SP1. o Western blot results of β-catenin, c-Myc, MMP7, MMP2, E-cad, and N-cad levels in each group. * P < 0.05, ** P < 0.01 . n.s. meant no significance. Error bar denotes Mean ± S.D. Later, we examined whether DOCK9-AS2 cooperated with SP1 to regulate CTNNB1 transcription. We discovered that silencing DOCK9-AS2 reduced SP1binding with CTNNB1 promoter, but did not affect SP1 protein expression ( Fig. 4k and S4F). Meanwhile, SP1 silence hindered the interaction between CTNNB1 promoter and DOCK9-AS2 ( Fig. 4l and S4G), without any influences on DOCK9-AS2 expression ( Fig. 4m and  S4H). Then, we found that overexpressing SP1 only partially restored DOCK9-AS2 silence-lowered CTNNB1 expression (Fig. 4n and S4I). Furthermore, the impact of DOCK9-AS2 deficiency on the levels of β-catenin, c-Myc, MMP7, MMP2, and N-cad, and E-cad, were partly counteracted by SP1 overexpression (Fig. 4o). Altogether, DOCK9-AS2 regulated CTNNB1 expression and Wnt/β-catenin pathway partly through SP1-mediated CTNNB1 transactivation.

Discussion
The functional role of exosomal lncRNAs has been reported in cancer development, including in PTC 36 . Herein, we first uncovered the correlation between DOCK9-AS2 and PTC by identifying its upregulation in PTC tissues and cell lines. Notably, we discovered the existence of DOCK9-AS2in exosomes of PTC patients and cell lines. Besides, we unveiled the tumorcontributing effect of DOCK9-AS2 on PTC cell proliferation, motility, EMT, and stemness, indicating a novel thought to hinder PTC progression.
Former studies have illustrated that lncRNAs promoted PTC development and by regulating stemness [37][38][39] . CSCs are identified as crucial participants in tumor progression 16,17 . Importantly, considering the highly dynamic equilibrium state of CSCs and non-CSCs 18,19 , non-CSCs could dedifferentiate into CSCs 20 . Increasing reports suggested the function of exosomes in transmitting molecules between cells, especially between non-CSCs and CSCs 40,41 . Presently, we validated that exosomes from PTC-CSCs increased DOCK9-AS2 level and facilitated the progression in PTC both in vitro and in vivo. These findings suggested that DOCK9-AS2 might influence the exosome-mediated cross-talk between non-CSCs and CSCs in PTC to drive PTC progression.
Wnt/β-catenin pathway is a key pathway responsible for cancer development, PTC included 42,43 . It is known that activation of Wnt/β-catenin pathway depends on nuclear translocation of β-catenin 44 , and GSK-3β is a regular inhibitor of β-catenin phosphorylation 45 . Our data first supported that DOCK9-AS2 positively regulated Wnt/ β-catenin activity by increasing β-catenin level but did not influence GSK-3β and p-GSK-3β.
Intriguingly, here we disclosed that DOCK9-AS2 located in both nucleus and cytoplasm of PTC cells. In nucleus, lncRNAs can interact with TFs to modulate transcription of target genes 5 . Consistently, SP1, a well-known oncogenic TF in cancers 46,47 was identified as the TF targeting CTNNB1 in PTC. Previously, the relation of SP1 to Wnt/β-catenin pathway has already been suggested 48,49 , but the direct interaction between SP1 and CTNNB1 was first revealed by our study. More significantly, we unmasked that DOCK9-AS2 activated CTNNB1 transcription by cooperating rather than regulating SP1. Interestingly, we also found that cytoplasmic DOCK9-AS2 regulated CTNNB1 through acting as a ceRNA by sequestering miR-1972. MiR-1972 is found as a tumor-suppressor in osteosarcoma 50 . Moreover, miR-1972 inhibition plus SP1 overexpression fully rescued Wnt/ β-catenin signaling activity in DOCK9-AS2-silenced PTC cells, therefore recovering cellular progression. These findings indicated that DOCK9-AS2 activated Wnt/β-catenin pathway through SP1 and miR-1972-regulated CTNNB1, resulting in accelerated PTC progression.

Conclusions
This study first revealed that DOCK9-AS2 was an exosomal lncRNA derived from PTC-CSCs and upregulated in PTC. Functionally, PTC-CSCs transferred DOCK9-AS2 to naïve PTC cells to facilitate cellular progression in PTC. Mechanistically, DOCK9-AS2 increased CTNNB1 expression by SP1 and miR-1972 to activate Wnt/β-catenin pathway (Fig. 8). These findings indicated that DOCK9-AS2 was a promising target for PTC therapy. However, this study is limited because of the small number of clinical samples. Therefore, we will dedicate ourselves to enlarge the clinical significance of DOCK9-AS2 in PTC. (see figure on previous page) Fig. 7 Exosomal DOCK9-AS2 derived from PTC CSCs induced tumorigenesis and metastasis of PTC in vivo. The exosomes from TPC1-CSCs transfected with sh-NC or sh-DOCK9-AS2#1 were extracted and incubated with naïve TPC1 cells. a Transfected TPC1 cells were subcutaneously inoculated into mice. Tumor growth volume was detected 4 days a time until day 28 to generate growth curve. b Tumor weight was tested at day 28 after sacrificing the mice in each group. c RT-qPCR data of DOCK9-AS2 and CTNNB1 expression levels in xenografts of each group. d Western blotting for β-catenin, c-Myc, MMP7, MMP2, N-cad, E-cad, CD133, Nanog, OCT4, and SOX2 in tumors from mice of each group. e, f The IHC pictures and quantification of stain positivity of Ki67, PCNA, E-cad, and N-cad in mice of each group. Scale bar: 100 μm. g Transfected TPC1 cells were intravenously injected into mice to form lung metastasis. Pictures of HE staining for the metastatic nodules in each group were taken and the nodules of the lung in mice were counted manually. Scale bar: 100 μm. ** P < 0.01. Error bar denotes Mean ± S.D. Fig. 8 Graphical abstract. Exosomal lncRNA DOCK9-AS2 derived from PTC-CSCs induced CTNNB1 transcription by interacting with SP1 and sponged miR-1972 to induce CTNNB1 level to activate Wnt/β-catenin and PTC proliferation, migration, invasion, and stemness.