Long non-coding RNA SOX2OT promotes the stemness phenotype of bladder cancer cells by modulating SOX2

Fresh bladder cancer tissue samples and pair-matched normal tissue samples were obtained from patients who underwent radical cystectomy. After resection, fresh bladder cancer tissue and pair-matched normal adjacent bladder tissue obtained from the same patient were snap-frozen in liquid nitrogen immediately. Each patient included in this study signed an informed consent form, and this study was approved by Institutional Review Board of Peking University First Hospital Biomedical Research Ethics Committee of Peking University First Hospital, Beijing, China. The normal urothelial cell line SV-HUC-1 and the bladder cancer cell lines SW780, 5637, J82, UM-UC-3, T24, UM-UC-14, HT1367, TUCCUP, RT4, BIU87 and EJ were used in this study. SV-HUC-1, HT1367, T24, J82 and UMUC3 cells were cultured in DMEM (Corning, USA) supplemented with 10% foetal bovine serum (FBS; Biological Industries, USA), while the SW780, 5637, UM-UC-14, TUCCUP, RT4, BIU87 and EJ cells were cultured in RPMI 1640 (Corning, USA) supplemented with 10% FBS (Biological Industries, USA). BCSC-SW780 and BCSC-5637 cells were cultured in DMEM/F-12 supplemented with 20 ng/mL EGF, 20 ng/mL bFGF and 2% B27. The plates were incubated at 37 °C in a humidified 5% CO₂ atmosphere.

BCSCs were isolated from bladder cancer cells (BCCs) using flow cytometry based on the stem cell markers CD44 and ALDH1. The BCSCs were resuspended in DMEM/F-12 supplemented with 20 ng/mL EGF, 20 ng/mL bFGF and 2% B27 and then cultured in RPMI 1640 supplemented with 10% FBS. The cells were incubated at 37 °C in a humidified 5% CO₂ atmosphere. Cell apoptosis was determined by flow cytometry. Briefly, cells were cultured in normal medium and transfected with the corresponding shRNA. Cells were collected after transfection for 48 h. Cell apoptosis was determined by PE Annexin V apoptosis detection kits (BD Pharmingen, San Diego, CA, USA). Finally, cell apoptosis was determined using flow cytometry (EPICS, XL-4, Beckman, CA, USA). Experiments were repeated at least three times.

The plasmid vectors PLKO.1-puro and pLVX-EF1α were purchased from BioVector NTCC, Inc., Guangzhou, China. The microRNA mimic (agomir) and the microRNA inhibitor (antagomir) were purchased from RiboBio, Guangzhou, China. Before transfection, the cells were cultured for 24 h. Then, the cells were transiently transfected with the corresponding vector using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 48 h, cells transfected with the corresponding vector were harvested for quantitative real-time PCR. The stable cell line was established by lenti-virus infection accordingly. Lenti-virus was produced using three vectors system: transfer vector, viral packaging (psPAX2) and viral envelope (pMD2G) at 4:3:1 ratio transfected into 293 T cells. Then, the bladder cancer cells were infected by lentiviruses according to the MOI value (the number of lentiviruses per number of cells). The knockdown and overexpressed stable cell lines were selected with puromycin (2 μg/mL) and blasticidin (10 μg/mL), respectively. Total RNA from the tissues and cells was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The cDNA was reverse transcribed from the total RNA by the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Japan). Quantitative real-time PCR was performed using SYBR Premix Ex Taq II (TaKaRa, Japan) and the 7500 Fluorescent Quantitative PCR System (Applied Biosystems Life Technologies, USA), and the results were normalized to β-actin or U6 small nuclear RNA. The detailed primer sequences are listed in Additional file 4: Table S2.

Total cell lysates were prepared as previously described. Total proteins were separated by 12% SDS–PAGE and transferred to PVDF membranes. The PVDF membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with the primary antibody anti-SOX2 (1:1000; Abcam, USA) or E-cadherin/N-cadherin/vimentin (1:2000; Cell Signaling Technology, USA). The membranes were then incubated with a secondary antibody (1:5000; Abcam, USA) and visualized with enhanced chemiluminescence using an ECL kit (Beyotime Biotechnology, China).

BCSC proliferation was determined by an ethynyl-2-deoxyuridine (EdU) incorporation assay using an EdU Apollo DNA in vitro kit (RiboBio, Guangzhou, China) following the manufacturer’s instructions. For the EdU incorporation assay, 24 h after transfection, the cells were incubated with 100 μl of 50 μM EdU per well for 2 h at 37 °C. Finally, cell fluorescence was visualized using fluorescence microscopy. BCSC proliferation was determined by a colony-formation assay. For the colony-formation assay, BCSCs were seeded in 6-well plates (2 × 10²). After incubation for 7 days at 37 °C in a humidified 5% CO₂ atmosphere, the cells were stained with 0.5% crystal violet and imaged. Finally, their absorbances were determined using a microplate reader (Bio-Rad, USA).

The migratory abilities and invasive abilities of BCSCs were determined using wound-healing assays and transwell assays, respectively. For the wound-healing assay, after transfection with the corresponding vector, the cells were incubated for 24 h; then, a wound was created using a sterile 200-μL pipette tip. Finally, cell migration was monitored under an optical microscope (Olympus, Japan), and the migration distance was calculated by HMIAS-2000. For the transwell assay, 5 × 10⁴ cells were seeded into the upper chamber with serum-free medium, and medium with 10% FBS was added into the lower chamber. After incubation for 24 h, the cells remaining in the upper chamber were wiped off, and the cells that had migrated to the bottom surface were fixed with 4% paraformaldehyde and imaged.

RNA FISH was performed using a fluorescent in situ hybridization kit (RiboBio, China) following the manufacturer’s instructions. The lncRNA SOX2OT FISH probes were also designed and synthesized by the RiboBio Company. Briefly, BCSCs were collected after transfection with the corresponding vector for 48 h and subsequently seeded on glass coverslips. Finally, fluorescence detection was performed with a confocal laser-scanning microscope (Leica, Germany).

Dual-luciferase reporter assays were performed using a Dual-Luciferase Reporter Assay System (Promega, USA) according to the manufacturer’s instructions. Briefly, SOX2OT-WT/MUT and SOX2-WT/MUT were constructed and co-transfected into BSCS-SW780 cells along with agomir-200c/agomir-NC using Lipofectamine 3000 (Invitrogen, USA) and incubated for 48 h. Finally, the luciferase activities were measured using a microplate reader (Bio-Rad, Hercules, CA, USA).

The spheroid-formation ability of BCSCs were determined using tumour sphere formation assays and single-cell tumour sphere formation assays, respectively. For the tumour sphere formation assays, BCSCs were collected after transfection with the corresponding vector for 48 h; then, 1 × 10² BCSCs were seeded on an ultra-low attachment surface 24-well plates (Corning, USA). BCSCs were resuspended in DMEM/F-12 supplemented with 20 ng/mL EGF, 20 ng/mL bFGF and 2% B27 and incubated for 7 days at 37 °C. Finally, the spheres were visualized under an optical microscope (Olympus, Japan). For the single-cell tumour sphere formation assays, BCSCs were collected after transfection with the corresponding vector for 48 h; then, one BCSCs were seeded on an ultra-low attachment surface 96-well plates (Corning, USA). BCSCs were resuspended in DMEM/F-12 supplemented with 20 ng/mL EGF, 20 ng/mL bFGF and 2% B27 and incubated for 7 days at 37 °C. Finally, the spheres were visualized under a confocal laser-scanning microscope (Leica, Germany).

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Peking University First Hospital (Beijing, China) and conducted in accordance with its recommendations and ethical regulations. For the tumour xenograft implantation experiment, 1 × 10⁵ SW780 cells were injected subcutaneously into 5-week-old male BALB/c nude mice (Vital River, Beijing, China), which were subsequently sacrificed 8 weeks later. For the metastasis experiment, 1 × 10⁵ 5637-Luc cells were suspended in 200 μL PBS and injected into the lateral tail veins of 5-week-old male B-NDG mice (BIOCYTOGEN, Beijing, China). Four weeks after the injection, mice were anaesthetized with isoflurane (YIPIN Pharmaceutical CO., LTD., Hebei, China). Ten minutes after D-luciferin was injected, sodium salt (150 mg/kg) was injected intraperitoneally, and cancer cells were detected with an in vivo imaging system, Xenogen IVIS (PerkinElmer, MA, USA). The total flux in photons per second was calculated and reported for each mouse’s lung and liver region using Living Image 4.3.1 (PerkinElmer/Caliper).

For immunohistochemistry, immunostaining was performed on tissue sections collected from nude mice. For immunofluorescence, BCSCs were seeded on glass coverslips after transfection with the corresponding vector for 48 h. Sections were then incubated with primary antibodies against E-cadherin/N-cadherin/vimentin (1:200; Cell Signaling Technology, USA) followed by incubation with the appropriate secondary antibody. Finally, the sections were visualized under an optical microscope (Olympus, Japan) or under a fluorescence microscope (Olympus, Japan). Using computer multimedia technology and fluorescence microscopy, we established a pathology organization analysis and a living cell fluorescence trace system.

SOX2OT expression was determined by RT-qPCR in bladder cancer tissues and cell lines. SOX2OT expression was upregulated in 71.7% (76/106) of bladder cancer tissues compared with in the corresponding normal tissue samples (Fig. 1a and b). Moreover, elevated SOX2OT expression was associated with a high histological grade, advanced TNM stage (Fig. 1c) and a poor prognosis (Fig. 1d). SOX2OT expression was upregulated in BC cell lines compared with in the normal urothelial cell line SV-HUC-1 (Fig. 1e). Flow cytometry based on the stem cell markers CD44 and ALDH1 was used to isolate BCSCs from BCCs (Fig. 1f). SOX2OT and SOX2 expression levels were significantly upregulated in BCSCs compared with bladder cancer non-stem cells (BCNSCs) (Fig. 1g). The correlations between SOX2OT expression and the clinical pathological characteristics of patients with urothelial carcinoma of the bladder (UCB) are shown in Table 1. The clinicopathological features of the patients are shown in Additional file 3: Table S1.

We further determined whether SOX2OT regulates the BCSC self-renewal ability. We downregulated SOX2OT expression in BCSCs using SOX2OT-specific shRNAs (Fig. 2a). The cell proliferation of BCSCs were determined using EdU incorporation and colony-formation assays. Cell proliferation inhibition induced by silencing SOX2OT was observed in BCSCs (Fig. 2b-e). Moreover, we found that knockdown of SOX2OT decreased the number and size of tumorspheres (Fig. 2f-i). These results demonstrated that SOX2OT promotes BCSC self-renewal.

We further determined whether SOX2OT regulates BCSC apoptosis. The changes in bladder cell apoptosis were determined using flow cytometry. Regrettably, there was no difference in the apoptosis of BCSCs transfected with the corresponding specific shRNA (Fig. 2j and k). The results indicated that SOX2OT does not affect BCSC apoptosis.

The migratory abilities of BCSCs were determined using a wound-healing assay. Inhibited cellular migration was observed in BCSCs after silencing SOX2OT (Fig. 3a and b). The invasive abilities of BCSCs were determined using transwell assays. Inhibited cell invasion was observed in BCSCs after silencing SOX2OT (Fig. 3c and d). We further determined whether SOX2OT regulates EMT in BCSCs. The expression levels of EMT markers were determined using RT-qPCR, western blotting and immunofluorescence analyses. Knockdown of SOX2OT increased E-cadherin expression and decreased N-cadherin/vimentin expression in BCSCs (Fig. 3e-h). The results indicated that SOX2OT promotes BCSC migration, invasion and EMT.

To investigate the underlying mechanisms of SOX2OT-mediated biological processes, we performed comprehensive transcriptional analysis by using TCGA and CCLE datasets. The results revealed that SOX2OT expression positively correlated with SOX2 expression in BC, and this correlation was also demonstrated in our tissue samples (Fig. 4a). SOX2 expression was upregulated in our tissue samples (Additional file 1: Figure S1a and S1b), and elevated SOX2 expression was associated with a high histological grade and advanced TNM stage (Additional file 1: Figure S1c). Moreover, Higher SOX2 expression is related to bladder cancer patients’ shorter overall survival (OS) and disease-free survival (DFS) in TCGA-BLCA (Additional file 1: Figure S1d). The correlations between SOX2 expression and the clinical pathological characteristics of UCB patients are shown in Table 2. Furthermore, we found that knockdown of SOX2OT inhibited SOX2 expression (Fig. 4b) and decreased the expression of SOX2 target genes (TP63, ST6GAL1, PCDH18, MSI2, CCND3, CDC25C, and EPHA7) (Fig. 4c). We further determined whether SOX2OT regulates the stemness phenotype of BCSCs in a SOX2-dependent manner. Our results showed that the SOX2-specific vector significantly reversed SOX2 expression in BCSCs (Fig. 4d) transfected with sh-SOX2OT, and SOX2 overexpression significantly reversed the inhibition of BCSC self-renewal (Fig. 4e-h and Additional file 2: Figure S2), migration (Fig. 4i and j) and invasion (Fig. 4k and l) induced by silencing SOX2OT. The results indicated that SOX2OT promotes the stemness phenotype of BCSCs in a SOX2-dependent manner.

The subcellular localization of lncRNAs is closely related to their biological function and potential molecular roles. First, we detected the subcellular localization of SOX2OT using RNA FISH. The RNA FISH results showed that SOX2OT was distributed mostly in the BCSC cytoplasm (Fig. 5a). Through searching in online bioinformatics database, bio-information analysis predicted that SOX2OT and SOX2 have common putative binding sites within the miR-200 cluster (Fig. 5b). The detailed microRNA prediction results are shown in Additional file 5: Table S3. Then, we found that knockdown of SOX2OT increased the expression level of miR-200c in BCSCs (Fig. 5c), and miR-200c expression negatively correlated with SOX2OT and SOX2 expression in BC (Fig. 5d). The results of dual-luciferase reporter assay showed that the co-transfection of SOX2OT/SOX2-Wt with Agomir200c significantly inhibited the luciferase activity, but the co-transfection of SOX2OT/SOX2-Mut with Agomir200c failed to affect the luciferase activity (Fig. 5e). Furthermore, knockdown of SOX2OT decreased the luciferase activity of cells transfected with SOX2-Wt (Fig. 5f). We further determined whether SOX2OT regulates SOX2 expression in BCSCs in a miR-200-dependent manner. We found that miR-200c overexpression inhibited SOX2 expression in BCSCs (Fig. 5g). Moreover, knockdown of miR-200c reversed the SOX2 expression inhibition of BCSCs induced by silencing SOX2OT (Fig. 5h). Moreover, knockdown of miR-200c significantly reversed the spheroid-formation ability inhibition induced by silencing SOX2OT (Fig. 5i and j). These results indicated that SOX2OT positively regulates SOX2 expression by sponging miR-200c in BCSCs.

The cell growth of different treatment groups in vivo was determined using xenograft generation. Tumours collected from mice were exhibited and measured (Fig. 6a). The tumour weight of the sh-NC treatment group was greater than that of the sh-SOX2OT group (Fig. 6b). Tumour growth in the sh-NC treatment group was faster than that in the sh-SOX2OT group (Fig. 6c). The proportion of tumour-free mice in the sh-SOX2OT group was higher than that in the sh-NC treatment group (Fig. 6d). We found that knockdown of SOX2OT inhibited SOX2 expression and downregulated the expression of SOX2 target genes (TP63, ST6GAL1, PCDH18, MSI2, CCND3, CDC25C, and EPHA7) in vivo (Fig. 6e). Furthermore, knockdown of SOX2OT inhibited BCC EMT in vivo (Fig. 6e and f). Moreover, we found that knockdown of SOX2OT inhibited SOX2 expression (Fig. 6g and h) and ki67 expression (Fig. 6i) in BCCs and SOX2OT and SOX2 were colocalized in BCCs (Fig. 6g) in vivo. The cell tumourigenicity of cells in different treatment groups was also determined using a cell fluorescence trace system in vivo. As shown in Fig. 6j, we determined the effects of SOX2OT on BCSC tumourigenicity using a cell fluorescence trace system. We found that knockdown of SOX2OT significantly decreased the proportion of sh-SOX2OT cells in xenografts (Fig. 6k and l). The results indicated that SOX2OT promotes BCSC growth and tumourigenicity in vivo.

We determined the role of SOX2OT in pulmonary tumour colonization using a whole-body fluorescence imaging system. We found that luciferase signals in the sh-SOX2OT group were remarkably lower than those in the sh-NC group and knockdown of SOX2OT reduced the incidence of bilateral pulmonary metastasis (Fig. 7a and b). There was no significant difference between the mouse weight of two treatment group (Fig. 7c). Haematoxylin-eosin staining was performed on the lung tissue to observe the metastases in the groups. We found that knockdown of SOX2OT significantly reduced the number and size of pulmonary metastases (Fig. 7d and e). Moreover, we found that knockdown of SOX2OT inhibited SOX2 expression (Fig. 6f and h) and inhibited EMT in BCCs in vivo (Fig. 7g). The results indicated that SOX2OT promotes BCSC metastasis and EMT in vivo. As shown in Fig. 7i, SOX2OT functions as a miRNA sponge to positively regulate SOX2 expression by sponging miR-200c and subsequently promotes the stemness phenotype of BCSCs, thus playing an oncogenic role in bladder cancer pathogenesis.