LncRNA TMPO-AS1 promotes esophageal squamous cell carcinoma progression by forming biomolecular condensates with FUS and p300 to regulate TMPO transcription

Esophageal squamous cell carcinoma (ESCC) is one of the most life- and health-threatening malignant diseases worldwide, especially in China. Long noncoding RNAs (lncRNAs) have emerged as important regulators of tumorigenesis and tumor progression. However, the roles and mechanisms of lncRNAs in ESCC require further exploration. Here, in combination with a small interfering RNA (siRNA) library targeting specific lncRNAs, we performed MTS and Transwell assays to screen functional lncRNAs that were overexpressed in ESCC. TMPO-AS1 expression was significantly upregulated in ESCC tumor samples, with higher TMPO-AS1 expression positively correlated with shorter overall survival times. In vitro and in vivo functional experiments revealed that TMPO-AS1 promotes the proliferation and metastasis of ESCC cells. Mechanistically, TMPO-AS1 bound to fused in sarcoma (FUS) and recruited p300 to the TMPO promoter, forming biomolecular condensates in situ to activate TMPO transcription in cis by increasing the acetylation of histone H3 lysine 27 (H3K27ac). Targeting TMPO-AS1 led to impaired ESCC tumor growth in a patient-derived xenograft (PDX) model. We found that TMPO-AS1 is required for cell proliferation and metastasis in ESCC by promoting the expression of TMPO, and both TMPO-AS1 and TMPO might be potential biomarkers and therapeutic targets in ESCC.

h. qPCR detection of TAS1 expression in 10 ESCC cell lines and 2 normal esophageal epithelial cells.
a. Statistical analysis of the popliteal lymph node weight in the indicated groups (n=6).
b. Representative images of H&E staining of the dissected popliteal lymph nodes from the indicated groups. The metastatic micro nodules were marked. Scale bar, 100 μm.
i. Immunoblotting analysis showed the expression of TAS1-regulated genes in KYSE150 and TE-11 cells with TAS1 knockdown with or without TMPO overexpression.

RNA isolation and quantitative real-time PCR (qPCR) assays
Total RNA was extracted from cultured cells using an EZ-press RNA Purification Kit (EZBioscience, Shanghai, China) according to instructions. qPCR was performed to determine relative gene expression according to the manufacturer's instructions. The primer sequences used are listed in Supplementary Table 2. Expression efficiency was assessed by qPCR and WB analysis.

Cytosolic/nuclear fractionation
Relative TAS1 expression in cytoplasmic and nuclear fractions was determined using a Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek Corp, Canada) according to the manufacturer's instructions. RNA was extracted from the cytoplasmic and nuclear fractions and subjected to qPCR analysis as described. β-Actin was used as a cytosolic marker, and U6 was used as a nuclear marker.

Fluorescence in situ hybridization (FISH) assay and immunofluorescence (IF) staining
FISH assays were performed using a lncRNA FISH Kit (RiboBio, Guangzhou, China) according to the manufacturer's instructions. Briefly, ESCC cells were fixed and permeabilized. Next, TAS1 FISH probes designed by RiboBio were added, and hybridization was carried out overnight in a dark humidified chamber at 37°C. All images were obtained with a Zeiss LSM880 high-resolution laser confocal microscope (Germany). Cy3 and DAPI channels were used for detection. 18S and U6 were used as markers for the cytosol and nucleus, respectively. IF was performed according to manufacturer's instructions, and anti-FUS antibody (5 µg/ml, Abcam, ab70381) was used.
Forty-eight hours after transfection, living cells were irradiated with 254 nm UV light at 400 mJ per cm 2 for crosslinking. Then, the cell lysates were collected. FLAG-tagged MCP-MS2-TAS1 was immunoprecipitated with anti-FLAG tag antibody (Cell Signaling Technology, 14793S) and protein A/G magnetic beads (MedChemExpress, HY-K0202). After three washes with low-salt wash buffer, the proteins were eluted with SDS loading buffer and detected by WB analysis.

MTS assay, BrdU cell proliferation assay
ESCC cells were seeded in 96-well culture plates at 800 cells per well. Cell viability was assessed daily for 5 consecutive days using MTS (Qiagen, Hilden, Germany) following the manufacturer's guidelines. The absorbance was measured at a wavelength of 490 nm on a Synergy Multi-Mode Microplate Reader (Biotek, Vermont, USA). A BrdU cell proliferation assay was performed using a BrdU Cell Proliferation ELISA Kit (Abcam, ab126556) according to the instructions.

Transwell migration assay and Matrigel invasion assay
Migration and invasion assays were performed using 24-well plates and 8-μm pore size Transwell filter inserts (Corning, NY, USA) with or without precoating with Matrigel (Corning, NY, USA).
ESCC cells (2 × 10 5 ) in FBS-free medium were added to the upper chamber, while the bottom chamber contained medium supplemented with 20% FBS. After incubation at 37°C for 24 h (migration) or 48 h (invasion), the membrane was washed, fixed and stained with crystal violet (Sangon Biotech, China). Then, invading or migrating cells on the underside of the membrane were counted in five random fields under a microscope.

Western blotting (WB) analysis
WB analysis was performed according to the instructions. Cells were lysed in RIPA lysis buffer.
The protein concentrations were calculated using a BCA assay kit (Thermo Fisher Scientific, US).

Immunohistochemistry (IHC) assays
For the IHC assays, staining and analysis were performed according to the manufacturer's

Cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models
For CDX models, 1 × 106 ESCC cells expressing the control shRNA (shCtrl) or TAS1-targeting sh#1 or sh#2 were injected subcutaneously into the dorsal flanks of 4-week-old female BALB/c nu/nu mice (five mice per group). Tumor growth was monitored every 3 days after transplantation using calipers. Mice bearing xenografts were euthanized at the endpoint, and tumor weights were measured. PDX models were constructed as described previously, and fresh ESCC samples obtained from patients were immediately subcutaneously inoculated into both flanks of nude mice. When the first generation of established PDXs (P1) reached~500 mm3, the tumors were cut into pieces of~10 mm3 and transplanted into other mice (P2). Ultimately, mice bearing P3 grafts were used to assess the therapeutic effects of TAS1 in vivo using antisense oligonucleotides (ASOs). When the P3 grafts reached~500 mm3 at approximately twenty-one days after transplantation, we began to inject 5 nmol of scrambled or in vivo-optimized TAS1 ASOs (RiboBio) per intratumoral injection every 3 days for 4 continuous doses. The target sequence is provided in Supplementary Table 1. Tumor sizes were measured every 3 days just before the next injection. The mice were euthanized at the endpoint, the tumors were dissected, and tumor weights were measured. All CDX and PDX tissues underwent subsequent pathological analyses.

In vivo metastasis models
For the lung metastasis model, 1 × 106 ESCC cells expressing luciferase and transfected with shCtrl or TAS1-targeting sh#1 or sh#2 were intravenously injected into 4-week-old female BALB/c nu/nu mice (six mice per group) through the tail vein. In vivo bioluminescence imaging was performed every four weeks after inoculation. The mice were euthanized 8 weeks after the injection. The lungs were dissected and fixed with 4% paraformaldehyde.
Subsequently, consecutive tissue sections were obtained and stained with hematoxylin-eosin (H&E) to observe the metastatic nodules in the lungs under a microscope. The number of lung nodules was measured.
For the inguinal sentinel lymph node metastasis model, 1 × 106 ESCC cells transfected with shCtrl or TAS1-targeting sh#1 or sh#2 were injected into the left footpads of 4-week-old female BALB/c nu/nu mice (six mice per group). Eight weeks after the injection, the mice were euthanized, the corresponding inguinal areas were dissected, and the lymph nodes were collected and fixed with 4% paraformaldehyde. Consecutive slices were made and stained with H&E to observe metastatic micro nodules under a microscope. The metastatic positive lymph node was determined when at least one spot of metastatic micro nodules was observed.
The number of metastasis-positive lymph nodes was measured.

Nuclear run-on (NRO) assay
The NRO assay was performed as described. Nuclei from 4 × 10 6 ESCC cells were freshly isolated with NP-40 lysis buffer and incubated ice before use. Nuclear pellets were resuspended in 40 µl of nuclei storage buffer and used for the transcription assay. Then, the suspended nuclei were incubated with 60 µl of transcription reaction buffer cocktail and 0.