Novel long noncoding RNA LINC02820 augments TNF signaling pathway to remodel cytoskeleton and potentiate metastasis in esophageal squamous cell carcinoma

Abstract

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors in China. However, there are no targets to treat ESCC because the molecular mechanism behind the cancer is still unclear. Here, we found a novel long noncoding RNA LINC02820 was upregulated in ESCC and associated with the ESCC clinicopathological stage. Through a series of functional experiments, we observed that LINC02820 only promoted the migration and invasion capabilities of ESCC cell lines. Mechanically, we found that LINC02820 may affect the cytoskeletal remodeling, interact with splice factor 3B subunit 3 (SF3B3), and cooperate with TNFα to amplify the NF-κB signaling pathway, which can lead to ESCC metastasis. Overall, our findings revealed that LINC02820 is a potential biomarker and therapeutic target for the diagnosis and treatment of ESCC.

Fig. 1. LINC02820 is upregulated in ESCC.

A Heatmap of 27 differentially expressed lncRNAs in three paired ESCC tissues and adjacent normal tissues. B The LINC02820 levels in different cancers. C LINC02820 RNA levels were detected by qRT-PCR in 86 pairs of ESCC and adjacent normal tissues. D Receiver operating characteristic (ROC) curve analysis of LINC02820 in ESCC (n = 86). E The overall survival analysis of 42 ESCC patients (n = 42, p = 0.059). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2. The construction of functional cell lines and LINC02820 does not affect proliferation in ESCC Cells.

A The expression of LINC02820 in ESCC cell lines. B Analysis of LINC02820 in ESCC cells transfected with siRNA(SiLINC02820) or negative control (SiNC), and transfected with overexpression plasmid (OE- LINC02820) or empty vector plasmid (Vector). C Analysis of LINC02820 in ESCC cells transfected with LINC02820 suppression plasmid (sg-1, sg-2, sg-3, and sg-4) or control plasmid (lenti-guide or guide). D Schematic diagram of CRISPRi/dCas9 and the fluormicrographs of K180/K410 cells transfected with LINC02820 suppression plasmid. E The proliferation of K180, K410, K30, and EC109 cells by MTS assay and colony formation experiment. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3. LINC02820 promotes metastasis in ESCC via cytoskeletal remodeling, not EMT.

A Analysis of the migration and invasion ability of K180 cells transfected with SiLINC02820 or siNC (scale bar, 100 μm). B Analysis of the migration and invasion ability of K30 cells transfected with OE- LINC02820 or Vector (scale bar, 100 μm). C The migration and invasion ability of K180 cells transfected with LINC02820 suppression plasmid (sg-3 and sg-4) or control plasmid (Guide) (scale bar, 100 μm). D, E K30 Vector and K30 OE-LINC02820 cell lines were injected in mice footpads and the popliteal lymph node of nude mice was shown, and the metastasis of the popliteal lymph node. F The protein levels of EMT markers when LINC02820 are inhibited or increased. G The levels of N-WASP, p-N-WASP, and Cortactin about invasive pseudopodia. H Inhibition of LINC02820 reduced the F-actin in K180 and K410 cells (scale bar, 10 μm). I Overexpression of LINC02820 increased the F-actin in K30 and EC109 cells (scale bar, 10 μm). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4. LINC02820 is mainly distributed in the nucleus and involved in TNF/NF-κB signaling pathways.

A, B Subcellular separation experiment for K180 and K410 cells. GAPDH (cytoplasmic control), U6 (nuclear control). C RNA-FISH was performed to determine LINC02820 location in ESCC cells (scale bar, 10 μm). D The top 20 KEGG enrichment pathway analyses between K180 cells transfected with SiLINC02820 or SiNC based on RNA-seq (|FC|≥1.2). E The top 20 KEGG enrichment pathway analyses between K30 cells transfected with vector or OE-LINC02820 based on RNA-seq (|FC|≥1.2). F The top 20 KEGG enrichment pathway analyses between K30 cells transfected with vector or OE-LINC02820 based on RNA-seq (|FC|≥2.0). G Downstream genes expression of NF-κB signaling pathway. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5. LINC02820 is involved in the TNF/NF-κB signaling pathway.

A The expression of upstream genes of the NF-κB signal pathway. B The changes of LINC02820 affected the expression of p65 and its phosphorylation. C, D Nuclear and cytoplasmic protein fractions were isolated from ESCC cell lines. E, F The expression of p65 and p-p65 in K180 and K30 cells in response to TNFα (20 ng/mL).

Fig. 6. LINC02820 cooperates with TNFα to amplify the NF-κB Signaling Pathway.

A, B The expression of p65 and p-p65 in K180 and K30 cells in response to TNFα (50 ng/mL). C The migration and invasion of K180 cells transfected with SiLINC02820 or siNC in response to TNFα (scale bar, 100 μm). D The migration and invasion of K30 cells transfected with OE- LINC02820 or Vector in response to TNFα (scale bar, 100 μm).

Fig. 7. LINC02820 cooperated with TNFα to promote p-p65 into the nucleus and reconstruct the cytoskeleton.

A The expression of p-p65 and F-actin in K180, K410, K30, and EC109 cells under TNFα (20 ng/mL) (scale bar, 10 μm).

Fig. 8. LINC02820 interacts directly with SF3B3 in ESCC.

A The integrity of RNA probes was detected by formaldehyde denaturing gel electrophoresis. B, C Eluates were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify immunoprecipitated proteins. D Analysis of SF3B3 in K180 cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS). E The binding between LINC02820 and SF3B3 was confirmed by western blot. F The result of RNA immunoprecipitation. G The correlation analysis between LINC02820 and SF3B3 or SF3B3 and p65(RELA). H Proposed model for LINC02820 in ESCC. *p < 0.05, **p < 0.01, ***p < 0.001.