A novel lncRNA TCLlnc1 promotes peripheral T cell lymphoma progression through acting as a modular scaffold of HNRNPD and YBX1 complexes

Abstract

Long noncoding RNAs (lncRNAs) play an essential role in tumor progression. Few researches focused on the clinical and biological relevance of lncRNAs in peripheral T cell lymphoma (PTCL). In this research, a novel lncRNA (ENST00000503502) was identified overexpressed in the main subtypes of PTCL, and designated as T cell lymphoma-associated lncRNA1 (TCLlnc1). Serum TCLlnc1 was associated with extranodal involvement, high-risk International Prognostic Index, and poor prognosis of the patients. Both in vitro and in vivo, overexpression of TCLlnc1 promoted T-lymphoma cell proliferation and migration, both of which were counteracted by the knockdown of TCLlnc1 using small interfering RNAs. As the mechanism of action, TCLlnc1 directly interacted with transcription activator heterogeneous nuclear ribonucleoprotein D (HNRNPD) and Y-box binding protein-1 (YBX1) by acting as a modular scaffold. TCLlnc1/HNRNPD/YBX1 complex upregulated transcription of TGFB2 and TGFBR1 genes, activated the tumor growth factor-β signaling pathway, resulting in lymphoma progression, and might be a potential target in PTCL.

Fig. 1. ENST00000503502 was overexpressed and related to oncogenes in peripheral T cell lymphoma (PTCL).

A LncRNA and mRNA expression in reactive hyperplasia (RH, n = 10) and peripheral T cell lymphoma (PTCL, n = 10) as revealed by lncRNA and mRNA microarray. B Volcano plot images of differentially expressed genes in PTCL and RH. C The gene signatures of ENST00000503502-coexpressed mRNAs in PTCL and RH.

Fig. 2. TCLlnc1 was clinically relevant in PTCL.

A Expression of TCLlnc1 in tumors of anaplastic large-cell lymphoma (ALCL)-anaplastic lymphoma kinase (ALK)-positive (ALK⁺ALCL, n = 9), ALCL-ALK negative (ALK⁻ALCL, n = 6), angioimmunoblastic T cell lymphoma (AITL, n = 23), and peripheral T cell lymphoma-not otherwise specified (PTCL-NOS, n = 32), as compared to RH (n = 16) by quantitative real-time polymerase chain reaction (qRT-PCR). B Correlation between TCLlnc1 expression in tumor and in serum of PTCL patients (n = 70). C Expression of TCLlnc1 in serum of RH (n = 10), ALK⁺ALCL (n = 18), ALK⁻ALCL (n = 12), AITL (n = 53), and PTCL-NOS (n = 55) in comparison with healthy volunteers (HV, n = 10). TCLlnc1 expression was quantified by qRT-PCR. D, E Progression-free survival (PFS, D) and overall survival (OS, E) of PTCL patients according to TCLlnc1 expression in serum (n = 138). F, G Univariate and multivariate analysis for PFS (F) and OS (G) in PTCL (n = 138).

Fig. 3. TCLlnc1 promoted T-lymphoma cell proliferation and migration in vitro.

A Expression of TCLlnc1 in Jurkat and Hut78 cells by qRT-PCR. B Overexpression of TCLlnc1 on Jurkat cells. C–E Effect of TCLlnc1 overexpression on cell proliferation (C), cell cycle (D), and cell migration (E) of Jurkat cells. Representative fields in transwell assay were captured using an inverted microscope (left) and the total number of migrated cells in the lower compartments was counted by flow cytometry (right). The scale bar represents 100 μm. F Knockdown of TCLlnc1 on Jurkat cells. G–I Effect of TCLlnc1 knockdown on cell proliferation (G), cell cycle (H), and cell migration (I) of Jurkat cells. Representative fields in transwell assay were captured using an inverted microscope (left) and the total number of migrated cells in the lower compartments was counted by flow cytometry (right). The scale bar represents 100 μm.

Fig. 4. TCLlnc1 promoted T-lymphoma cell proliferation in vivo.

A Tumor volumes and weights of mice injected with pLenti-Vector- and pLenti-TCLlnc1-transfected Jurkat cells. B Immunohistochemistry analysis of Ki-67 in tumors of mice injected with pLenti-Vector- and pLenti-TCLlnc1-transfected Jurkat cells. C Tumor volumes and weights of mice injected with pLenti-shRNA-ct- and pLenti-shRNA-TCLlnc1-transfected Jurkat cells. D Immunohistochemistry analysis of Ki-67 in tumors of mice injected with pLenti-shRNA-ct- and pLenti-shRNA-TCLlnc1-transfected Jurkat cells.

Fig. 5. TCLlnc1 activated TGF-β signaling and increased cytokine production.

A RNA-seq data of tumors of PTCL patients (n = 40) according to TCLlnc1 expression. B Gene ontology terms of differentially expressed genes according to TCLlnc1 expression in tumors. C Gene set enrichment analysis (GSEA) according to TCLlnc1 expression in tumors. D Correlation of TCLlnc1 expression with cytokines levels in serum of PTCL patients (n = 138).

Fig. 6. TCLlnc1 physically interacted with heterogeneous nuclear ribonucleoprotein D (HNRNPD) and Y-box binding protein-1 (YBX1).

A Nuclear location of TCLlnc1 in Jurkat cells (red). Nuclei were stained by 4′, 6-diamidino-2-phenylindole (DAPI, blue). B TCLlnc1-binding proteins detected by mass spectrometry. C Western blot validation of proteins pulled down with TCLlnc1. D Interaction of TCLlnc1 with HNRNPD and YBX1 detected by RNA immunoprecipitation (RIP) assay. The fold enrichment of TCLlnc1 in RIP is relative to its matching IgG control RIP. LncRNA linc00152 was referred as a negative control. E Expression of TCLlnc1, HNRNPD, and YBX1 after indicated RNA knockdown detected by qRT-PCR and western blot. F Western blot analysis of HNRNPD and YBX1 pulled down by full-length or truncated TCLlnc1. G Interaction of HNRNPD and YBX1 after TCLlnc1 knockdown detected by co-immunoprecipitation (co-IP).

Fig. 7. TCLlnc1 provoked lymphoma progression via HNRNPD and YBX1.

A Expression of TGFB2 and TGFBR1 after overexpression of TCLlnc1, HNRNPD, or YBX1 detected by qRT-PCR. B Expression of TGFB2 and TGFBR1 after knockdown of TCLlnc1, HNRNPD, or YBX1 detected by qRT-PCR. C Luciferase activity assay of TGFB2 and TGFBR1 promoter in cells transfected with HNRNPD siRNA, YBX1 siRNA, or NC siRNA. D Proliferation of Jurkat cells after transfection of HNRNPD siRNA or the YBX1 siRNA, plasmid encoding TCLlnc1, or control vector. E Migration of Jurkat cells after transfection of HNRNPD siRNA or YBX1 siRNA, plasmid encoding TCLlnc1, or control vector. F A schematic model of lncRNA TCLlnc1 function in PTCL.