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

Ping Zhao, Meng-Meng Ji, Ying Fang, Xiao Li, Hong-Mei Yi, Zi-Xun Yan, Shu Cheng, Peng-Peng Xu, Anne Janin, Chao-Fu Wang, et al.

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## To cite this version:

Ping Zhao, Meng-Meng Ji, Ying Fang, Xiao Li, Hong-Mei Yi, et al. A novel lncRNA TCLlnc1 promotes peripheral T cell lymphoma progression through acting as a modular scaffold of HNRNPD and YBX1 complexes. Cell Death and Disease, 2021, 12 (4), 10.1038/s41419-021-03594-y. hal-03258381

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# A novel IncRNA TCLnc1 promotes peripheral T cell lymphoma progression through acting as a modular scaffold of HNRNPD and YBX1 complexes

Ping Zhao<sup>1,2</sup>, Meng-Meng Ji<sup>1</sup>, Ying Fang<sup>1</sup>, Xiao Li<sup>1</sup>, Hong-Mei Yi<sup>2</sup>, Zi-Xun Yan<sup>1</sup>, Shu Cheng<sup>1</sup>, Peng-Peng Xu<sup>1</sup>, Anne Janin<sup>3,4</sup>, Chao-Fu Wang<sup>1,2</sup>, Li Wang<sup>1,3</sup> and Wei-Li Zhao<sup>1,3</sup>

**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 (TCLnc1). Serum TCLnc1 was associated with extranodal involvement, high-risk International Prognostic Index, and poor prognosis of the patients. Both in vitro and in vivo, overexpression of TCLnc1 promoted T-lymphoma cell proliferation and migration, both of which were counteracted by the knockdown of TCLnc1 using small interfering RNAs. As the mechanism of action, TCLnc1 directly interacted with transcription activator heterogeneous nuclear ribonucleoprotein D (HNRNPD) and Y-box binding protein-1 (YBX1) by acting as a modular scaffold. TCLnc1/HNRNPD/YBX1 complex upregulated transcription of *TGFB2* and *TGFB1* genes, activated the tumor growth factor-β signaling pathway, resulting in lymphoma progression, and might be a potential target in PTCL.

**Introduction**

Peripheral T cell lymphomas (PTCL) encompass a heterogeneous group of neoplasm derived from T cell lineages and represent ~10–15% of non-Hodgkin lymphoma¹, mainly including anaplastic large-cell lymphoma (ALCL)-anaplastic lymphoma kinase (ALK)-positive (ALK+ALCL), ALCL-ALK-negative (ALK-ALCL), angioimmunoblastic T cell lymphoma (AITL), and PRCL-not otherwise specified (PTCL-NOS)². PTCLs are characterized by advanced disease stage at diagnosis³, and

dismal prognosis, with a 5-year patients survival rate of <30% (except for ALK+ALCL)⁴⁻⁶. Therefore, prognostic biomarkers closely related to lymphoma progression and expressed generally in the main histological subtypes need to be further investigated in PTCL.

To date, studies have focused on the alterations in protein-coding genes that serve as diagnostic and therapeutic targets of PTCL⁷. Growing evidence suggests that noncoding RNAs are also critically involved in tumor progression⁸,⁹. Long noncoding RNAs (lncRNAs) belong to a class of noncoding sequences >200 nucleotides in length¹⁰ and are emerging as essential factors in the tumorigenesis and metastasis of various cancers, including hematological malignancies¹¹,¹². The expression of HOTAIR enhances B-lymphoma cell growth through mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase signaling pathway¹³. LncRNA metastasis-associated lung adenocarcinoma transcript 1 is also overexpressed in hematological

Correspondence: Chao-Fu Wang (wangchaofu@126.com) or Li Wang (wl_wangdong@126.com) or Wei-Li Zhao (zhao.weili@yahoo.com)

¹Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China

²Department of Pathology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China

Full list of author information is available at the end of the article

These authors contributed equally: Ping Zhao, Meng-Meng Ji

Edited by G. Calin

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malignancies¹⁴, rendering tumor cell resistant to chemotherapeutic agents¹⁵. As a mechanism of action, lncRNAs exhibit diverse aspects regarding tumor progression through regulating chromatin organization and gene transcription in the nucleus. LncRNAs also modulate messenger RNA (mRNA) stability, translation, and post-translational modification in the cytoplasm¹⁶,¹⁷. However, pathogenic role and underlying molecular mechanism of lncRNAs remain largely unknown in PTCL.

In the present study, we performed genome-wide lncRNA expression assay in PTCL and identified a novel lncRNA (ENST00000503502) associated with lymphoma progression and poor prognosis in patients, designating it as T cell lymphoma-associated lncRNA1 (TCLlnc1). The biological function of TCLlnc1 on T-lymphoma cell proliferation and migration was evaluated in vitro and in vivo.

# Materials and methods

## Patients and clinical specimens

Patients with newly diagnosed, histologically confirmed PTCL treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)-based chemotherapy between June 2013 and October 2019 were eligible and enrolled in this study. Exclusion criteria of patients were as previously described¹⁸. One hundred and thirty-eight PTCL patients were included and their characteristics were summarized in Supplementary Table 1. The study was approved by the Shanghai Ruijin Hospital Review Board with informed consent obtained from all patients in accordance with the Declaration of Helsinki. Pathological diagnosis was established according to the World Health Organization classification¹⁹.

## Transcriptomic sequencing

Total RNA was extracted from high-quality frozen tumor samples of 40 PTCL cases (from 70 PTCL cases detected for TCLlnc1 levels) using RNA RNeasy Mini Kit (Qiagen, Dusseldorf, German) for RNA sequencing (RNA-seq) and analyzed as previously described²⁰⁻²⁵.

## Cell lines and culture conditions

T-leukemia/lymphoma cell lines Jurkat, Hut78, and embryonic kidney cell line HEK-293T were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured as previously described²⁶.

## Cell cycle assay

Jurkat and Hut78 cells were transfected with TCLlnc1 plasmid or small interfering RNA (siRNA). A total of 1 × 10⁶ Jurkat and Hut78 cells were harvested after 72 h of incubation, stained with propidium iodide (MultiSciences Bio- tech, Hangzhou, China), and analyzed by flow cytometry²⁶.

## Cell proliferation assay

A total of 5 × 10³ Jurkat and Hut78 cells were seeded onto 96-well plates per well. After incubation for the indicated time (0, 24, 48, and 72 h), proliferation was measured using Cell Counting Kit-8 (Dojindo, Japan) according to the manufacturer's protocol²⁷.

## Transwell assay

Transwell assay was performed as previously described²⁸, the migrated cells in the lower compartments were counted by flow cytometry, and images of the cells fixed on the bottom surface of the filter were taken under the microscope.

## Quantitative real-time PCR

RNA expression levels were measured using quantitative real-time polymerase chain reaction (qRT-PCR) as previously described²⁶. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control and Hut78 cells for calibration²⁹⁻³¹. The primers (Supplementary Table 2) were provided by Biosune Bio- technology Company (Shanghai, China).

## Western blot

Western blot analysis was performed as previously described³². Rabbit anti-heterogeneous nuclear ribonucleoprotein D (HNRNPD) antibody (12382S, Cell Signaling Technology, Danvers, MA, USA) and rabbit anti-Y-box binding protein-1 (YBX1) (9744S, Cell Signaling Technology) antibody were used for western blot. The dilution of primary antibodies was 1:1000. An anti-GAPDH antibody was used as the endogenous loading control.

## Fluorescence in situ hybridization (FISH)

The expression of TCLlnc1 in Jurkat cells was examined by using in situ hybridization assay³³, with anti-TCLlnc1 oligodeoxynucleotide probe sets (lncRNA FISH Probe Mix, Sangon Biotech, Shanghai, China).

## In vitro transcription

Full length of TCLlnc1 and antisense TCLlnc1 were cloned from the plasmid of TCLlnc1 into downstream of the T7 promoter using Phanta EVO HS Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China). TCLlnc1 and antisense TCLlnc1 with T7 promoter was purified with Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). In vitro transcription assays were performed using MEGAscript™ T7 Transcription Kit according to the manufacturer's instructions (Invitrogen).

## RNA pull-down assay

RNA pull-down assays were performed with Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo) according to the manufacturer's instructions³⁴.
RNA immunoprecipitation

RNA immunoprecipitation (RIP) experiments were
performed using the Magna RIPTM Kit (Millipore, Bill-
erica, MA, USA). The co-precipitated RNAs were detec-
ted by RT-PCR. To demonstrate that the detected RNA
signals specifically bind to either HNRNPD or YBX1, the
total RNA (input controls) and normal rabbit IgG con-
trols were simultaneously assayed.

Plasmids, lentivirus construction, and small interfering
RNAs

Full-length TCLnc1 was cloned into the vector pLenti
CMV-MCS-EF1α-ZsGreen1-PGK-Puro for over-
expression (Lingke Biotech, Shanghai, China). All short
hairpin RNA (shRNA) lentivirus and siRNAs involved in
the study were constructed by Lingke Biotech. All siRNA
sequences are provided in Supplementary Table 3.

Co-immunoprecipitation (Co-IP)

Manual immunoprecipitation was performed according
to Pierce™ Classic Magnetic IP/Co-IP Kit User Guide
(Thermo). Both the input and co-IP samples were ana-
lyzed by western blot analysis using anti-HNRNPD or
anti-YBX1 antibodies (Cell Signaling Technology).

Luciferase reporter assay

The designated combinations of indicated plasmids and
other relevant siRNAs plus pRL-TK-Renilla were trans-
fected into HEK-293T cells with the Lipofectamine 3000
reagent (Invitrogen), and luciferase activity was detected
using the Dual-Luciferase Reporter Assay System
(Promega).

In vivo experiments

Female BALB/c nude mice 5 to 6 weeks of age were
purchased from the Shanghai Laboratory Animal Center
(Shanghai, China). All experiments were performed in
accordance with relevant institutional and national
guidelines and the regulations of the Shanghai Medical
Experimental Animal Care Commission. The subject was
approved by the Laboratory Animal Ethics Committee of
Ruijin Hospital affiliated with Shanghai Jiao Tong Uni-
versity School of Medicine. In vivo experiments were
performed as previously described²⁶.

Statistical analysis

All statistical analyses were performed using SPSS 21.0
(IBM, Armonk, NY, USA). The significance of the dif-
ferences between groups was estimated using either the
Student's *t* test, *χ*² test, or Mann–Whitney *U* test.
Progression-free survival (PFS) and overall survival (OS)
were calculated using the Kaplan–Meier method with the
log-rank test for comparisons. The Cox regression and
proportional hazards models were used for univariate/

multivariate analysis. A value of *P* < 0.05 indicated a sig-
nificant difference.

Results

lncRNA candidate ENST00000503502 is clinically relevant
in PTCL

LncRNA expression profile was examined in tissue
samples of ten PTCL and ten reactive hyperplasia (RH)
using Arraystar Human LncRNA Microarray v2.0. In total,
33,045 lncRNAs and 30,215 mRNAs were collected from
the authoritative databases RefSeq, UCSC Known Genes
dataset, Ensembl, and related literatures, as per the man-
ufacturer's instructions. Heatmap displayed differentially
expressed lncRNAs and mRNAs in PTCL, as compared to
RH (Fig. 1A). To identify potential oncogenic lncRNAs,
more stringent filtering criteria (raw signal intensity >2000,
fold change >2, P < 0.01, 200 nt < RNA length < 1000 nt,
Fig. 1B) were used and 54 upregulated lncRNAs were
obtained; lncRNA-mRNA co-expression network analysis
was performed using the Cytoscape software. The top
five lncRNAs (ENST00000394174, ENST00000432567,
ENST00000503502, ENST00000456305, and NR_033390)
with more co-expressed oncogenes (according to the
Catalogue Of Somatic Mutations In Cancer [COSMIC,
https://cancer.sanger.ac.uk] database) in PTCL than in RH
were identified as candidate lncRNAs and assessed by qRT-
PCR (Supplementary Fig. 1A). As shown in Supplementary
Fig. 1B, ENST00000503502 expression level was sig-
nificantly higher in PTCL tissues compared with RH tis-
sues (P = 0.0004). Thus, among the candidate lncRNAs,
ENST00000503502 presented a significant increase in
PTCL and had the strongest correlation with the expression
of oncogenes, which mainly involved in the cell cycle,
cytokine-related pathways, metastasis, and chemotherapy
resistance (Fig. 1C); thereby, ENST00000503502 was desig-
nated TCLlnc1, and further exploration of clinical significance
and biological functions should be explored. TCLnc1 was
located in chromosome 4 (chr4:139,546,266–139,546,727)
with a 462 nt length and identified as a lncRNA rather than a
protein-coding transcript by CPC (Coding Potential Calcul-
lator, http://cpc.cbi.pku.edu.cn). An analysis was performed
on the coding potential using CPAT (Coding Potential
Assessment Tool, http://lilab.research.bcm.edu/cpat), and the
results indicated that TCLnc1 was unlikely to encode any
protein product (Supplementary Fig. 1C).

In order to validate the microarray data, TCLlnc1
expression was detected by qRT-PCR in tumor samples of
70 PTCL patients, including 9 cases of ALK+ALCL, 6
cases of ALK-ALCL, 23 cases of AITL, and 32 cases of
PTCL-NOS, as well as 16 cases of RH. Compared with RH
(0.224 ± 0.101), the expression level of TCLnc1 was sig-
nificantly increased in the main subtypes of PTCL
(ALK+ALCL, 0.292 ± 0.077, P = 0.0272, ALK-ALCL,
2.075 ± 0.682, P = 0.0005, AITL, 0.982 ± 0.379, P =
<figure><img src="image_4.png" /></figure>

0.0005, and PTCL-NOS, 1.386 ± 0.365, *P* = 0.0002, Fig. 2A). Moreover, the expression of TCLlnc1 in serum positively correlated with TCLlnc1 in tumors of PTCL patients (*P* < 0.0001, Fig. 2B). TCLlnc1 expression was then assessed in enlarged serum samples of 138 PTCL patients, including 18 cases of ALK<sup>+</sup>ALCL, 12 cases of ALK<sup>-</sup>ALCL, 53 cases of AITL, and 55 cases of PTCL-NOS, as well as in 10 cases of RH and 10 cases of healthy volunteers (HVs). Serum TCLlnc1 was significantly higher in PTCL patients (ALK<sup>+</sup>ALCL, 0.651 ± 0.134, *P* < 0.0001,

ALK<sup>-</sup>ALCL, 3.940 ± 1.625, *P* < 0.0001, AITL, 1.879 ± 0.335, *P* < 0.0001, and PTCL-NOS, 2.986 ± 0.457, *P* < 0.0001) than in HV (0.076 ± 0.030), no significant difference was observed between RH and HV (*P* = 0.5288, Fig. 2C).

As shown in Table 1, 138 PTCL patients were divided into high- and low-expression groups based on the median value of TCLlnc1. High TCLlnc1 expression group was significantly associated with extranodal involvement (*P* = 0.0152), high-risk International Prognostic Index (IPI) (*P* = 0.0063), and poor chemotherapy
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<table><caption>**Table 1** Clinicopathological characteristics of PTCL patients according to TCLlnc1 expression in serum (n = 138)ª.</caption><thead><tr><th>Variables</th><th>Low TCLlnc1<br>(n = 69)</th><th>High TCLlnc1<br>(n = 69)</th><th>P value</th></tr></thead><tbody><tr><td>Gender</td><td></td><td></td><td>0.4650</td></tr><tr><td>Female</td><td>20 (29%)</td><td>24 (35%)</td><td></td></tr><tr><td>Male</td><td>49 (71%)</td><td>45 (65%)</td><td></td></tr><tr><td>Age (years)</td><td></td><td></td><td>0.1677</td></tr><tr><td>&lt;60</td><td>44 (64%)</td><td>36 (52%)</td><td></td></tr><tr><td>≥60</td><td>25 (36%)</td><td>33 (48%)</td><td></td></tr><tr><td>ECOG</td><td></td><td></td><td>0.8127</td></tr><tr><td>0-1</td><td>58 (84%)</td><td>59 (86%)</td><td></td></tr><tr><td>≥2</td><td>11 (16%)</td><td>10 (14%)</td><td></td></tr><tr><td>LDH level</td><td></td><td></td><td>0.4823</td></tr><tr><td>Normal</td><td>24 (35%)</td><td>28 (41%)</td><td></td></tr><tr><td>Elevated</td><td>45 (65%)</td><td>41 (59%)</td><td></td></tr><tr><td>Extranodal<br>involvement</td><td></td><td></td><td>0.0152</td></tr><tr><td>&lt;2</td><td>48 (70%)</td><td>34 (49%)</td><td></td></tr><tr><td>≥2</td><td>21 (30%)</td><td>35 (51%)</td><td></td></tr><tr><td>Ann Arbor stage</td><td></td><td></td><td>0.2359</td></tr><tr><td>I-II</td><td>20 (29%)</td><td>14 (20%)</td><td></td></tr><tr><td>III-IV</td><td>49 (71%)</td><td>55 (80%)</td><td></td></tr><tr><td>IPI</td><td></td><td></td><td>0.0063</td></tr><tr><td>Low risk</td><td>45 (65%)</td><td>29 (42%)</td><td></td></tr><tr><td>High risk</td><td>24 (35%)</td><td>40 (58%)</td><td></td></tr><tr><td>Response to<br>treatment</td><td></td><td></td><td>0.0006</td></tr><tr><td>CR</td><td>41 (59%)</td><td>21 (30%)</td><td></td></tr><tr><td>Non-CR</td><td>28 (41%)</td><td>48 (70%)</td><td></td></tr></tbody></table>

ECOG Eastern Cooperative Oncology Group, LDH Lactic dehydrogenase, IPI International Prognostic Index, CR complete remission.

ªχ² test.

response (P = 0.0006). Accordingly, median PFS and OS of the patients in the high TCLlnc1 expression group were 10 and 20 months, respectively; significantly shorter than those of the low TCLlnc1 expression group (34 months, P = 0.0003, Fig. 2D, and not arrived, P < 0.0001, respectively, Fig. 2E). Univariate analysis showed that multiple extranodal involvements, advanced stage, and high levels of serum TCLlnc1 were adverse prognostic factors for both PFS and OS. When using multivariate analysis, high level of serum TCLlnc1 and advanced stage were independent adverse prognostic factors for both PFS and OS (Fig. 2F, G).

## TCLlnc1 promotes T-lymphoma cell proliferation and migration in vitro

To further determine the biological function of TCLlnc1 in PTCL, overexpression and knockdown of TCLlnc1 were performed using TCLlnc1 plasmid and siRNA on T-lymphoma cell line Jurkat and Hut78. The expression level of TCLlnc1 was measured by qRT-PCR, and Jurkat cells expressed a higher level of TCLlnc1 than those of Hut78 cells (0.076 ± 0.004 vs. 0.033 ± 0.003, P = 0.0013, Fig. 3A). Jurkat and Hut78 cells transfected with TCLlnc1 plasmid showed higher expression levels of TCLlnc1 than cells transfected with vectors (Jurkat: 0.401 ± 0.090 vs. 0.076 ± 0.004, P = 0.0230, Fig. 3B; Hut78: 0.474 ± 0.038 vs. 0.033 ± 0.003, P = 0.0003, Supplementary Fig. 2A). After a 72-h culture, overexpression of TCLlnc1 significantly increased cell proliferation, as compared to vector cells (Jurkat: 1.471 ± 0.085 vs. 0.927 ± 0.150, P = 0.0341, Fig. 3C; Hut78: 2.230 ± 0.042 vs. 1.602 ± 0.136, P = 0.0116, Supplementary Fig. 2B). Flow cytometric analysis revealed that TCLlnc1 decreased tumor cell arrest in the G0/G1 phase (Jurkat, TCLlnc1 vs. control vector: 41.240 ± 0.413% vs. 46.980 ± 0.927%, P = 0.0048; Hut78, TCLlnc1 vs. control vector: 22.810 ± 0.550% vs. 31.170 ± 0.260%, P = 0.0002), with an obvious increase in S-phase cells (Jurkat, TCLlnc1 vs. control vector: 46.350 ± 1.524% vs. 40.000 ± 1.176%, P = 0.0299, Fig. 3D; Hut78, TCLlnc1 vs. control vector: 51.250 ± 1.407% vs. 46.260% ± 0.806%, P = 0.0372, Supplementary Fig. 2C). Meanwhile, overexpression of TCLlnc1 significantly promoted cell migration, as compared to vector cells (Jurkat, 12190 ± 1067 vs. 8672 ± 1060, P = 0.0341, Fig. 3E; Hut78, 26,500 ± 4162 vs. 12,702 ± 1179, P = 0.0332, Supplementary Fig. 2D). T-lymphoma cells with TCLlnc1 knockdown had significantly lower TCLlnc1 expression levels than siRNA-NC cells (Jurkat, 0.023 ± 0.002 vs. 0.063 ± 0.005, P = 0.0020, Fig. 3F; Hut78, 0.005 ± 0.001 vs. 0.014 ± 0.001, P = 0.0001, Supplementary Fig. 2E). Knockdown of TCLlnc1 significantly reduced cell proliferation (Jurkat, 0.927 ± 0.076 vs. 1.710 ± 0.104, P = 0.0036, Fig. 3G; Hut78, 1.320 ± 0.141 vs. 1.845 ± 0.058, P = 0.0261, Supplementary Fig. 2F), decreased S-phase cells (Jurkat, 25.200 ± 1.199% vs. 44.700 ± 3.794%, P = 0.0080, Fig. 3H; Hut78, 34.250 ± 1.831% vs. 43.610% ± 1.717%, P = 0.0203, Supplementary Fig. 2G) and retarded cell migration (Jurkat, 21,070 ± 8331 vs. 51,844 ± 6899, P = 0.0117, Fig. 3I; Hut78, 5647 ± 1150 vs. 13673 ± 2442, P = 0.0410, Supplementary Fig. 2H).

## TCLlnc1 promotes T-lymphoma cell proliferation in vivo

In order to search for in vivo evidence of TCLlnc1 on lymphoma progression, murine xenograft models were established with subcutaneous injection of Jurkat cells stably transfected with pLenti-TCLlnc1 or pLenti-Vector (as control), as well as pLenti-shRNA-TCLlnc1 or pLenti-
<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 4** TCLinc1 promoted T-lymphoma cell proliferation in vivo. A Tumor volumes and weights of mice injected with pLenti-Vector- and pLenti-TCLinc1-transfected Jurkat cells. B Immunohistochemistry analysis of Ki-67 in tumors of mice injected with pLenti-Vector- and pLenti-TCLinc1-transfected Jurkat cells. C Tumor volumes and weights of mice injected with pLenti-shRNA-ct- and pLenti-shRNA-TCLinc1-transfected Jurkat cells. D Immunohistochemistry analysis of Ki-67 in tumors of mice injected with pLenti-shRNA-ct- and pLenti-shRNA-TCLinc1-transfected Jurkat cells.</figcaption></figure>

shRNA-ct (as control). The sizes and weights of pLenti-TCLinc1 tumors were significantly larger than those of pLenti-Vector tumors (Fig. 4A). Tumors of pLenti-TCLinc1 exhibited increased positivity of Ki-67 (Fig. 4B). Accordingly, knockdown of TCLinc1 reduced tumor growth, thus affecting the weight of pLenti-shRNA-TCLinc1 tumors, as compared to pLenti-shRNA-ct tumors (Fig. 4C). Tumors of pLenti-shRNA-TCLinc1 exhibited decreased positivity of Ki-67 (Fig. 4D).

TCLinc1 activates tumor growth factor (TGF-β) signaling and regulates cytokine production

To better understand the underlying molecular
mechanism of TCLinc1, we performed RNA-seq analysis to

obtain the transcriptional profiles in tumor samples of
PTCL patients according to TCLinc1 expression. Forty
PTCL patients were divided into high- and low-expression
groups based on the median value of TCLinc1. A total of
1115 differentially expressed transcripts (fold change ≥1.5,
P < 0.05) were identified; these included 372 upregulated
and 743 downregulated transcripts (Fig. 5A). Gene ontol-
ogy (GO) analysis showed that the most significantly
altered biological processes included pathways involved in
cytokine production, cell–cell adhesion, and cytokine
secretion (Fig. 5B). Gene set enrichment analysis (GSEA)
revealed that the gene sets were significantly related to
regulation of tumor necrosis factor (TNF) biosynthetic
process, cellular response to TGF-β stimulus, and
<figure><img src="image_8.png" /></figure>

regulation of epithelial to mesenchymal transition (Fig. 5C). Clinically, the correlation between the expression levels of TCLnc1 and cytokines, such as interleukin-6 (IL-6), IL-10, and TNF in serum (Fig. 5D) of PTCL patients indicated that TCLnc1 activated TGF-β signaling pathway and regulated cytokine production in PTCL.

## TCLnc1 interacts with HNRNPD and YBX1 as a modular scaffold

LncRNA exerts biological function by physically interacting with transcriptional factors, histone regulators, and

other cellular factors³⁵. FISH assay showed that TCLnc1 was mainly located in the nucleus of Jurkat cells (Fig. 6A), suggesting that TCLnc1 may function at the transcriptional level via interacting with nucleus molecules or proteins. To further explore the underlying molecular mechanism, biotinylated TCLnc1 and antisense TCLnc1 RNA (negative control) were incubated with total protein extracts from Jurkat cells and pulled down with streptavidin. Only HNRNPD and YBX1 were detected by mass spectrometry from three independent RNA pull-down assays (Fig. 6B) and immunoblotting (Fig. 6C).
<figure><img src="image_9.png" /></figure>
Physical interaction of TCLnc1 with HNRNPD and YBX1 was validated by RIP using anti-HNRNPD antibody and anti-YBX1 antibody, with linc00152 as a negative control. TCLnc1 is specifically bound to HNRNPD and YBX1 in Jurkat cells (Fig. 6D). In addition, qRT-PCR and western blot assays showed that knockdown of TCLnc1 had no effect on HNRNPD and YBX1 mRNA expression or protein expression. The knockdown of HNRNPD and YBX1 had no effect on TCLnc1 expression (Fig. 6E). These data indicated that TCLnc1 physically interacted with HNRNPD and YBX1. Subsequently, a series of TCLnc1 deletion mutants were constructed to map the precise binding regions for HNRNPD and YBX1. According to in vitro RNA pull-down assays, segment 1–324 nt of TCLnc1 interacted with HNRNPD, while segment 216–462 nt of TCLnc1 interacted with YBX1 (Fig. 6F). Moreover, as revealed by co-IP, HNRNPD, and YBX1 interacted with each other in Jurkat cells and could be attenuated by knockdown of TCLnc1 (Fig. 6G). Together, TCLnc1 was important for HNRNPD and YBX1 complex formation as a modular scaffold.

## TCLnc1 functions with HNRNPD and YBX1 complex in regulating TGF-β signaling pathway

Because lncRNA regulates downstream genes expression by interacting with transcription factors, we next investigated which of the TGF-β signaling pathway genes are regulated by the TCLnc1, HNRNPD and YBX1 complex. As shown by qRT-PCR in Jurkat cells, the relative mRNA expression level of *TGF-β2* (*TGFB2*) and *TGF-β receptor I* (*TGFBR1*) was significantly upregulated by overexpression of TCLnc1 (*TGFB2*: 1.004 ± 0.064 vs. 2.471 ± 0.350, *P* = 0.0146; *TGFBR1*: 1.049 ± 0.044 vs. 2.105 ± 0.323, *P* = 0.0316), HNRNPD (*TGFB2*: 1.004 ± 0.064 vs. 2.311 ± 0.229, *P* = 0.0054; *TGFBR1*: 1.049 ± 0.044 vs. 1.653 ± 0.061, *P* = 0.0013), and YBX1 (*TGFB2*: 1.004 ± 0.064 vs. 2.777 ± 0.240, *P* = 0.0020; *TGFBR1*: 1.049 ± 0.044 vs. 1.920 ± 0.087, *P* = 0.0009) (Fig. 7A), but was downregulated by knockdown of TCLnc1 (*TGFB2*: 1.004 ± 0.065 vs. 0.338 ± 0.093, *P* = 0.0042; *TGFBR1*: 1.000 ± 0.225 vs. 0.446 ± 0.060, *P* = 0.0010), HNRNPD (*TGFB2*: 1.004 ± 0.065 vs. 0.620 ± 0.023, *P* = 0.0051; *TGFBR1*: 1.000 ± 0.225 vs. 0.548 ± 0.016, *P* < 0.0001), and YBX1 (*TGFB2*: 1.004 ± 0.065 vs. 0.509 ± 0.034, *P* = 0.0025; *TGFBR1*: 1.000 ± 0.225 vs. 0.4777 ± 0.047, *P* = 0.0006) (Fig. 7B). Other key genes of TGF-β signaling pathway were not altered by TCLnc1, HNRNPD, or YBX1, such as *TGF-β1* (*TGFB1*), *TGF-β3* (*TGFB3*), *TGF-β receptor II* (*TGFBR2*), and *SMAD2*, *SMAD3*, *SMAD4* (Supplementary Fig. 3A). qRT-PCR analysis of *TGFB2* and *TGFBR1* expression was performed in the pLenti-Vector, pLenti-TCLnc1-, pLenti-shRNA-NC-, and pLenti-shRNA-TCLnc1-transfected Jurkat cells, revealing similar results in vivo (Supplementary Fig. 3B).

To finally confirm the direct regulation of TCLnc1/HNRNPD/YBX1 complex to *TGFB2* and *TGFBR1* expression, luciferase assays were performed. As expected, HNRNPD and YBX1 activated *TGFB2* and *TGFBR1* transcription by binding to the promoters of *TGFB2* and *TGFBR1* (Fig. 7C). The knockdown of HNRNPD or YBX1 reversed TCLnc1-mediated cell proliferation (Fig. 7D) and migration (Fig. 7E). These data demonstrated that TCLnc1 exerted its function by binding to HNRNPD and YBX1. TCLnc1/HNRNPD/YBX1 complex regulated key gene transcription to activate TGF-β signaling pathway, induce cytokine production, and promote cell proliferation and invasion in PTCL (Fig. 7F).

## Discussion

LncRNAs are aberrantly expressed in various cancers and actively participate in disease progression¹². In the present study, TCLnc1 was identified as a lncRNA overexpressed in the main PTCL histological subtypes linked to both lymphoma cell proliferation and in vitro and in vivo migration. Of note, circulating lncRNAs have potential utility as noninvasive and blood-based biomarkers³⁶. Accordingly, we revealed that serum TCLnc1 expression correlated with tumor TCLnc1 expression and was significantly associated with extranodal involvement, high-risk IPI, and independently indicated poor patients' clinical outcomes. Therefore, not only do our findings support the essential role TCLnc1 plays during tumor progression, but we also provide a potential biomarker for PTCL disease prognosis prediction.

TGF-β signaling pathway represents one of the best-recognized cascades modulating tumor proliferation and migration³⁷. We showed that TCLnc1 overexpression led to the activation of TGF-β signaling pathway and production of cytokines, such as IL-6, IL-10, and TNF. The results are consistent with the previous study regarding cutaneous T cell lymphoma, stating that TGF-β and IL-10 are hallmarks of advanced stages, increased cell migration, and decreased antitumor responses³⁸. TGF-β is composed of three isoforms, TGFB1, TGFB2, and TGFB3. In order to activate the TGF-β signaling pathway, TGF-β isoforms bind to TGFB2, thereby recruiting TGFB1 and upregulate downstream cascade³⁷. Here, *TGFB2* and *TGFBR1* were potential targets of TGF-β signaling activation provoked by TCLnc1 overexpression. Galuniertib, a small-molecular inhibitor of TGFB1, has recently been reported to possess efficient tumor inhibitory activity in breast cancer³⁹ and multiple myeloma⁴⁰. Based on the fact that TCLnc1 promoted both tumor growth and the migration of PTCL via TGF-β signaling activation, therapeutic targeting of TGF-β may counteract tumor progression in PTCL with TCLnc1 overexpression.

LncRNAs interact with RNA-binding proteins to form nuclear ribonucleoprotein complexes, which are crucial
<figure><img src="image_10.png" /></figure>
regulators of transcriptional programs in the nucleus<sup>41,42</sup>. Our results demonstrated that TCLlnc1 directly inter-
acted with HNRNPD and YBX1 and acts as a modular
scaffold in PTCL. HNRNPs represent an RNA-binding
ribonucleoprotein family of transcription activators that
are implicated in RNA stability and gene expression reg-
ulation in various cancers<sup>41</sup>. In colorectal cancer,
HNRNPD interacts with LINC01354 and contributes to
proliferation and metastasis through the activation of the
Wnt/β-catenin signaling pathway<sup>43</sup>. YBX1 is also a tran-
scription activator of the RNA- and DNA-binding
domain. The RNA-binding domain can bind to lncRNA
or mRNA, whereas the DNA-binding domain binds to a
consensus sequence in the target genes promoter, impli-
cating numerous cellular processes, particularly tumor-
igenesis<sup>44</sup>. More importantly, in multiple cancer types, S-
phase-enriched lncRNAs interact with HNRNPK-YBX1
complex and affect cancer cell hallmarks through reg-
ulating phosphatidylinositol-3-kinase/AKT and MAPK
signaling activation<sup>45</sup>. Our findings indicated that
HNRNPD and YBX1 are required for TCLlnc1-mediated
regulation of TGF-β signaling pathway, as well as T-
lymphoma cell proliferation and migration. The results
further strengthen the understanding of lncRNA as an
oncogenic driver on PTCL progression.

Conclusions

TCLlnc1 is a clinically, functionally, and mechanistically
oncogenic lncRNA in PTCL. TCLlnc1 and its down-
stream signaling pathway may be meaningful for risk
stratification and targeted therapy in patients with PTCL.

Acknowledgements

We appreciate the effort of the physicians in enrolling patients and thank all the patients involved for allowing us to analyze their clinical data.

Author details

¹Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China. ²Department of Pathology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China. ³Pôle de Recherches Sino-Français en Science du Vivant et Génomique, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China. ⁴U1165 Inserm/Université Paris 7, Hôpital Saint Louis, Paris, France

Author contributions

P.Z. and M.-M.J. carried out the experiments and wrote the manuscript. Y.F. performed qRT-PCR and analyzed data. X.L. and Z.-X.Y. prepared biological samples. S.C., P.-P.X., and A.J. gathered detailed clinical information for the study and analyzed the data. H.-M.Y. and C.-F.W. performed pathological research. C.-F.W., L.W., and W.-L.Z. conceived the study, directed and supervised research, and wrote the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported, in part, by research funding from the National Natural Science Foundation of China (81670176, 81520108003, and 81830007); the Chang Jiang Scholars Program; the Shanghai Municipal Education Commission Gaofeng Clinical Medicine Grant Support (20152206 and

20152208); Clinical Research Plan of Shanghai Hospital Development Center (SHDC2020CR1032B); Shanghai Science and Technology Development fund (19MC1911000); the Multicenter Clinical Research Project by Shanghai Jiao Tong University School of Medicine (DLY201601); the Collaborative Innovation Center of Systems Biomedicine; and the Samuel Waxman Cancer Research Foundation.

Data availability

The datasets used and/or analyzed during the current study are available from
the corresponding author on reasonable request.

Consent for publication

All authors have agreed to publish this manuscript.

Conflict of interest

The authors declare no competing interests.

Ethics approval and consent to participate

The study was approved by the Shanghai Ruijin Hospital review board with
informed consent obtained in accordance with the Declaration of Helsinki.
Animals were used according to the protocols approved by Shanghai Ruijin
Hospital Animal Care and Use Committee.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s41419-021-03594-y.

Received: 7 October 2020 Revised: 8 February 2021 Accepted: 22 February 2021

Published online: 25 March 2021

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