The long non-coding RNA LINC01013 enhances invasion of human anaplastic large-cell lymphoma

Anaplastic large-cell lymphoma (ALCL) is a rare type of highly malignant, non-Hodgkin lymphoma (NHL). Currently, only studies on the chimeric oncogene NPM-ALK have reported a link to ALCL progression. However, the specific molecular mechanisms underlying the invasion of ALCL are still unclear. Here, we sought to investigate differentially expressed, long non-coding RNAs (lncRNAs) in ALCL and their potential biological function. Our microarray analyses revealed that LINC01013, a novel non-coding RNA gene, was highly expressed in clinical specimens of ALCL and was significantly upregulated in invasive ALCL cell lines. Knockdown of LINC01013 suppressed tumor cell invasion; conversely, its overexpression enhanced tumor cell invasion. LINC01013-induced invasion was mediated by activation of the epithelial-to-mesenchymal transition (EMT)-associated proteins, snail and fibronectin. Specifically, LINC01013 induced snail, resulting in activation of fibronectin and enhanced ALCL cell invasion. Collectively, these findings support a potential role for LINC01013 in cancer cell invasion through the snail-fibronectin activation cascade and suggest that LINC01013 could potentially be utilized as a metastasis marker in ALCL.

expressed in various kinds of cancer, and the expression levels of certain lncRNAs are associated with disease progression or diagnosis, or might serve as potential therapeutic targets [12][13][14] . Further, the clinical significance of the contribution of lncRNAs to ALCL development and the key roles of lncRNA functional networks are currently unknown.
In this study, we investigated the invasion activity involving ALK and the molecular mechanisms underlying the pathogenesis of ALK(+) ALCL. We found that several lncRNA genes were highly expressed in ALCL clinical specimens and hypothesized that their functions were associated with cancer progression. Specifically, our microarray analysis revealed that LINC01013 (long intergenic non-protein-coding RNA 1013), a novel lncRNA that has not been linked to human cancer in the literature, is overexpressed in clinical specimens of ALCL and is significantly up-regulated in invasive ALK(+) ALCL cell lines. We further investigated and characterized this aberrantly expressed lncRNA in ALCL to determine whether it can be used as a novel biomarker and/or therapeutic target for ALCL.

LINC01013 depletion suppresses ALCL cell invasion.
To determine the effects of LINC01013 on cell invasion, we established LINC01013-knockdown KASPAS-299 and KASPAS-invasive cell lines. Notably, KASPAS-299 and KASPAS-invasive cell lines depleted of LINC01013 displayed significantly decreased invasion (~2-fold) compared with control cells (Fig. 3a). The epithelial-to-mesenchymal transition (EMT)-associated markers, snail and fibronectin, were also significantly decreased in LINC01013-depleted cells compared with control cells (Fig. 3b,c). Snail, a transcription factor associated with cancer metastasis, regulates fibronectin to promote lymphoma cell invasion. Similarly, depletion of snail in KASPAS-299 and KASPAS-invasive cells decreased invasion ability compared with control cells (Fig. 4a), and markedly down-regulated fibronectin (Fig. 4b,c). The expression levels of other Snail targets or EMT regulators have no effect between control and snail-depleted cells (Fig. S1) or control and LINC01013-depleted cells (Fig. S2). Taken together, these data confirm the requirement of LINC01013 to accelerate tumor cell invasion and implicate the snail-fibronectin cascade in this process.
LINC01013 promotes ALCL cell invasion by activating the snail pathway. To further confirm the involvement of snail activation in LINC01013-induced phenotypic changes, we performed invasion assays using LINC01013-overexpressing cells. Notably, LINC01013-overexpressing SR-786 cells (SR-786-LINC01013) displayed significantly increased invasion (~2-3-fold) compared with parental SR-786 cell controls ( Fig. 5a,b, left panel), and this phenotype was attenuated by snail depletion (Fig. 5a, right panel). SR-786-LINC01013 cells also exhibited marked upregulation of snail and fibronectin, an increase that was specifically blocked by depletion of snail (Fig. 5b, right panel, and c). Collectively, these results support a potential role of LINC01013 in promoting cancer cell invasion through activation of the snail-fibronectin cascade in ALCL (Fig. 5d).

Discussion
No recent studies have reported an association between lncRNAs and ALCL progression or, by extension, the involvement of an lncRNA-mediated mechanism in the invasion of ALCL. To identify specific pathways underlying ALCL carcinogenesis, we used an oligonucleotide microarray analysis of ncRNA genes to identify lncRNAs that were differentially expressed in ALCL. We further evaluated clinical specimens for aberrantly expressed lncR-NAs to identify potential biomarkers of ALCL invasion. Notably, we found that the novel lncRNA, LINC01013, is highly expressed in ALCL specimens and showed that its expression is positively correlated with the invasivity of ALK(+) cells. Gain-and loss-of-function experimental strategies clearly demonstrated that LINC01013 enhanced ALK(+) ALCL cell invasion. Thus, LINC01013 may be a novel oncogene that could serve as an invasion marker for ALK(+) ALCL metastases.
LncRNAs are important regulators of gene expression and are thought to have a wide range of functions in cellular and developmental processes [16][17][18] . The functions of a few lncRNAs have been experimentally defined 19,20 . These studies have demonstrated the involvement of lncRNAs in fundamental processes of gene regulation, including chromatin modification, direct transcriptional regulation, RNA processing, post-translational regulation of protein activity or localization, and miRNA modulation. In the current study, we demonstrated that LINC01013 promotes an increase in mRNA and protein expression levels of snail and fibronectin, indicating the involvement of lncRNA in the transcriptional regulation of downstream genes.
Scientific RepoRts | 7: 295 | DOI:10.1038/s41598-017-00382-7 The mesenchymal marker snail is a core transcription factor in the EMT process that stimulates expression of the downstream target gene encoding fibronectin to enhance cancer invasion 21 . To date, few studies have linked the regulation of lncRNA and snail/fibronectin in lymphoma. LINC01013 enhances invasion abilities in ALK(+) ALCL cell lines and may stimulate metastasis through activation of snail-fibronectin components. Our findings are consistent with the conclusion that activation of these mesenchymal markers leads to ALCL cell metastasis.
Previous studies of ALCL progression have focused on NPM1-ALK translocation and its relative contribution to pathogenesis 22 . Fusions of ALK have oncogenic potential because their aberrant tyrosine kinase activity enhances cell proliferation and survival and promotes cytoskeletal earrangement 23 . NPM1-ALK-interacting molecules ultimately lead to the activation of key pathways, including RAS/ERK (extracellular signal-regulated kinase), phospholipase C (PLC-γ), phosphoinositide 3-kinase (PI3K), and JAK/STAT (signal transducer and activator of transcription) pathways 24,25 . Activation of STAT3 is associated with a specific signature that includes several transcription factors (e.g., CEBP/β), cell cycle (e.g., cyclin D, c-myc) and survival/apoptosis molecules (e.g., Bcl-A2, Bcl-XL, survivin, MCL-1), and cell-adhesion proteins 26 . These studies have demonstrated the regulation of coding genes and their cross-talk in ALCL tumor formation. Several reports have shown that miRNAs, a type of short non-coding RNA, regulate ALCL malignancies. For example, miR-29a expression was found to modulate apoptosis through inhibition of MCL-1 expression in ALCL 27 ; ectopic expression of miR-150 inhibits proliferation and blocks S-phase entry of ALK(+) cells 28 ; and downregulation of miR-16 induces vascular endothelial growth factor (VEGF) expression in ALK(+) ALCL 29 . However, the detailed role of long non-coding genes in the pathogenesis of ALCL is still unclear and may represent an important avenue for future research.
In conclusion, this study is the first to demonstrate the involvement of an lncRNA in ALCL tumor progression. The results provide new insights into the mechanism by which the lncRNA, LINC01013, contributes to the promotion of ALCL cell invasion, showing that it acts through activation of the snail-fibronectin cascade. Our findings collectively support a potential role of LINC01013 in ALCL progression and suggest that LINC01013 expression could be effectively utilized as a metastatic marker in ALCL.

Materials and Methods
Ethics statement. All   Immunoblot analysis. Total cell lysates and conditioned media were prepared, and protein concentrations were determined using a Bradford assay kit (Pierce Biotechnology, Rockford, IL, USA). Equivalent amounts of protein were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel. Separated proteins were transferred to a nitrocellulose membrane (pH 7.9; Amersham Biosciences Inc., Piscataway, NJ, USA), blocked with 5% non-fat powdered milk, and incubated with specific anti-snail (Cell Signaling Technology, Cell Signaling, Danvers, USA; #6615) and anti-fibronectin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-9068) primary antibodies at 4 °C overnight. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse, anti-rabbit or anti-goat IgG secondary antibody, as appropriate, for 1 h at room temperature. Immune complexes were visualized using an enhanced chemiluminescence (ECL) detection kit (Amersham) and Fuji X-ray film. Establishing SR-786 cell lines stably overexpressing LINC01013. The SR-786 cell line, grown in 10-cm cell culture dishes, was transfected with the LINC01013 expression plasmid using the Lipofectamine reagent (Invitrogen). After 24 h, transformants were selected from transfected cells by growing in medium containing the antibiotic neomycin G418 (400-800 μg/ml) for 2-3 weeks. Survival clones were pooled and used to western blot or functional assay. Expression levels of LINC01013 RNA in the selected clones were determined using quantitative reverse transcription-PCR (q-RT-PCR).
shRNA-mediated LINC01013 knockdown. Short hairpin RNA (shRNA) sequences targeting LINC01013 were purchased from the National RNAi Core Facility (Institute of Molecular Biology, Academia Sinica, Taiwan). The KARPAS-299 and KARPAS-invasive cell lines were transiently transfected with shRNA targeting the endogenous LINC01013 gene using the TurboFect reagent (Invitrogen). LINC01013 repression was confirmed by q-RT-PCR. shRNA-mediated snail knockdown. Short hairpin RNA (shRNA) sequences targeting snail were purchased from the National RNAi Core Facility (Institute of Molecular Biology, Academia Sinica, Taiwan). The SR-786, SR-786-LINC01013, KARPAS-299 and KARPAS-invasive cell lines were transiently transfected with shRNA targeting endogenous snail mRNA using the TurboFect reagent (Invitrogen). Snail repression was confirmed by Western blot analysis.
Invasion assays. The influence of LINC01013 on invasion ability in vitro was determined by Transwell assay (Falcon BD, Franklin Lakes, NJ) using LINC01013-depleted KARPAS-299 or LINC01013-overexpressed SR-786 cells, as described previously 15 . Briefly, cell density was adjusted to 10 5 cells/ml, and 100 μl of the suspension was seeded into upper chambers of the Transwell plate, either coated (invasion) or not coated (migration) with Matrigel (Becton-Dickinson). For both assays, the pore size of the upper chamber was 8 mm. The medium in the upper chamber was serum-free DMEM, and the lower chamber contained DMEM supplemented with 20% FBS, included as a chemoattractant. After incubation for 24 h at 37 °C, cells traversing the filter from the upper to lower chamber were stained with crystal violet and counted. Experiments were repeated at least three times.
Statistical analysis. Data are expressed as mean values ± SEM of at least three experiments. Statistical analyses were performed using Student's t test and one-way analysis of variance (ANOVA). Where appropriate, the Mann-Whitney U test or Fisher's exact test was used to compare two groups; a Kruskal-Wallis test or Pearson's χ 2 test was used if more than two groups were compared. The relationship between the results of two different examinations was analyzed with Spearman's correlation test. P-values < 0.05 were considered statistically significant.