LINC00152 expression in normal and Chronic Lymphocytic Leukemia B cells

Abstract Long non‐coding RNAs are emerging as essential regulators of gene expression, but their role in normal and neoplastic B cells is still largely uncharacterized. Here, we report on the expression pattern of the LINC00152 in normal B cells and Chronic Lymphocytic Leukemia B cell clones. Higher LINC00152 levels were consistently observed in memory B cell populations when compared to naïve B cells in the normal tissues analyzed [peripheral blood (PB), tonsils, and spleen]. In addition, independent stimulation via Immunoglobulins (IG), CD40, or Toll‐like Receptor 9 (TLR9) upregulated LINC00152 in PB B cells. The expression of LINC00152 in a cohort of 107 early stage Binet A CLL patients was highly variable and did not correlate with known prognostic markers or clinical evolution. TLR9 stimulation, but not CD40 or IG challenge, was able to upregulate LINC00152 expression in CLL cells. In addition, LINC00152 silencing in CLL cell lines expressing LINC00152 failed to induce significant cell survival or apoptosis changes. These data suggest that, in normal B cells, the expression of LINC00152 is regulated by immunomodulatory signals, which are only partially effective in CLL cells. However, LINC00152 does not appear to contribute to CLL cell expansion and/or survival in a cohort of newly diagnosed CLL patients.


| INTRODUCTION
Non-coding RNAs (ncRNAs) constitute more than 90% of the RNAs transcribed from the human genome. 1 The vast majority of the annotated ncRNAs have been discovered only in the past 10 years and are still largely uncharacterized (1)(2)(3). Once considered a byproduct of transcriptional noise, ncRNAs possess instead critical regulatory functions in gene expression. [1][2][3] Among ncRNAs, long non-coding RNAs (lncRNAs) are arbitrarily classified as >200 nucleotides long species, account for most of this pervasive transcription, [1][2][3] and are strongly cell-and tissue-restricted although not well evolutionarily conserved. 1,3,4 LncRNAs exert their roles in many cellular functions operating through different mechanisms. Their versatility mainly depends on their subcellular localization and the adoption of various structural modules with interacting partners. 2,5 By regulating gene expression at epigenetic, transcriptional, and post-transcriptional levels, lncRNAs are involved in fundamental cellular processes such as proliferation and apoptosis, development and differentiation, as well as human diseases, including cancer. 6 Chronic Lymphocytic Leukemia (CLL) accounts for 30% of all cases of leukemia and is the most common form in adults in Western countries. 7 CLL is characterized by the proliferation and accumulation of CD5-positive B-lymphocytes in the peripheral blood (PB), bone marrow (BM), lymph nodes (LN), and spleen. 8 Clinically, CLL occurs in two forms: indolent, characterized by mutated immunoglobulin heavy chain (IGHV), and aggressive identified by unmutated IGHV genes. 9,10 Additionally, more than 80% of CLL cases present either genomic aberrations or mutations in select genes (e.g., NOTCH1, TP53, BCL2, and ATM). 11 Few studies have investigated the role of lncRNAs in CLL (reviewed in Ref. 12 ). A recent report revealed that the lncRNA treRNA is overexpressed in CLL and is associated with poor prognosis. 13 Similarly, Croce and coworkers reported a strong correlation between lncRNA MIAT expression and disease aggressiveness since lncRNA upregulation protects CLL lymphocytes from apoptosis, ultimately sustaining monoclonal malignant B cell proliferation. 14 Conversely, the expression of lincRNA-p21 is significantly reduced in CLL patients, and low expression of this lncRNA demarcates a more aggressive form of CLL with a poor prognosis. 15 LincRNA-p21 and the paraspeckleenriched lncRNA NEAT1 are involved in the induction of cell death after DNA damage 16 ; however, we recently observed that the expression of NEAT1 is quite heterogeneous in B cells derived from CLL patients irrespectively of cytogenetic groups or clinical outcome. 17 LINC00152, also known as CYTOR (cytoskeleton regulator RNA), is an intergenic lncRNA initially reported to control cell cycle progression through the interaction with a network of proteins associated with the M phase of the cell cycle, thus acting as a noncoding oncogene. 18 Indeed, LINC00152 is significantly overexpressed in the vast majority of human cancers according to the TCGA datasets (http://ualcan.path.uab.edu/cgi-bin/Pan-cancer-lncRNA.pl? genenam=CYTOR), and several studies demonstrated that its overexpression facilitates tumor progression and invasion (reviewed in Ref. 19 ). On this basis, it has been proposed that LINC00152 may represent an effective target for diagnostic, prognostic, and therapeutic purposes in human cancers.
The main aim of this study was to investigate the LINC00152 expression in normal and CLL B cells in response to immunomodulatory stimuli. As an ancillary purpose, the potential prognostic role of LINC00152 was tested in a prospective cohort of newly diagnosed early stage Binet A CLL patients.  Table S1.   The diagnosis was confirmed by flow cytometry analysis together with the determination of CD38 and ZAP-70 expression, IGHV mutational status, and cytogenetic abnormalities as previously described. 23 TP53 and NOTCH1 mutational status was determined by PCR as previously described. 24,25 PBMCs from patients with CLL were isolated as previously reported. 26 In all instances, the percentage of purified B cells (CD19+) exceeded 95%.

| Expression and silencing of LINC00152
Total RNA was isolated using the TriPure reagent (Roche) and retrotranscribed (50 ng) using Transcriptor Reverse Transcriptase (Roche) and random hexamers according to the manufacturer's instructions.
Transfections were carried out for 48 h using Kit V for Nucleofector II (Amaxa) according to the manufacturer's instructions.

| Statistical analysis
The student's t-test was performed for statistical analysis (GraphPad Prism v.6). Mann-Whitney unpaired t-test was used for nonparametric comparisons of data sets (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). The predictive value of LINC00152 as continuous value for discriminating patients who needed therapy from those who did not was investigated by the ROC curve analysis.
An area under the ROC curve close to 0.5 indicates the complete lack of prognostic value of LINC00152. Time to First Treatment (TTFT) and overall survival (OS) analyses were performed using the Kaplan-Meier method. Statistical significance of associations between LINC00152 and TTFT or OS was calculated using the log-rank test.

| Expression of LINC00152 is induced upon stimulation in normal B cells
To assess whether immune-modulatory signals could modify the  Figure 1C).

| Expression of LINC00152 in CLL cell clones
Several lines of evidence indicate that CLL clones have the features of memory B cells. 28,29 Based on this notion, we evaluated the expression of LINC00152 in 107 purified CLL clones collected from a cohort of newly diagnosed Binet stage A patients (O-CLL1 protocol).
The expression of LINC00152 in CLL clones was compared with CD19 + /CD27 − (naïve) and CD19 + /CD27 + (memory) B cells derived from PB. As shown in Figures 2 and S1, the expression of LINC00152 was highly variable among CLL samples and ranged from CLL clones in which LINC00152 expression was higher than in memory B cells to CLL clones in which LINC00152 expression was lower than in naïve B cells. The latter were the majority of the patients studied. Variability of LINC00152 expression was observed both in IGHV-mutated (M)and in IGHV-unmutated (U)-CLL. Although U-CLL clones averaged a higher expression level of LINC00152 than M-CLL, differences were not statistically significant (see Figure S2).

| Expression of LINC00152 does not predict Time to First Treatment (TTFT)
Next, we investigated whether the different amounts of LINC00152    Figure S3 and Table S2). However, in the M-CLL group, CD40L/IL4 stimulation more effectively induced the expression of LINC00152.

| Silencing LINC00152 does not induce cell death in CLL cell line(s)
OSU-CLL cell line, derived from the EBV infection of a leukemic clone of a CLL patient, 27  Under these experimental conditions, despite the specific downregulation of LINC00152, we could not observe any relevant changes in cell viability and apoptosis compared with controls ( Figure 5). Similar results were obtained using the cell line MEC1, derived from a CLL clone ( Figure S4). -45 4 | DISCUSSION LINC00152 has been reported to be abnormally expressed in several types of human cancers (reviewed in Ref. 19 ). In most cases, this information is related to solid cancers, while fewer reports present data regarding the expression of LINC00152 in hematological malignancies. [29][30][31] In addition, to our knowledge, no information is available as to the role and expression of LINC00152 in normal lymphocytes.

F I G U R E 3 (A) Receiver
We first addressed the expression of LINC00152 in B cell subpopulations derived from peripheral blood, tonsils, and spleens of normal donors. In addition, the expression of LINC00152 was investigated in leukemic clones derived from a cohort of newly diagnosed CLL patients.