Downregulation of the lncRNA RP11-432I5.4 inhibits tumorigenesis in the JAK2V617F-positive classic myeloproliferative neoplasms CURRENT STATUS: POSTED

Background: Although aberrant expression of long non-coding RNA (lncRNA) is associated with many human cancers, little is known about its role in the JAK2V617F-positive classic myeloproliferative neoplasms (cMPNs). Methods: In this study, we performed a comprehensive analysis of lncRNAs in human cMPNs cells using a lncRNA cDNA microarray and identified the lncRNA RP11-432I5.4 acted as an effective factor in JAK2V617F-positive cMPN cells. Results: The lncRNA RP11-432I5.4 showed higher expression in cMPN patients, especially higher in JAK2V617F-positive cells compared with JAK2V617F-negative cells. Overexpression of lncRNA RP11-432I5.4 increased the proliferation of JAK2V617F-positive cells while downregulation of it decreased proliferation, promoted apoptosis and triggered S phase arrest of JAK2V617F-positive cells. Furthermore, in a mouse xenograft model, the silencing of lncRNARP11-432I5.4 repressed tumor formation in vivo . Conclusions: Taken together, these results revealed that lncRNA RP11-432I5.4 plays an important role in cMPN tumorigenesis and may be a potential novel target for treatment of JAK2V617F-positive cMPN patients. them, lncRNA RP11-432I5.4 (NCBI accession number HG508247), was identified to be greatly upregulated in JAK2V617F-positive MPN samples. Loss- or gain-of-function analysis indicated that lncRNA RP11-432I5.4 promoted tumorigenesis and progression, playing a vital role in JAK2V617F-positive cMPNs. This study demonstrated the mechanism of how lncRNA RP11-432I5.4 influences JAK2V617F-positive cMPNs and provides a potential strategy for the treatment of JAK2V617F-positive cMPNs.

Mammalian genomes are pervasively transcribed to produce thousands of long non-coding RNAs (lncRNAs) (12,13). LncRNAs are transcripts longer than 200 nucleotides that have little or no proteincoding capacity (14,15). Nuclear lncRNAs can silence or activate gene expression in cis or in trans manner (16), guide DNA methylation and histone modification (17), interplay with nuclear domains (18), perform gene-control through sequestration (19) and regulate higher-order chromosomal interactions (20). Aberrant expression of many lncRNAs is involved in the progression of CML, a BCR-ABL-positive MPN. For example, in CML, lncRNA-BGL3 functions as a competitive endogenous RNA for binding microRNAs to cross-regulate phosphatase and tensin homolog (PTEN) expression, and Bcr-Abl-mediated cellular transformation critically requires silence of tumor-suppressor lncRNA-BGL3 (21).
Overexpression of lncRNA-H19 contributes to the progression of CML by enhancing cell survival, colony formation, and inhibiting cell apoptosis (22). LncRNA X-inactive specific transcript (Xist) is required for hematopoietic stem cell survival and function. Deleting Xist causes marrow fibrosis, leukemia, and histiocytic sarcoma (23). However, whether lncRNAs contribute to the pathogenesis and progression of BCR-ABL-negative cMPNs, especially JAK2V617F-positive cMPNs, remains to be explored. Hence, we undertook a comprehensive study examining lncRNA expression in JAK2V617Fpositive cMPN patients and querying the functional consequences of lncRNA expression.
In this study, using lncRNA microarray analysis, we found that numerous lncRNAs are differentially expressed in JAK2V617F-positive cMPN patients compared to their JAK2V617F-negative counterparts.

Patients and Cell lines
A total of 12 samples from cMPN patients (six JAK2V617F-positive and six JAK2V617F-negative) were included in the lncRNA microarray experiment. All patients were recruited from Shanghai Tongji Hospital. The cMPN diagnosis was defined according to World Health Organization (WHO) criteria (24).
All subjects had given written informed consent in accordance with the Declaration of Helsinki.
Three cell lines were used in the study. HEL, a human JAK2V617F-expressing cell line, and K562 (a human wild type JAK2-expressing cell line) were cultured in RPMI-1640 and IMDM medium (Gibco, Grand Island, NY, USA) respectively with 10% fetal bovine serum at 37℃ in a humidified atmosphere of 5% CO 2 . 293T cells were cultured in high-glucose Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO 2 .

LncRNA microarray analysis
The lncRNA cDNA microarray was from Arraystar (Arraystar, Rockville, MD, USA). Bone marrow mononuclear cells aspirated from the 12 cMPN patients were isolated by Ficoll-Hypaque density gradient centrifugation. Total RNA of every patient was extracted from bone marrow mononuclear cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instruction.
Integrity and concentration of RNA were assessed after RNA extraction and prior to sample labeling. Agilent Quick Amp Labeling Kit was used for sample labeling. Hybridization was performed in Agilent's SureHyb Hybridization Chambers. After washing, slides were scanned with the Agilent DNA Microarray Scanner. Data was extracted using Agilent Feature Extraction software. Further data analysis was performed using Agilent GeneSpring GX v11.5.1 software. Subgroup analysis was performed using home-made scripts. The cDNA synthesis, labelling, hybridization, and data analysis were carried out as previously described (25).
Quantitative real-time polymerase chain reaction (qRT-PCR) Specific quantitative real-time PCR (qRT-PCR) assays for lncRNA RP11-432I5.4 were performed and used GAPDH as the internal control. Reverse transcription was performed to synthesize cDNA. qRT-  shRNA lentiviral or the control lentiviral were subcutaneously injected into both back flanks of male nude mice (6 weeks old of age and around 20 g in weight). Tumor growth was monitored and measured in weight at the indicated days post-inoculation by a caliper. Thirty-four days after injection, mice were sacrificed and xenograft tumor weight was measured.

Statistical analysis
All data represented are the mean ± standard deviation from at least 3 independent experiments. The data was analyzed with two-tailed Student's t tests, and differences with p < 0.05 were considered statistically significant.

Results
The

Consent for publication
Not applicable.

Availability of data and materials
The data used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.