Cell isolation and culture

Bone marrow samples were collected from four MLL leukemia patients at the time of their initial diagnosis, with informed consent obtained from patients treated at the First Affiliated Hospital of Sun Yat-sen University. Ethical approval for sample collection was granted by the Hospital’s Protection of Human Subjects Committee, and detailed clinicopathological characteristics of the patients can be found in Additional file 1: Table S1.

Human THP1 and MOLM-13 cells (ATCC, USA) were cultured in RPMI-1640 medium (HyClone, USA), while MV4-11 cells (ATCC, USA) were cultured in IMDM (HyClone, USA). All cell cultures were supplemented with 10% fetal bovine serum (HyClone, USA) and maintained at 37 °C in a 5% CO2 atmosphere. The primary cells cultured in IMDM (HyClone) supplemented with 20% FBS.

RNA isolation and quantitative real-time PCR (RT-PCR)

Total RNA was extracted from bone marrow and cell samples using an Invitrogen™ TRIzol™ Kit (Thermo Fisher, USA) according to the manufacturer’s instructions and was stored at -80 °C before RT-PCR. RNA was reverse-transcribed into cDNA with the PrimeScript® RT reagent Kit (Takara, DDR047A, Japan). Quantitative RT-PCR for lncRNA and mRNA was performed using the SYBR Premix ExTaq real-time PCR Kit (Takara, Japan) according to the manufacturer’s instructions. All of the data were normalized to GAPDH expression as a control. The expression level for each lncRNA and mRNA was determined using the 2−△△Ct method. All primers were confirmed by sequencing the PCR product fragments, as shown in Additional file 1: Table S2.

RNA interference and lentivector expression systems

RNA interference using siRNA (Ribobio, China) was performed to knockdown LAMP5-AS1 and LAMP5. Transient transfections of siRNAs were performed using the Neon Transfection System (Invitrogen, USA) with 10 µl reactions according to the manufacturer’s guidelines. Lentiviral shRNA templates were cloned into the pGreenPuro™ shRNA Cloning and Expression Vector (System Biosciences, Germany), and pSIH1-H1-siLuc-copGFP was used as negative control. Tandem mRFP-GFP-LC3 plasmid for autophagy flux was constructed as previously described [31,32,33]. For stable expression assays, lentiviral pGreenPuro™ shRNA vectors and pCDH-CMV-MCS-EF1-Puro eukaryotic expression vector were packaged into lentiviruses using Lentivector Expression Systems (System Biosciences, Germany) consisting of pPACKH1-GAG, pPACKH1-REV, and pVSV-G. Finally, the lentiviruses were transformed into THP1, MV4-11 and MOLM-13 cells, and the transformed cells were then selected with puromycin. The primers for siRNAs and shRNAs were described in shown in Additional file 1: Table S2.

Protein extraction and immunoblotting

Total protein was extracted from bone marrow samples and cells using RIPA lysis buffer (Beyotime Biotechnology, China) with 1× Thermo Scientific™ Halt™ Protease Inhibitor Cocktail (Thermo Fisher, USA) according to the manufacturer’s instructions. For tumor samples, the tumor cells were first homogenized from the tumor tissues by a freezing grinder (LUKYM-II, China), and then, the total protein was extracted from the tumor cells.

For Immunoblotting, all protein samples were suspended in 5x loading buffer and then denatured for 5 min at 100 °C, separated via SDS-PAGE, transferred to PVDF membranes, and blotted. The antibodies are as fellow: MLL1 (BETHYL, A300-086 A-3); LC3B (Sigma, L7543); LAMP5(Sigma, A78501); β-tubulin (Invitrogen, 32-2600); β-actin (Sigma, A2228); GAPDH (Proteintech, 10494-1-AP); DOT1L (CST, 90,878 S); H3(Abcam, Ab1791); H3K79me2(Abcam, Ab3594); H3K79me3(Abcam, Ab2621); IgG (CST, 3900); Goat Anti-Mouse IgG HRP (Thermo Fisher, H10007); Goat Anti-Rabbit IgG HRP (Thermo Fisher, A18903); Goat Anti-Rabbit IgG H&L DyLight® 594(Abcam, ab96885);Goat Anti-Rabbit IgG H&L DyLight® 647 (Abcam, ab150115).

RNA pull-down

We performed pull-down assays as previously described [24, 34]. Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher, USA) was used to perform the pull-down assays with the biotinylated LAMP5-AS1 and LAMP5-AS1 antisense. Finally, the proteins enriched by the biotinylated LAMP5-AS1 and LAMP5-AS1 antisense were resolved via SDS-PAGE and western blotting.

Chromatin immunoprecipitation

ChIP analysis is performed on chromatin extracts from THP1 and MOM-13 cells using a Magna ChIP™ G-Chromatin Immunoprecipitation Kit (17–611) (Merck Millipore, Germany) with dimethyl- and trimethyl-Histone H3 (Lys79) according to the manufacturer’s standard protocol. Rabbit IgG served as the negative control. The fold-enrichment of H3K79me2/3 was quantified by quantitative RT-PCR and calculated relative to the input chromatin. The primers used for ChIP-qPCR analysis were listed in Additional file 1: Table S2.

RNA and DNA fluorescence in situ hybridization (FISH) and DOT1L immunofluorescence

To detect the subcellular location of LAMP5-AS1 RNA on LAMP5 locus, we carried out FISH in THP1 cells using the RiboTM Fluorescent In Situ Hybridization Kit (RiboBio, China). Cells were washed briefly with PBS and then fixed in 4% formaldehyde for 15 min at room temperature. Cells were permeabilized in PBS containing 0.5% Triton X-100 on ice for 5 min and then blocked in the preliminary hybridization solution for 30 min at room temperature after three washes with PBS for 10 min each. Hybridization was carried out using the Cy3-labeling LAMP5-AS1 FISH Probe Mix (RiboBio, China) and Digoxigenin (DIG) -labeling LAMP5 DNA primers (the DNA primers were labeled DIG using the DIG High Prime DNA Labeling and Detection Starter Kit II, Roche, Switzerland) in a humidified chamber at 37 °C for 12–16 h. Cells were rinsed with SSC buffer in accordance with the order 4×, 2×, and 1×. For co-localization studies, after RNA and DNA FISH, cells were fixed again for 5 min in 2% formaldehyde and subjected to immunofluorescence with DOT1L primary antibody (CST, 90,878 S) and followed by DIG-FITC (Abcam, ab119349) and Goat Anti-Rabbit IgG H&L DyLight® 647 (Abcam, ab150115). Finally, cells were observed on a Zeiss7 DUO NLO confocal laser microscope (Carl Zeiss, Germany).

Transmission electron microscopy

TEM images of autophagosomes were obtained from thin sections using a JEM1400 electron microscope (JEOL, Japan). The autophagy fluorescence signals were obtained using an anti-LC3B antibody (Novus Biologicals, Taiwan of China) analyzed by Zeiss7 DUO NLO confocal laser microscope (Carl Zeiss, Germany).

Chromatin isolation by RNA purification (ChIRP) assay

ChIRP assay was performed as previously described [35, 36]. First, a pool of 13 probes would cover the whole LAMP5-AS1 for achieving the maximal capture ratio. All of the single stranded LAMP5-AS1 DNA probes with 3’-Biotin TEG modification. Random DNA probes with 3’-Biotin as the negative control. A total of 200 million cells were harvested and cross-linked by 3% formaldehyde. After wash by PBS, the cells were lysed by the lysis buffer (with fresh Protease Inhibitor, PMSF, and RNasin) to each tube and resuspend the pellet (avoid clumps), proceed immediately to sonication. The samples were added the hybridization buffer and followed by ChIRP procedure. Finally, the RNA, DNA and protein are eluted and detected.

Flow cytometric analysis

For cell apoptosis assay, cells were stained with Annexin V/FITC and propidium iodide (PI) (Lianke, China) and then analyzed by flow cytometry (BD, USA). For GFP positive cells in mice samples, cells were first separated from mice organs such as blood, bone marrow, spleen and liver. These mice-derived cells were washed with cooling PBS and then were analyzed on a BD FACSCelesta analyzer (BD Biosciences, USA).

Animal model

The xenotransplantation experiments were performed as previously described [24, 37, 38]. Five-week-old male NOD/SCID mice were maintained under specific pathogen-free conditions in the Laboratory Animal Center of Sun Yat-sen University. All experimental procedures were performed according to the institutional ethical guidelines for animal experiments. Mice were randomly assigned to three groups of ten mice each. In each group, lentiviral stably transduced sh-NC, sh-LAMP5-AS1 (2 × 106 MOLM-13) were subcutaneously injected into the mice; while 2 × 106 MV4-11 cells transduced sh-NC, sh-LAMP5-AS1 were intravenously (tail vein) implanted mice. For subcutaneously implanted mice, tumor growth was monitored for 2 days each. Three weeks after inoculation, the intravenously implanted mice were sacrificed for analysis. Human cell engraftment (GFP + cell populations) in the bone marrow, peripheral blood, spleen, and liver was evaluated by flow cytometry. The remaining mice were performed the survival assay.

Statistical analysis

Spearmen and Pearson’s correlation coefficient was used to determine the correlation between LAMP5-AS1 and LAMP5 expression. Fisher’s exact test was used to determine the significance of two groups. Mean ± SEM was used to analyzed the three independent experiments. Two-tailed tests were used for univariate comparisons. Kaplan-Meier method with a log-rank test is used to analyze the mice survival. p < 0.05 was considered statistically significant.