Prediction of lncRNA HCP5 encoding products using bioinformatics analysis
To identify the lncRNA HCP5 encoded peptides or proteins in breast cancer, we integratively analyzed HCP5 open reading frame (ORF), ribosome profile (Ribo-seq) and MS/MS data of MDA-MB-231 cell line (Fig. S1). LncRNA HCP5 sequence (GRCh38 fasta format) was obtained from NCBI Gene . Ribo-seq data (GSE69923) and MS/MS data (PXD008222) were downloaded from Gene Expression Omnibus (GEO) database  and EMBI-EBI-PRIDE database , respectively.
HCP5 ORFs were identified by ORFfinder  in NCBI based on HCP5 exon sequence. We screened ORFs with start codon “ATG” in the positive strand as candidates. Then, we obtained Ribo-seq files of SRA format and converted them to fastq format by using fastq-dump tool (https://ncbi.github.io/sra-tools/fastq-dump.html). Next, adaptor sequences were trimmed using Trim Golare (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Reads shorter than 25 bp after adaptor trimming were discarded. rRNA sequences were filtered by using RNAcentral database . Furthermore, the remaining reads were aligned to reference genome GRCh38 using TopHat2  and bam format file was obtained. The ORFs fewer than 400 nt with reads mapped were regarded as convinced HCP5 small ORFs (sORFs). In addition, MS/MS raw data was converted to mgf format by MSConvert . Then, Peppy was used to get amino acid (aa) sequences for peptides or proteins and aligned to GRCh38. Finally, the convinced HCP5 sORFs with MS/MS peptides mapped were validated as highly convinced HCP5 sORFs.
Breast tumor tissue microarrays (TMA) were obtained from Shanghai Outdo Biotech Co. (Shanghai, China). The TMA HBre-Duc140Sur-01 contains 140 cases of invasive ductal carcinomas and 45 pared precancerous breast tissues from the regions around cancers. No patients received adjuvant radiotherapy, chemotherapy, or immunotherapy before surgery. The experimental protocols were approved by The Human Research Ethics Committee from Medical College of Southeast University.
Immunohistochemistry staining (IHC)
The tissue sections were dried at 60°C for 1 h then dewaxed in xylene and rehydrated through graded alcohol concentrations using standard procedures. Antigen retrieval was performed in citrate buffer (pH 6.0) and autoclave at 121°C for 90 s. After washing in PBS (3 min × 3), sections were blocked with goat serum (Boster, Wuhan, China) in the room temperature for 30 min. Then each section was treated with HCP5-132aa mouse polyclonal antibodies (B01, Abnova, Inc.; at a dilution of 1:200 solution) at 4°C overnight. After washing in PBS (5 min × 3), each section was incubated with Polink-1 HRP DAB Detection System One-step polymer detection system for mouse antibody (ZSGB-BIO, Beijing, China) at room temperature for 20 min. After washing in PBS (3 min × 3), the slides were counterstained with hematoxylin. For negative controls, the primary antibody was substituted with PBS.
Reagents and cell viability assay
Ferrostatin-1 (Fer-1; SML0583) was obtained from Sigma-Aldrich, Liproxstatin-1 (Lip-1; S7699), RLS3 (S8155), Sulfasalazine (SULF; S1576) and Erastin (S7242) was obtained from Selleck. cn. MDA-MB‐231 cells (5×104/mL) were seeded in 96‐wells plates with 100 µL per well for 24 hs, then 0.099 µM, 0.197 µM, 0.375 µM, 0.625 µM, 1.25 µM, 2.5 µM, 5 µM, 10 µM and 20 µM RLS3, Sulfasalazine and Erastin were added in to plates. After 16 h, 24 h and 48 h, 10 µL of the Cell Counting Kit‐8 (CCK‐8) reagent was added into each well and incubated in 37°C, 5% CO2 for 2 hours. Then cell growth was detected and calculated the inhibition rate.
Cell culture and lentivirus-mediated transduction of shRNA
Human breast cancer cell lines MDA-MB-231 and MDA-MB-468 cell lines were cultured in L-15 medium with 10% fetal bovine serum (FBS), in 37°C incubator without CO2.
The procedure of lentivirus infection is as follows: the plate containing cells was added with appropriate amount of lentivirus in concentration gradient, followed by adding 1/1000 polybrene to enhance infection. The sequence of the HCP5-132aa shRNA was showed in Table S1. Lentivirus vector LV5 containing full-length HCP5-132aa or empty vector were purchased from GenePharma Co., Ltd. (Shanghai, China).
Cell proliferation assays
MDA-MB‐231 cells and MDA‐MB‐468 cells were seeded in 96‐wells plates and transfected with HCP5-132aa, Vector, HCP5-132aa shRNA and NCs, respectively. After transfection for 48 hours, 10 µL of the CCK‐8 reagent was added into each well, incubated in 37°C, 5% CO2 for 2 hour, and cell growth was detected by an enzyme labeling instrument at 450 nm.
Colony formation assays
For colony formation assays with monolayer culture, MDA-MB‐231 cells (0.4 × 103/well) and MDA‐MB‐468 cells (0.4 × 103 /well) were plated in a 6‐wells plates for two weeks. After fixed with methanol, the cells were stained with 0.1% crystal violet 30 min and then the colonies were imaged and counted.
For transwell migration, 1 × 105 /ml cells were suspended in 200 µl medium without bovine serum albumin into upper chamber of 24-well transwell plates (8 µm pore size; Corning), and 500 µl of medium containing 10% FBS was added to the lower chambers. After 24 h co-culture, the cells on the lower surface of membrane were fixed in 4% paraformaldehyde, stained by 0.1% crystal violet. The stained cells were then counted under light microscope. Photographs of random fields across three replicate wells by 200 times magnification were captured for analysis.
RNA extraction and quantitative reverse transcription PCR
TRIzol (Invitrogen, USA) was used to isolate the total RNA of tissues, and cDNA was synthesi-zed with PrimeScriptTM RT Master Mix (Takara Biomedical Technology Co., Ltd, Beijing, China). A qTOWER2.0 Real-Time PCR System (Analytik Jena AG) with ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China) was used to perform quantitative PCR (qPCR). Relative mRNA expression was standardized using the housekeeping GAPDH. The following human primers purchased from Sangon Biotech. (Shanghai, China) were as follows: HCP5-132aa R: 5’- GAGGCATGGCTGCTGTCACAC-3’, F: 5’- TGGCTGGACGATTCTCCTCACAC-3’; GAPDH R: 5’-TCTCGCTCCTGGAAGATGGTGAT-3’, F: 5’- CGGAGTCAACGGATTTGGTCG-3’. The procedures were performed in triplicate.
MDA-MB-231 and MDA-MB-468 cells transfected with vector, HCP5-132aa, shHCP5-132aa or shNC were harvested and lysed with cell lysis buffer for western blotting (Beyotime, Shanghai, China). The proteins (30 µg per lane) were separated on 12% SDS-polyacrylamide gels and transferred into polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Primary antibodies for mouse-anti-AMID/AIFM2 (B-6) (1:500) (sc-377120, Santa cruze, Inc.), mouse-anti-FTH1 (1:500) (sc-376594, Santa cruze, Inc.), mouse-anti-ACSL4 (1:500) (sc-365230, Santa cruze, Inc.), rabbit-anti-GPX4 (1:1000) (ab125066, Abcam, USA); HCP5-132aa (1:1500) (Huabio, China) and rabbit-anti-β-actin (1:1000) (Bioss Antibodies, 10 Inc.) were incubated at 4°C overnight. Binding of the primary antibody was detected using an enhanced chemiluminescence kit (ECL Amersham). The Image J software was used to quantify and analyze each specific western blot band.
Lipid ROS assays
Lipid ROS levels were determined using C11-BODIPY dye (D3861, ThermoFisher Scientific) according to the manufacturer’s instructions. Cells were seeded in six-wells plates overnight, then the culture medium was replaced with Erastin (15 µM), RSL3 (1.25 µM), Liproxstatin-1 (50 µM) or Ferrostatin-1 (1 µM) treatment for 48h. Then the medium was replaced with 5µM C11-BODIPY-containing medium for 1 h. Later, the cells were harvested by trypsin and washed three times with ice-cold PBS followed by re-suspending in PBS plus 1% BSA. The amount of ROS within cells was examined by flow cytometry analysis (FACSCantoTM II, BD Biosciences).
For the iron assay, we used an Iron Assay Kit (MAK025-1KT, Sigma Aldrich) to measure Fe2+ or total iron in each cell line. First, 2 × 106 of cells was rapidly homogenized in 4 ~ 10 volumes of Iron Assay buffer. Samples were centrifuged at 13,000 × g for 10min at 4°C to remove insoluble material. To measure ferrous iron, 5 µl of iron assay buffer was added to each well. Samples were mixed well using a horizontal shaker or by pipetting and the reactions were incubated for 30 min at room temperature in dark conditions. Then, 100 µL of Iron Probe was added to each well containing standard or test samples. Samples were mixed well using a horizontal shaker or by pipetting and the reactions were incubated for 60 min at room temperature in dark conditions. Finally, the absorbance was measured at 593 nm (A593).
Cells were fixed in 4% formaldehyde for 30 min at room temperature before cell permeabilization with 0.1% Triton X-100 (4°C, 10 min). Cells were saturated with PBS containing 2% bovine serum albumin for 1 h at room temperature and processed for immunofluorescence with 1mg/ml C11-BODIPY followed by 10mg/ml Hoechst 33258 (Invitrogen, Carlsbad, CA, USA). Between all incubation steps, cells were washed three times for 3 min with PBS containing 0.2% bovine serum albumin. Fluorescence signals were analyzed using an Olympus Fluoview 1000 confocal microscope (Olympus Corp, Tokyo, Japan).
Transmission electron microscopy (TEM)
Cells were collected and fixed in 2.5% glutaraldehyde for at least 3 h. Then the cells were treated with 2% paraformaldehyde at room temperature for 60 min and 0.1% glutaraldehyde in 0.1 M sodium cacodylate for 2 h, followed by post-fixing with 1% OsO4 for 1.5 h. After a second washing, cells were dehydrated with graded acetone and finally embedded in Quetol 812. Ultrathin sections were observed under an H7500 electron microscope (Hitachi, Tokyo, Japan).
Xenograft assay in Nude mice
For each experiment, 24 mice were randomly divided into the following four groups: (1) control shRNA model receiving DMSO (vehicle); (2) control shRNA model receiving Erastin; (3) HCP5-132aa shRNA model receiving DMSO (vehicle) and (4) HCP5-132aa shRNA model receiving Erastin. Indicated subcutaneously injected into the dorsal flanks right of the midline in nude mice (female, 4 ~ 6 weeks). At day seven, mice were intraperitoneal injected with Erastin (50 mg/kg i.v., two times a week) for three weeks. Erastin was dissolved in the vehicle (2% DMSO and 98% PBS) and prepared by Ultrasonic Cleaner (Fisher Scienti c, Hampton, NH). A final volume of 300 µL of Erastin was applied via intraperitoneal injection. And 16 mice were randomly divided into the following two groups: (1) HCP5-132aa shRNA model receiving DMSO (vehicle) and (2) HCP5-132aa shRNA model receiving RSL3. At day seven, mice were intraperitoneal injected with RSL3 (100 mg/kg i.v., two times a week) for three weeks. RSL3 was dissolved in the vehicle (2% DMSO, 2% Tween80, 30% PEG300 and 66% PBS) and prepared by Ultrasonic Cleaner (Fisher Scienti c, Hampton, NH). A final volume of 200 µL of RLS3 was applied via intraperitoneal injection. Tumors were measured twice a week. The volumes were calculated using the following formula: volume (mm3) = length × width2 × π/6.
All statistical analyses were performed using SPSS 19.0 software (IBM Corp, Armonk, NY, USA). Results are expressed as the mean ± standard deviation (SD). Group means were compared using Student’s t-test for independent data. All P-values are two-tailed, and P < 0.05 was considered to indicate statistical significance. The chi-square test was used to compare HCP5-132aa expression between breast cancer tissues and paired breast tissues and the association with clinicopathologic parameters. Survival analyses were estimated using the Kaplan–Meier method.