Cell culture and human tissues
A549, MCF-7, HCT116, Eca109, and CCC-HIE-2 cells were maintained in DMEM (Biological Industries, #01-052-1ACS; Beit Haemek, Israel) supplemented with 10% fetal bovine serum (FBS, Biological Industries, #04-001-1A; Beit Haemek, Israel) and 1% penicillin-streptomycin (Biological Industries, #03-031-1BCS,Beit Haemek, Israel). H1299, NCI-H1975 and NCI-H226 cells were cultured in RPMI-1640 (Biological Industries, #01-100-1ACS; Beit Haemek, Israel) with 10% FBS and 1% penicillin-streptomycin. CCC-HSF-1 cells were cultured in DMEM/F12 (Biological Industries, #01-172-1ACS; Beit Haemek, Israel) supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were cultured in a humidified incubator at 37 °C and 5% CO2. All cell lines were verified to be free of mycoplasma contamination every 3 months. Individual cell line authentication was confirmed using the AmpFISTR Identifiler PCR Amplification Kit (ThermoFisher Scientific, #4427368) from Applied Biosystems and GeneMarker V1.91 software (SoftGenetics LLC). Information on cell lines is provided in Supplementary Table 6. Formalin-fixed paraffin-embedded (FFPE) normal colon mucosa, colon adenoma, COAD, LUAD and LUSC tissues were retrieved from archives of the Department of Pathology at Shanxi Cancer Hospital (Taiyuan, China). Studies using human tissues were approved by the Human Research Ethics Committees of the Shanxi Cancer Hospital in agreement with the guidelines set forth by the Declaration of Helsinki.
Antibodies and reagents
Information on antibodies and reagents used in this study is provided in Supplementary Tables 7 & 8, respectively.
SiRNAs and short hairpin RNA (shRNA) Oligos
SiRNAs were obtained from GenePharma (Shanghai, China) and transfected using the lipofectamine 3000 Transfection Kit (ThermoFisher Scientific, #L3000-015). ShRNA oligos were purchased from TSINGKE Biological Technology (Beijing, China).
Plasmids
The FH1-tUTG plasmid was a kind gift from A/Professor M. J. Herold (Walter and Eliza Hall Institute of Medical Research, Australia). The pcDNA3.1(+), pGL4.73[hRluc/SV40] and pSin-3×Flag-E2F1 plasmids were kind gifts from Professor Mian Wu (Translational Research Institute, Henan Provincial People’s Hospital and People’s Hospital of Zhengzhou University, Zhengzhou, China). The pEGFP-C1 plasmid was a kind gift from A/Professor Yongyan Wu (Department of Otolaryngology, Shanxi Key Laboratory of Otorhinolaryngology Head and Neck Cancer, the first affiliated hospital, Shanxi Medical University, Taiyuan, China). The pMDLg/pRRE plasmid (#12251), pMD2.g plasmid (#12259) and pRSV-Rev plasmid (#12253) were purchased from Addgene. The pGL3-PLANE-promoter and the pGL3-PLANE-promoter-∆E2F1-BR were purchased from Sangon Biotech (Shanghai, China). Other plasmids used in this study were generated by inserting the PCR products to the pcDNA3.1(+) or pEGFP-C1 vectors. Primers used in the fusion PCR are shown in Supplementary Table 9.
Quantitative PCR (qPCR)
Total RNA was extracted from cultured cells using the Gene JET RNA Purification Kit (ThermoFisher Scientific, #K0731) according to the manufacturer’s instructions. cDNA was synthesized from 1 mg of total RNA using the PrimScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, #RR047A; Dalian, China). Of the resultant cDNA, 12.5 ng was used in the 20 μl qPCR mix, containing 10 ml of TB Green Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa, #RP820A; Dalian, China) and 0.4 mM of each primer. Samples were amplified for 40 cycles using a StepOnePlusTM Real-Time PCR System (ThermoFisher Scientific). 2−ΔΔCT method was used to calculate the relative gene expression levels normalized to the GAPDH housekeeping control. Primer sequences are listed in Supplementary Table 10.
Chromatin Immunoprecipitation (ChIP)
ChIP assays were performed using the ChIP Assay Kit (Beyotime, #P2078; Shanghai, China) according to the manufacturer’s instructions. Briefly, cells were cross-linked with a final concentration of 1% formaldehyde in growth medium for 15 min at 37 °C and quenched by the addition of glycine solution for 5 min at room temperature (RT). Then cells were harvested, lysed and sonicated. After being cleared by centrifugation at 12,000 × g for 10 min at 4 °C, the cell lysate was subjected to a 1:10 dilution and rotated with E2F1, H3K4me3 and H3K27me3 antibodies or corresponding mouse/rabbit normal immunoglobulin (IgG) antibodies at 4 °C overnight. Then, 60 ml of protein A/G agarose beads was added to the antibody-lysate mixture and rotated at 4 °C for an additional 1 hr. Beads were washed, and DNA fragments were eluted, purified and subjected to PCR analysis using the specific primers. PCR products were separated by gel electrophoresis on the 2% agarose gel. Information on antibodies and primers used in this study are shown in Supplementary Tables 7 & 11, respectively.
Luciferase reporter assays
A549 and H1299 cells were transfected with pGL3-PLANE-promoter reporters or pGL3-PLANE-promoter-∆E2F1-BR reporters together with pGL4.73[hRluc/SV40] reporters expressing the renilla luciferase. After 48 hr, firefly and renilla luciferase activities were examined by a Dual-Luciferase® Reporter Assay System (Promega, #E910) with a VARIOSKAN LUX microplate reader. The renilla luciferase activity was used to normalize the firefly luciferase activity.
Colony formation
Cancer cells were seeded in six-well plates at 2,000 cells/well. After growing for further two weeks, cells were fixed with methanol and staining with 0.5% crystal violet. The images were captured with a Bio-Rad GelDoc™ XR + imaging system (Bio-Rad). The percentage and intensity of area covered by crystal violet-stained cell colonies were quantified using ImageJ-plugin “ColonyArea”.
Cell cycle analysis
Cell cycle analysis was performed using the Cell Cycle and Apoptosis Analysis Kit (Meilunbio, #MA0334; Dalian, China) according to the manufacturer’s instructions followed by flow cytometry. Briefly, A549 and H1299 cells transfected with PLANE siRNAs for 48 hr in 24-well plates were harvested and fixed in 75% ethanol at 4 °C overnight. After being centrifuged, cells were incubated in the staining solution at 37 °C in the dark for 30 min. Then cells were subjected to analysis using a flow cytometer (FACSAria, BD Biosciences).
Anchorage-independent cell growth
Cells carrying an inducible PLANE knockdown in response to Dox were seeded in the Ultra-Low attachment 6-well plate (Corning, #3471) at 2,000 cells/well. Cells with or without treatment with doxycycline (Dox) and cessation of Dox treatment were incubated at 37 °C in a humidified incubator until colonies were formed. Colonies were counted under a light microscope29.
In situ hybridization (ISH)
ISH assays were performed using the RNAscopeÒ 2.5 HD Detection Reagent-BROWN (Advanced Cell Diagnostics, #322310) according to the manufacturer’s instructions37, 58. Briefly, FFPE LUSC and LUAD as well as COAD tissue microarrays (#HLug-Squ150Sur-02, #HLugA180Su03, #HColA180Su12) purchased from the Shanghai Outdo Biotech Co., Ltd (China) were deparaffinized in xylene for 5 min at RT twice, followed by dehybridization in 100% alcohol. After being air-dried, the tissue sections were incubated with hydrogen peroxide for 10 min at RT and washed in the distilled water for five times. Then the sections were heated in target retrieval reagent to 100 °C for 20 min, followed by being treated with proteinase K and incubated in hybridization buffer containing probes (Advanced Cell Diagnostics, #570031) at 40 °C for 3 hr. After being washed, the sections were incubated with 3,3′-diaminobenzidine (DAB), and counterstaining was carried out using hematoxylin.
The percentage of positive cells was ranged from 0 to 100%. The intensity of staining (intensity score) was judged on an arbitrary scale of 0 to 4: no staining (0), weakly positive staining (1), moderately positive staining (2), strongly positive staining (3) and very strong positive staining (4). A reactive score (RS) was derived by multiplying the percentage of positive cells with staining intensity divided by 10.
Immunofluorescence (IF)
Cells grown on coverslips were fixed in 4% formaldehyde for 10 min at RT. After being washed using PBS, cells were then permeabilized in blocking buffer for 60 min at RT. Antibodies diluted 1:500 in blocking buffer were incubated with cells overnight at 4 °C. Cells were washed in PBS and incubated with secondary antibodies diluted 1:200 in blocking buffer for 60 min at RT in the dark. After being washed, cells were mounted in the ProLongTM Glass Antifade Mountant with NucBlue reagent (ThermoFisher Scientific, P36981). Images were digitally recorded using a Leica SP8 confocal microscope. Information of antibodies used in this study was shown in Supplementary Table 7.
Subcellular fractionation
Cells were harvested by trypsinization and lysed in hypotonic buffer A (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.15% Triton X-100, cOmplete™, EDTA-free Protease Inhibitor Cocktail) on ice for 15 min. The supernatants after centrifugation at 12,000 × g for 3 min were collected as the cytoplasmic fractions and the pellets were subjected to the nuclear fractionation. The pellets were rinsed with cold PBS once and lysed in an equal volume of buffer B (20 mM Hepes pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5% Triton X-100, cOmplete™, EDTA-free Protease Inhibitor Cocktail) on ice for 15 min. Cytoplasmic and nuclear fractions were centrifuged at 16,000 × g for 20 min to remove the insoluble debris. The supernatants were collected for RNA isolation and immunoblotting analysis.
In vitro transcription
The DNA templates used for in vitro synthesis of PLANE, antisense PLANE and NCOR2 pre-mRNA were generated by PCR amplification from cDNAs using PrimerSTAR Max DNA Polymerase (TAKARA, #R045A; Dalian, China). Forward primers containing the T7 RNA polymerase promoter sequence and reverse primers without the promoter sequence were used for synthesizing PLANE, antisense PLANE, and NCOR2 pre-mRNA. After PCR amplification, the products were purified using a MiniBEST Agarose Gel DNA Extraction Kit (Takara, #9762; Dalian, China), and subjected to in vitro transcription using a TranscriptAid T7 High Yield Transcription Kit (ThermoFisher Scientific, #K0441) according to the manufacturer’s instructions. The in vitro-transcribed RNAs could be further labelled with biotin using a PierceTM RNA 3' End Desthiobiotinylation Kit (ThermoFisher Scientific, #20163). Primer sequences are shown in Supplementary Table 11.
Domain-specific chromatin isolation by RNA purification (dChIRP)
dChIRP assays were performed as previous described59. Briefly, A549 and H1299 cells were harvested and cross-linked in 1% glutaraldehyde for 10 min at RT with rotation. The cross-linked cells were lysed in lysis buffer (50 mM Tris-Cl [pH 7.0], 10 mM EDTA, 1% SDS, PMSF, Superase-in), followed by sonication. 4 µg antisense / sense biotin-labelled probes or 10 µg in vitro-transcribed biotin-labelled PLANE / antisense PLANE were rotated with cell lysates at 37 °C for 4 hr, followed by adding 100 µl C-1 magnetic beads (Invitrogen, #65002) to each sample and incubating at 37 °C for 30 min with rotation. Beads were then washed in wash buffer for five times, followed by RNA isolation. Probe sequences are shown in Supplementary Table 11.
Biotin RNA pull-down (RPD)
A549 and H1299 cells were harvested and washed in PBS for three times. Cell pellets were then lysed in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2.5 mM MgCl2, 1 mM EDTA, 10% Glycerol, 0.5% Nonidet P-40/Igepal CA-630, 1 mM DTT, cOmplete™ EDTA-free Protease Inhibitor Cocktail and RNase inhibitors) and sonicated. 4 µg antisense / sense biotin-labelled probes were incubated with lysates at 4 °C overnight before rotating with streptavidin beads (ThermoFisher Scientific, #20349) for additional 2 hr. Beads were then washed in lysis buffer for four times, followed by RNA isolation and immunoblotting analysis. Information of antibodies and probes is shown in Supplementary Tables 7 & 11, respectively.
Mass spectrometry (MS) analysis
Proteins co-pulled down with RNA using antisense / sense biotin-labelled probes were separated by 10% acrylamide gels and visualized by Coomassie brilliant blue staining. The specific protein band shown in the group using antisense probes along with the corresponding region in the group using sense probes were resected and digested, followed by the liquid chromatography–mass spectrometry (LS-MS) analysis using a mass spectrometer (ThermoFisher Scientific, EASY-nLC1000 & LTQ Orbitrap Velos Pro). Proteins identified from the mass spectrometry analysis are listed in Supplementary Table 4.
RNA immunoprecipitation (RIP)
RIP assays were performed using a Magna RIPTM Kit (Millipore, #17-700; Darmstadt, Germany) according to the instruction provided by the manufacturer. Briefly, cell lysates prepared in hypotonic buffer supplemented with RNase inhibitor and protease inhibitor were incubated with magnetic beads pre-incubated with hnRNPM antibodies at 4 °C overnight. After being washed with RIP wash buffer, the bead-bound immunocomplexes were subjected to immunoblotting analysis and RNA isolation. Information on antibody and primers used in this study is shown in Supplementary Tables 7 & 11.
Immunoprecipitation (IP)
Cells were collected with trypsinization and lysed with lysis buffer (20 mM Tris-HCl pH 8.6, 100 mM NaCl, 20 mM KCl, 1.5 mM MgCl2, 0.5% NP-40, cOmplete™ EDTA-free Protease Inhibitor Cocktail) on ice for 1 hr and centrifuged at 16,000 × g for 30 min. After quantification using a BCA protein assay kit (ThermoFisher, #23225), 3 mg of total protein were rotated with antibodies at 4 °C overnight. Protein-antibody complexes were then captured with the PierceTM Protein A/G Agarose (ThermoFisher Scientific, #20421) at 4°C for 2 hrs with rotation and beads were then rinsed with wash buffer (25 mM Tris, 150 mM NaCl, pH 7.2), boiled and subjected to immunoblotting analysis. Antibodies used in this study are shown in Supplementary Table 7.
Absolute quantification of PLANE
Absolute RNA quantification was performed using the standard curve method by qPCR. cDNA was synthesized using 1 mg of the total RNA extracted from a fixed cell number. Ten-fold serial dilutions of the pcDNA3.1-PLANE plasmid (102 to 107 molecules per ml) were used as a reference molecule for the standard curve calculation. Assays were reconstituted to a final volume of 20 µl using 5 µl cDNA from cells or 5 µl serial diluted pcDNA3.1-PLANE plasmid and cycled using a StepOnePlusTM Real-Time PCR System. Data calculated as copies per 5 µl cDNA were converted to copies per cell based on the known input cell equivalents. Primer sequences used are listed in Supplementary Table 10.
Inducible shRNA knockdown
The FH1-tUTG inducible knockdown vector was digested using BsmBII (NEW ENGLAND BioLabs, #R0580S) and XhoI (NEW ENGLAND BioLabs, #R0146S) enzymes, and the annealed shRNA oligos were inserted into the digested vector using the T4 DNA ligase (ThermoFisher Scientific, #EL0014). The lentiviral particles were packaged via co-transfection of FH1-tUTG vector inserted with shRNA oligos (44 µg), pMDLg/pRRE plasmid (22 µg), pMD2.g plasmid (13.2 µg) and pRSV-Rev plasmid (11 µg) plasmids into HEK293T cells60. A549 or H1299 cells were transduced with the lentiviral particles in 6 cm cell culture dishes to establish inducible knockdown cell sublines. The knockdown of PLANE was induced in response to doxycycline treatment. ShRNA sequences are shown in Supplementary Table 12.
Xenograft mouse model
A549 cells expressing the inducible PLANE shRNAs were subcutaneously injected into the dorsal flanks of 4-week-old female nude mice (6 mice per group, Shanghai SLAC Laboratory Animal Co. Ltd., China). Tumor growth was measured every 3 days using a calliper. Mice were sacrificed after 33 days of cancer cell transplantation. Tumors were excised and measured. Studies on animals were conducted in accordance with relevant guidelines and regulations and were approved by the Animal Research Ethics Committee of the first affiliated hospital, Shanxi Medical University and Shanxi Cancer Hospital and Institute (China). All mice were housed in a temperature-controlled room (21-23 °C) with 40–60% humidity and a light/dark cycle of 12 h/12 h.
Statistical Analysis
Statistical analysis was carried out using the GraphPad Prism 8 to assess differences between experimental groups. Statistical differences were analyzed by two-tailed Student’s t-test or one-way ANOVA test followed by Tukey’s multiple comparisons. P values lower than 0.05 were considered to be statistically significant.
Data Availability
The RNA sequencing data have been deposited in the NCBI Gene Expression Omnibus database under the accession code GSE162215. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD022747. The long noncoding RNA expression data and E2F1 mRNA expression data referenced during the study are available in a public repository from the Cancer RNA-seq Nexus dataset (http://syslab4.nchu.edu.tw/). The cancer patient survival data referenced during the study are available in a public repository from the GEPIA website (http://gepia.cancer-pku.cn/) under the accession codes TCGA-LUSC, TCGA-COAD, TCGA-KIRC and TCGA-UCEC. The gene amplification frequency data referenced during the study are available in a public repository from the cBioPortal website (https://www.cbioportal.org/) under the accession code TCGA PanCancer Atlas Studies.