LncRNA expression data were downloaded from TCGA-LUAD dataset (59 normal samples, 535 LUAD tissue samples). LINC01419 expression level analysis was performed using TCGA-LUAD dataset. The website (http://pridb.gdcb.iastate.edu/RPISeq/index.html) was utilized to predict a binding relationship between LINC01419 and EZH2. The binding sites of EZH2 on the promoter region of FBP1 were predicted using the hTFtarget database.
Cell culture and transfection
LUAD cell lines A549 (BNCC337696), HCC78 (BNCC338064), H1975 (BNCC340345), and normal human bronchial epithelial cell line BEAS-2B (BNCC101953) were purchased from BeNa Culture Collection (China). Among them, LUAD cells were cultured using RPMI-1640 medium, and BEAS-2B was cultured using DMEM medium. All the mediums were supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Then the cells were cultured in a 37 °C constant temperature incubator containing 5% CO2.
LUAD-CSCs cell sorting: To investigate the expression of LINC01419 in CSCs, we isolated CD44+ (LUAD stem cells) or CD44− (LUAD non-stem cells) cell population from A549 and HCC78 cell lines using CD44 MicroBead kits (cat. no. 130–095-194, Miltenyi Biotec), which were performed according to the operating instructions provided by the manufacturer .
To investigate the effect of abnormal expression of LINC01419, EZH2, and FBP1 on the biological function of LUAD cells, we constructed overexpression vectors oe-LINC01419, oe-EZH2, and the corresponding negative control oe-NC. To silence expression of LINC01419, EZH2, and FBP1, we constructed sh-LINC01419, sh-EZH2, sh-FBP1 and the corresponding negative sh-NC; all sequences in this study were synthesized by Shanghai Sangon (Shanghai, China). LUAD cells were seeded in 6-well plates and cultured to about 60%, and then the above vectors were transfected into the corresponding cells using Lipofectamine 2000 kit, and finally the relevant indicators were detected.
Transfected LUAD cells were seeded into 96-well culture plates (1 × 103 cells/well). After 0, 24, 48, and 72 h of cell culture, 10 μL of CCK-8 detection reagent was added and the incubation was continued for 2 h. Absorbance values were detected by a microplate reader at 450 nm.
Cell sphere-forming assay
1 × 103 LUAD cells were seeded in 6-well plates (plates were pretreated with polyHEMA sterile solution) and were placed in standard CSCs medium (DMEM/F12 + 2% B27 + 100 U/ml penicillin + 100 ng/ml streptomycin + 20 ng/ml human-derived EGF + 10 ng/ml human-derived bFGF). 14 days after transfection, and the number of tumor spheres was counted under an inverted microscope.
RNA immunoprecipitation (RIP)
RIP experiments were performed using an EZMagna RIP kit (Millipore, Billerica, MA, USA) on the basis of the instructions provided by the manufacturer . Briefly, cells were lysed with RIP lysis buffer, and the lysates were incubated with beads bound with antibodies, then the beads were washed with proteinase K to remove proteins, and finally the expression levels were measured by qRT-PCR.
Chromatin immunoprecipitation (ChIP)
In this study, ChIP experiments were conducted utilizing the Magna ChIP Kit (Millipore, Bedford, MA) under the instruction of the manufacturer . Cells were treated with 4% formaldehyde and the cell lysate was sonicated to interrupt intact chromatin into chromatin fragments (between 200 and 300 bp). Then, chromatin was immunoprecipitated with anti-EZH2 antibody from Abcam, and IgG was treated as a control. The precipitated chromatin DNA was recovered and measured using qRT-PCR. Among them, the primers used for ChIP-PCR are shown in Additional file 2: Table S1.
Total RNA extraction was conducted by using Trizol reagent, which was then reversely transcribed into cDNA using a reverse transcription kit. The SYBR Premix Ex Taq II kit was employed for conducting qRT-PCR, and qRT-PCR was operated utilizing Applied Biosystems 7500 Real Time PCR system taking GAPDH as internal reference. The primers used in this study are shown in the Additional file 3: Table S2.
Western blot assay
Total proteins extraction was performed by using RIPA lysate. BSA kit was utilized for examining total protein concentration. Then protein was separated by SDS-PAGE, and transferred onto PVDF membrane. Next, the membrane was blocked for 1 h and incubated with primary antibodies (EZH2 (ab191250, 1:5000), FBP1 (ab109020, 1:2000), CD44 (ab243894, 1:1000), CD133 (ab222782, 1:2000), ALDH-1 (ab177463, 1:2000), GAPDH (ab181602, 1:10,000), IgG (ab109489, 1:2000)) overnight at 4 °C. After being washed three times, the membrane was added with horseradish peroxidase (HRP) secondary antibody. Then it was incubated overnight at 4 °C. Finally, the brightness of the protein bands was detected , and the above antibodies were purchased from Abcam (UK).
Animal model construction
All animal experiments were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Medical Laboratory Animal Care Committee of Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University. For xenograft model construction, a total of 10 male BALB/c nude mice (6-week-old, 20–22 g) were purchased, and the mice were randomly divided into 2 groups (n = 5/group): sh-NC group and sh-LINC01419 group. HCC78 cells transfected with sh-NC and HCC78 cells transfected with sh-LINC01419 (cell concentration of 2 × 106 cells/200 μL) were subcutaneously injected into the flanks of the mice, respectively. During the normal feeding period, the subcutaneous tumor growth of mice was observed and recorded every 7 days, and the tumor weight, tumor length (L), and width (W) were measured and recorded. The tumor volume calculation formula was: tumor volume V = (L × W2)/2 mm3. 28 mice were fed normally. The mice were anesthetized with 2% pentobarbital sodium (50 mg/kg) and sacrificed by cervical dislocation. Subcutaneous tumors were excised and weighed. All mice were dissected and tumor tissues were collected for subsequent experimental analysis [22, 23].
In this study, immunohistochemical experiments were conducted using the method described in a previous article . The antibodies used in this study were as follows: FBP1, Ki67, and the above antibodies were purchased from Abcam (UK).
The FBP1 promoter region containing the binding site was cloned into pGL3-basic (Panomics, Fremont, CA, USA) and designated as FBP1 WT. All binding site mutated FBP1 promoter regions were cloned into pGL3-basic and designated as FBP1 MT. pRLSV40 (Promega, Fitchburg, WI, USA) was used as a control. To investigate the effect of silencing or overexpression of EZH2 on FBP1 transcriptional activity, sh-EZH2 or oe-EZH2 was co-transferred into LUAD cells and luciferase activity was measured using a dual-luciferase reporter assay system.
All data for this study are presented as mean ± standard deviation. All experiments were repeated at least three times. All data were analyzed using GraphPad Prism 8.0 (CA, USA) and compared between two or more groups using Student’s-t test or ANOVA test. P < 0.05 was considered statistically significant.