TCGA analysis
The differential level of ILF3-AS1 was detected through Cancer Genome Atlas (TCGA) and calculated by t-test.
Cell culture
H1299 and H358 cells were got from ATCC and maintained in RPMI-1640 with penicillin/streptomycin (10%/1%). A549 cells, also obtained from ATCC cell bank, were maintained in Ham's F-12K. All cells were cultivated at 37°C and 5% CO2.
Cell transfection
The transcript of ILF3-AS1 was amplified and inserted to pcDNA3.1 vector after digested with Hind III and Bam HI. The primer for ILF3-AS1 was forward 5’-CCCAAGCTTATCTTACGCCCGTCGCCCTGAG-3’ and reverse 5’-CGGGATCCGACACGGGAAACAGGAGGATTTA-3’.
The mimics, inhibitor, and corresponding NCs of MiR-185-5p (GenePharma, China) were transfected to H1299 and H358 cells by Lipofectamine 3000.
QRT-PCR
Total RNA preparation was performed by RNA extraction kit (Beijing ComWin Biotech Co.,Ltd). RNA (100 ng) was subjected to reverse transcription by Evo M-MLV transcription kit (AG, China). ILF3-AS1 forward 5’-TAAACCCCACTGTCTTCC-3’, reverse 5’-TTCCTTGCTCTTCTTGCTC-3’; miR-185-5p forward 5’-TGGAGAGAAAGGCAGT-3’, reverse 5’- TGTCGTGGAGTCGGC-3’; ING4 forward 5’-ATGGCTGCGGGGATGTATTTGGAAC-3′, reverse 5′-CTATTTCTTCTTCCGTTCTTGGGAGCAG-3′, U6 forward 5’- TCTTTCTCGAGCAAGGTCGGGCAGGAAGAGGGCCTA-3’, reverse 5’-AAAACTCGAGGAATTCGGGCCCGGTGTTTCGTCCTTTCCACAAG-3’; GAPDH forward 5′-GGAGCGAGATCCCTCCAAAAT-3′, reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′. U6 or GAPDH was regarded as loading control, and 2−ΔΔCT value was used for statistical analysis.
MTT assay
After transfection, cells were cultured in 96-well plates and maintained for 24 h. subsequently, cells were incubated with MTT (Sangon Biotech) for 4 h before termination of culture. Afterwards, DMSO was appended and OD value of cell culture was measured at 490 nm.
Annexin V-FITC/PI double stainings
Briefly, cells were re-suspended in appropriative buffer and stained with Annexin V-FITC at 26°C. Subsequently, cells were incubated with 5 µl PI, 5 min before flow cytometric analysis.
PI-FACS analysis
Cells were washed in D-Hanks at 4°C. Then, cells were fixed by 75% ethanol for 60 mins and re-suspended in D-Hanks containing RNase (10 µg/µl) and PI (2 µg/µl). The cell cycle was analyzed under the Agilent NovoCyte flow cytometry.
Transwell assay
The upper chambers were pre-coated with Matrigel or not for invasion and migration, respectively. Cells were suspended and inoculated into the upper chamber. Then, 10% FBS-RPMI was added to the inferior chamber. After incubated at 37°C for 24 h, cells at the lower chambers were stained with crystal violet, and photographed under the microscope with camera at 100× magnification.
Western blot
Cells were lysed using RIPA buffer (Beyotime Biotechnology, China) and separated by SDS-PAGE. separated using PAGE gel electrophoresis, the samples were transferred to PVDF. The identified proteins were studied by the antibodies against ING4 (1:1000, ABclonal) and GAPDH (1:5000, Proteintech) at 4°C. After a night, the corresponding secondary antibody (1:5000, ASGB) were performed to incubate membranes for 30 min at 26°C. Immune blots were visualized by ECL Kit (Servicebio, Wuhan, China). The protein bands were quantitatively analyzed by gel analysis software (Alpha Innotech Corporation).
Nude mouse subcutaneous xenograft model construction
Athymic nude mice (4 weeks old) were divided control, NC and si-ILF3-AS1 groups, n = 6 per group. Mice in different groups were subcutaneously implanted with 4×106 H1299 cells transfected with blank, si-NC and si-ILF3-AS1, respectively. H1299 cells transfected with blank, si-NC and miR-185-5p mimics were implanted in mice (n = 6 per group), respectively. Then, the volume and weight of tumor in mice were monitored weekly for consecutive 6 weeks.
Statistical analysis
Results were performed as mean ± SD and analysis achieved by GraphPad Prism 8. Student’s t test was used for two groups. Multi-groups comparison was performed by ANOVA. When p less than 0.05, significant differences were considered.