Bioinformatics predictions and screening.
The long non-coding RNAs that areexpressed at high levels in lung cancer were screened using StarBase V3.0 (http://starbase.sysu.edu.cn/).The miRNAsthat bind to the lncRNAs and the possible downstream targets of miR-218-5p were predicted by the most frequently used algorithms TargetScan, miRDB and PicTar, and we noticed the overlappingmiRNAs and targets identified by the three algorithms. The putative promoter of miR-218-5p was predictedusing Promoter Scan (http://www-bimas.cit.nih.gov/molbio/proscan/).
Cell line and human LUSC tissue
NCI-H520 cells were cultured with the RPMI-1640 (Gibco, Grand Island, NY, USA) containing10%foetal bovine serumand 1,000 U/mL P/S. The human LUSC cells were cultured at 37℃ in a humidified atmosphere with 5% CO2.Cells were transfectedusing Lipofectamine 2000 Reagent (Invitrogen, USA) according to the manufacturer’s instructions. The NCI-H520 cells and three pairs of human lung squamous cell carcinoma tissues were provided by the Department of Cancer Institute, North China University of Science and TechnologyAffiliated People’s Hospital. The number, gender, classification and age of the patients with LUSC are shown in Table 1. Informed consent was obtained from all subjects or their direct relatives.The cell and tissue studies weresubmitted to and approved by both the Ethics Committee of North China University of Science and Technology and the Ethics Committee of Hebei Medical University.
qRT-PCR.
Total RNA was extracted from lung cancer tissues and the cell line with the mirVana miRNA Isolation Kit (Ambion, USA) according to the manufacturer’s instructions. Onemicrogram of RNA was reverse transcribed into cDNAswith Moloney murine leukaemia virus reverse transcriptase (Takara, Japan). qRT-PCR was conducted with a SYBR® Taq™ kit (Takara Bio, Japan) and the iQ5 Real Time PCR Detection System. The level of hsa-miR-218-5p in the transcripts was normalized to U6 as the internal control. The levels of the RNASEH1-AS1, NET1 and POU2F1 mRNAs were normalized to β-actin as the internal control. The quantity of the negative control group was defined as 1, and the relative fold change of the experimental group was calculated. All the RT and qPCR primer sequences are listed in the Table 2.
Plasmid and miRNA mimics.
The miR-218-5p overexpression mimics and ASO-miR-218-5pplasmids were commercially synthesized. The sequencesare listed in Table 2.The sh-RNASEH1-AS1 and sh-POU2F1 pSilencer vectors were generated by annealing the sense and antisense strands of the hairpin RNA followed by insertion onto the pSilencer2.1-U6 neo vector between the BamHI and HindIII sites.
The wild type and mutant 3’-UTRs of the NET1 or POU2F1 genesharbouring the predicted miR-218-5p binding site were inserted into the 3' end of the reporter gene of the pmirGLO vector. Similarly, the putative promoter area and miR-218-5p binding site in lnc-RNASEH1-AS1 were amplified by PCR and inserted into the 3' end of the reporter gene of the pGL3-basic-luciferase vector.
All the insertions mentioned above were verified by DNA sequencing. All primers used in this study are listed in Table 2.
Dual-luciferase reporter assay.
NCI-H520 cells were cultivated in 48-well plates at a density of 6 × 104cells per well. The miR-218-5p transfection reagents were prepared at a final concentration of 20μM and incubated for 4h. After 24 h, pmirGLO-targetgene-3’UTR/mut transfection regents were added into cultured cells at a final concentration of 0.5μg. After transfection for 4 h, the transfection mixture was replaced with 300 μL of fresh complete 1640 medium. After cotransfection for 48 hours, the luciferase activity was measured usingthe Dual-Luciferase Reporter System (Thermo, USA) according to the manufacturer’s instructions.
Cell viability and proliferation assays.
In the MTT assay, NCI-H520 (1000 per well) cells were plated into 96-well plates. At 24, 48, and 72h after transfection, MTT was added to every well, and the plates were incubated for 4h. The absorbance was measured at a wavelength of 570 nm to evaluate cell viability.
In the colony formation assay, transfected NCI-H520 cells (200 cells per well) were trypsinized, plated into 12-well plates and cultured for 7 to14 days at 37 °C. The colonies were stained with a solution composed of 0.2% crystal violet and 20% methanol. Colonies with greater than 50 cells were counted and analysed. The colony formation rate was calculated using the following formula: colony formation rate = (number of colonies/number of seeded cells) × 100%. All cell experiments were conducted at least three times.
Transwell invasion and wound healing assays.
In the transwell invasion assay, NCI-H520 cells (1×105 per well) were seeded in the upper chamber of every insert (Millipore, USA) containing 50 μl of Matrigel (Millipore, USA). Eight hundred microliters of DMEM supplemented with 20%FBS (JIBCO, USA) were added to the lower chambers. After 72h, cells that had attached to the lower surface were stained f with crystal violet or 15 min. Afterwards, we captured images and counted the cells.
In the wound-healing assay, NCI-H520 cells were cultured in 12-well plates. When cell confluence reached 70-80%, scratches were generated with a 50 µl pipette tip, and non-adherent cells were removed by three washes with PBS. Wounded cells were cultured in medium lacking FBS for 0, 24, and 48 h. Images of three randomly selected fields of view were captured in each well.
Western blotting.
NCI-H520 cells were collected and lysed with lysis buffer (100 mM Tris-HCl, 2% SDS, 1 mM mercaptoethanol and 25%glycerol). Cell extracts were heated in loading buffer and the same amounts of cell extracts were separated on a 10% SDS-PAGE gels. After electrophoretic transfer to PVDF membranes (Millipore, USA), the protein bands were probed with its corresponding primary antibodies (anti-NET1, anti-POU2F1, Saier Biotechnology, China)overnight at 4 °C. The secondary antibody (anti-GAPDH, Saier Biotechnology, Tianjin, China) was added and incubated with the membrane at room temperature for 1.5 hour. The PVDF membranes were washed with PBS four times and the immunoreactive target bands were visualized using the chemiluminescence imaging system (Huqiu Image Instruments, Suzhou, China). Band intensities were quantified using LabWorks image analysis software (UVP, USA).
Animal model.
Twenty BALB/c-nu mice (female) aged 5 to 6 weekswere purchased from the Institute of Experimental Animals, Chinese Academy of Medical Sciences (Beijing).They were randomly divided into two groups.In total, 2×107 NCI-H520 cells were transfected with siR-RNASEH1-AS1 or siR-NC and suspended in 100 µl of serum-free RPMI1640 for every nude mouse. The cell suspension was injected directly into the left side of the back of the mice. The tumour volume was measured every three days after injection. Four weeks later, anaesthetized mice were sacrificed by cervical dislocation, and their tumourmasseswere harvested. No premature deaths were documented. The tumour weight was measured and the average tumour weight was calculated. The tumour tissues were stored at -80℃or used to perform haematoxylin-eosin (HE) and immunohistochemical staining.All studies were performed under the American Association for the Accreditation of Laboratory Animal Care guidelines and adhered to national and international standards. All animal experiments were approved by the Ethics Committee of Hebei Medical University.
CHIP assay.
The binding of POU2F1 to the lnc-RNASEH1-AS1 promoter was confirmed by ChIP assay according to the instructions of Chromatin Immunoprecipitation Kit (Millipore, USA). NCI-H520 cells were used in the cross-linking step after reaching 80-90% confluence. Isolated chromatin was sonicated to shear the DNA. Next, immunoprecipitation and elution of cross-linked protein/DNA were performed according to the manufacturers’ protocol.Three to five micrograms of anti-RNA polymerase or anti-Rabbit IgG were used as the positive and negative control groups, respectively.Three to five microgramsof anti-POU2F1was used in the experimental group. Cross-links of protein/DNA complexes were reversed to free DNA, and spin columns were used for DNA purification. Primers flanking the predicted POU2F1 binding site in the lnc-RNASEH1-AS1 promoter were used for PCR.The primers used in this studyare listed in Table 2.The LabWorks image acquisition and analysis system (UVP, USA) was used to capture images and quantify the intensities of target signals after gel electrophoresis. The quantity of the input group or anti-POU2F1-ChIP group was 3481or 8041, respectively. The quantity of the input group was defined as 1, and the fold change (2.30) in the anti-POU2F1-ChIP group was calculated.
Statistical evaluation.
The data were analysed using GraphPad Prism 6 Software (GraphPad Software, USA) with the two-tailed Student’s t test. The results are presented as the means±S.D. of three separate experiments. Unpaired Student’s t-test was used to compare the two groups. A p value less than 0.05 was regarded as a statistically significant difference (* p<0.05, ** p<0.01, and ***p<0.001).