Cell culture
Human lung cancer cell lines A549, A549/DDP, and human embryonic kidney cells HEK-293T were purchased from Shanghai Institute of Biosciences Cell Resource Center (Shanghai, China) and cultured in RPMI-1640 (Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries Israel Bei-Haemek Ltd., Israel), 100 U/mL of penicillin sodium, and 100 μg/mL of streptomycin sulfate (Sangon Biotech Shanghai, China) at 37 °C in a humidified atmosphere of 5% CO2 (Thermo Electron, USA)[26].
Construction of vectors
Construction of EGR1, lncRNA HOTAIR, and miR-6807-3p overexpression vectors
The full-length EGR1 cDNA was amplified from A549/DDP total cDNA using PCR with primers containing the EcoRI/HindIII restriction sites. The amplified product was purified and cloned into the pcDNA3.1 vector (Addgene, China) to generate the pcDNA3.1-EGR1 construct. The lncRNA HOTAIR sequence (NR: 003716.3) was searched according to the known HOTAIR gene sequence (GeneID: 100124700) in NCBI GenBank, and the full length lncRNA HOTAIR gene was chemically synthesized and then cloned into the pcDNA3.1 vector to construct pcDNA3.1-lncRNA HOTAIR. Full-length miR-6807-3p fragment was amplified by PCR from the total DNA of A549/DDP cells. The amplified product was purified and cloned into pcDNA3.1 vector to construct pcDNA3.1-miR-6807-3p. The empty pcDNA3.1 vector was used as a negative control. The primers used for plasmid construction are shown in Table 1.
Construction of RNAi vectors
Four short hairpin RNAs (shRNAs) targeting EGR1 were designed. The forward and reverse sequences were ligated to the circular loop sequence (5'-CGAA-3'), and the reverse sequence was ligated to the termination sequence (5'-TTTTTT-3') and the BamH I (Fermentas, China) restriction site at the 5' end. At the 3' end, there was an EcoRI (Fermentas, China) restriction site. The recombinant vectors were named shEGR1-1, shEGR1-2, shEGR1-3, and shEGR1-4. The pRNAT-H1.1/Shuttle-RFP (Scotch Plams, USA) empty plasmid was used as a control. To verify the specificity of the interfering fragment, the rescue vector pcDNA3.1-EGR1Δ was constructed.
Three short hairpin RNAs (shRNAs) targeting lncRNA HOTAIR were designed. The forward and reverse sequences were ligated to the circular loop sequence (5'-CGAA-3'), and the reverse sequence was ligated to the termination sequence (5'-TTTTTT-3'), and the BamHI (Fermentas, China) restriction site at the 5' end. At the 3' end, there was a HindIII (Fermentas, China) restriction site. The recombinant vectors were named shLncRNA HOTAIR-1, shLncRNA HOTAIR-2, and shLncRNA HOTAIR-3. The pRNAT-H1.1/Shuttle-RFP (Scotch Plams, USA) empty plasmid was used as a control. To verify the specificity of the interfering fragment, the rescue vector pcDNA3.1-LncRNA HOTAIRΔ was constructed. The sequences of the shRNAs are shown in Table 2.
Construction of luciferase reporter vectors
For the MRP1 promoter reporter gene assay, the MRP1 gene promoter (-560 to +176 bp) was PCR-amplified from A549/DDP genomic DNA and cloned into the pGL3-basal luciferase reporter plasmid (Promega, China) to generate pGL3-MRP1-600. Deletion mutagenesis of the predicted EGR1 binding site by nested PCR yielded pGL3-600-ΔE1 (AGGTGAGCGGGCGC), pGL3-600-ΔE2 (GGGGCGGGGGGG), pGL3-600-ΔE3 (TGGGGCGGGGC), and pGL3-600-ΔE4 mutant reporter genes; of these, pGL3-600-ΔE4 was simultaneously mutated at the three putative EGR1 binding sites.
For EGR1 3′UTR reporter assay, the entire 3′ UTR of EGR1 was chemically synthesized and cloned into psiCHECK-2 dual-luciferase reporter plasmid. (Promega, Madison, WI, USA) immediately downstream of the stop codon of the luciferase gene to generate psiCHECK-2- EGR1 3′ UTR-wt. The mutant EGR1 3′UTR reporter, designated as psiCHECK-2- EGR1 3′ UTR-mut, was created by mutating the seed region of the predicted miR-6807-3p binding site (ATGCAGT to TACGTCA) through chemical synthesis.
The predicted interaction between lncRNA HOTAIR and miR-6807-3p was analyzed with the help of the DIANA tool on the biometric prediction website. Dual luciferase reporter assays were performed to determine if miR-6807-3p is a direct target of lncRNA HOTAIR. The binding region of lncRNA HOTAIR was cloned and chemically synthesized. The primers used for plasmid construction are shown in Table 3. All recombinant vectors were verified by sequencing after construction in Sangon Biotech Co., Ltd. (Shanghai, China).
Real-time RT-PCR analysis
Total RNA was extracted from A549 and A549/DDP cells using EZ-10 RNA Mini-Preps KitS (BIO BASIC INC, China). Total RNA was reverse transcribed to cDNA using Bioteke super RT Kit (Bioteke Corporation, China). The real-time PCR reactions were carried out in a 96-well microtiter plate using the power 2×SYBR Real-Time PCR Premixture kit (Bioteke Corporation, China) and Real-Time PCR (Eppendorf, USA). All samples were analyzed in triplicate in three independent experiments. The fluorescence of the PCR products was detected by the same apparatus. The number of cycles for the amplification plot to reach the threshold limit (Ct value) was used for quantification. β-actin and U6 were used as endogenous controls. Using the Ct values obtained by the analysis software, the relative expression levels of EGR1 and MRP1 mRNAs, lncRNA HOTAIR, and miR-6807-3p were calculated through the 2-ΔΔCt algorithm[26-28]. The primers used in PCR are shown in Table 4.
Western blotting analysis
The indicated cells were lysed in precooled lysis buffer (Western and IP Cell Lysate Kit from Beyotime Biotechnology, China) containing 150 mM NaCl, 20 mM Tris-Cl (pH 7.5), 1% Triton X-100, and various inhibitors such as EDTA, sodium pyrophosphate, β-glycerophosphate, sodium orthovanadate, and leupeptin. The protein lysates were separated on 10 % SDS-PAGE gels and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Germany). After antigen blocking, the membranes were incubated with primary antibodies (anti-mouse IgG Bioss) overnight at 4 °C with gentle shaking. Membranes were probed with the following primary antibodies: MRP1 (1:300, Bioss, China), EGR1 (1:300, Bioss, China), and GAPDH (internal control, 1:300, Bioss, China). The secondary rabbit anti-mouse IgG (LI-COR Biotechnology, USA) antibody was added subsequently at a 1:1000 dilution and incubated at 4 °C for 1 hour with gentle shaking[29]. Bands were visualized with the LI-COR OdysseyH scanner and software (LI-COR Biotechnology, USA). Blots (and gels) were imaged using an Odyssey Infrared Imaging System Scan resolution.
Chromatin immunoprecipitation (ChIP)-PCR assay
Based on the prediction, three putative EGR1-binding sites were found in MRP1 promoter, namely EGR1-1, AGGTGAGCGGGCGC (-68/-55 bp); EGR1-2, GGGGCGGGGCGG (-53/-42 bp); and EGR1-3, TGGGGCGGGGC (-39~-27 bp); where +1 is the transcription start site. To study the interaction between these putative binding sites and EGR1, ChIP-PCR assay was performed as described[30]. Briefly, the A549/DDP cells were grown to approximately 80% confluence, treated with formaldehyde (Sigma, USA) for 10 minutes, and pulsed with ultrasonic waves on wet ice. Then, the samples were immunoprecipitated with control IgG (Bioss, China), anti-RNA polymerase II antibody, and anti-EGR1 antibody (Bioss, China). Afterwards, the protected DNA fragments were collected, and PCRs were performed using the following specific primer set: Forward Primer: GCCGAGAGGTGGCTGGTCC; Reverse Primer: CGCACCGCCGCCTGGTT. Finally, the products from PCRs were analyzed by 1.5% agarose gel electrophoresis.
Luciferasereportergeneassay
HEK-293T cells were grown in 24-well plates and co-transfected with 2 μL polyethylenimine (PEI; Sigma), 0.5 μg EGR1 overexpression vector, 0.5 μg of MRP1 promoter-luciferase plasmid DNA, and 0.01 μg of Renilla luciferase reporter plasmid DNA. After 4 h of incubation, the medium without fetal calf serum was replaced with 1640 complete medium. A549/DDP cells were grown in 24-well plates and co-transfected with 2 μL PEI (Sigma), 0.5 μg miR-6807-3p overexpression vector, and 0.5 μg of HOTAIR luciferase plasmid DNA. After 4 hours of incubation, the serum-free medium was replaced with 1640 complete medium. Firefly and Renilla luciferase activities were measured 48 hours after transfection, using a dual-luciferase reporter system (Promega, WI, USA) following the manufacturer's protocol[27]. The firefly luciferase activity was normalized to the Renilla luciferase activity. Each experiment was repeated at least three times.
Flow cytometry to determine the drug efflux of Rho-123
Fluorescence intensity of Rho-123 (99%, Shanghai Yuanyi Biotechnology Co., Ltd.) in A549/DDP transfected with the aforementioned expression vectors was detected using flow cytometry (BECKMAN COULTER, USA) to assess the change in MRP1 protein efflux capacity. The cells were collect in EP tubes containing 10 μg/mL Rho-123 and incubated for 30 minutes at 37 °C in a 5% CO2 cell incubator. Flow cytometry was used to measure the fluorescence intensity and emission wavelength of Rho-123 in cells with excitation wavelengths of 488 nm and 530 nm[26]. The subtraction of the fluorescence retained from the total fluorescence is the fluorescence index of Rho-123. This process was repeated three times and an average value was obtained to calculate the ability of Rho-123 to flow-out.
Drug sensitivity evaluation via MTT assay
In order to detect drug sensitivity, A549/DDP cells were exposed to cisplatin (DDP), Toosendanin (TSN), and 5-fluorouracil (5-FU). Thiazolyl blue tetrazolium bromide (MTT, Sangon Biotech shanghai, China) assay was used to detect changes in drug sensitivity and to measure half-inhibitory drug concentrations. A549/DDP cells were transfected with pRNAT-H1.1/Shuttle-RFP, pcDNA3.1, pRNAT-H1.1/Shuttle-RFP + pcDNA3.1, pRNAT-H1.1/Shuttle-RFP-shEGR1-3, or pcDNA3.1. The cells were cultured for 48 h with pcDNA3.1-Egr1Δ+ pRNAT-H1.1/Shuttle-RFP-shEGR1-3 and seeded in 96-well plates at a density of 5 × 105 cells per well. After 48 hours of incubation, 20 μL MTT (5 mg/mL) was added. After 5 hours of incubation, 180 μL of DMSO was added to each well. After incubation at 37 °C for 24 hours, the absorbance was measured at 570 nm using a microplate reader (TECAN, Switzerland)[26, 28].
Observation of cell morphological changes
According to the results of MTT assay, three anticancer drugs (DDP, TSN, and 5-FU) were added to A549/DDP cells transfected with different expression vectors. After 48 hours, acridine orange (AO) staining was performed. Cell morphology was observed under laser confocal microscope (Leica, Germany).
Statistical analysis
All results are expressed as means ± SD. Representative bands were selected from independent western blotting experiments. When data distributions approximated normality and two groups were compared, a Student’s t-test was performed to evaluate the significance of differences. Differences were regarded as significant at a level of P<0.05. All statistical tests were performed using Prism software (GraphPad).