Cell culture and collection
MCF-7 was obtained from the American Type Culture Collection (ATCC, USA). Cell lines were cultured in RPMI-1640 Medium (Thermo Fisher HyClone, USA) with 10% fetal bovine serum (FBS) (Gibco, MA, USA), and 1% Penicillin Streptomycin Mixture (Beijing solarbio Science﹠Technology Co., Ltd.) in 5% CO2 at 37℃. MCF-7 cell lines were divided into siRNA-5 group (knocked-down group, KD group) and negative control (NC) group. Following manufacturer’s instructions, small interfering RNA-5 (5′-3′sense: CUAUACGAAUGGGAUUUGATT; 5′-3′antisense: UCAAAUCCCAUUCGUAUAGTT), NC (5'-3'sense: UUCUCCGAACGUGUCACGUTT; 5′-3′antisense: ACGUGACACGUU CGGAGAATT) (GenePharma, Shanghai, China) and Lipofectamine 2000 (Invitrogen, USA) were individually mixed at 1:1(volume ratio) and stored for 20 minutes at room temperature. Two groups of mixed small interfering RNA and Lipofectamine 2000 were added to MCF-7 cells in the logarithmic growth phase, and cultivated in 5% CO2 for 24 hours at 37℃. The small interfering RNA had a final concentration of 100 nM [13, 14]. MCF-7 cells were washed with 0.01M PBS (phosphate buffer saline) to remove the medium. Afterwards, a lysis buffer (7 M urea and 4% SDS (sodium dodecyl sulfate)) containing 1 mM PMSF (Phenyl- methane-sulfonyl fluoride) was added to each group of cells (1×107cells). The cells were lysed on ice for 30 minutes. The supernatant of the mixed solution was collected for analysis.
SDS-PAGE and Protein Digestion
Total proteins of the above mixed solution from the two cell groups were extracted. Protein concentrations of isolated proteins in the above two group cells were determined by a BCA (bicinchoninic acid) protein assay kit (Appygen Technologies Inc., Beijing, China). Protein concentrations of samples were adjusted to a constant level using the dilution. Total protein (20 µg) per lane was separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in the gels were cut into gel slices, and the gel bands were digested by an in-gel trypsin method.
Reductive Alkylation and Protease Digestion
Dithiothreitol (20 mM final concentration) was added to extracted protein samples (100 µg/µL), and the samples incubated at 37℃for 60 minutes. Iodoacetamide (40 mM final concentration) was added, and the samples incubated in the dark at room temperature for 40 minutes. Pre-cooled acetone (volume ratio of 5:1) was added to the samples, and the mixture placed in - 20℃for 2 hours. The sediment was collected by 10,000g centrifugation at 4℃for 20 minutes and re-dissolved in 100 mM tetraethylammoniumbromide buffer. Trypsin (1 mg trypsin/50 mg protein) was added to the protein samples, and settled at 37℃for overnight.
Protease-digested proteins were labeled with 113,117, 114, and 118. The NC group was labeled with 113 and 117, whereas the siRNA-5 group was labeled with 114 and 118. These were the two biological replications. 100µg peptides were labeled with an iTRAQ reagent tube [15, 16].
First dimensional separation of peptides
Separation of peptides was performed by ultra-high pressure liquid chromatography (UPLC) (Waters Corp., Milford, MA, USA) with a 2.1×150 mm X Bridge BEH300 column (Waters Corp., Milford, MA, USA). The moving phase was a mixture of water (pH adjusted to 10 with ammonia and formic acid), and acetonitrile was isocratically transmitted using a pump at a flowrate of 0.4mL/min. The wavelength of the ultraviolet absorbance detector was 214/280 nm. The projects used for gradient elution are listed in Table 1. A total of 10 fractions were collected according to different retention times. Rotation vacuum concentrators (Christ RVC 2-25, Martin Christ, Germany) were used for concentration, and dissolved in buffer solution (pH adjusted to 10 with ammonia and formic acid) for further analysis.
Labeled peptides were analysed using a NanoAcquity UPLC system (Waters Corporation, Milford, MA) combined with a quadrupole- Orbitrap mass spectrometer (Q-Exactive) (Thermo Fisher Scientific, Bremen, Germany), incorporating a C18 column (75µm×25 cm, Thermo, USA). The mobile phase was a mixture of water, with 2% acetonitrile and 0.1% formic acid isocratically delivered using a pump at a flowrate of 300 nL/min. The schemes used for gradient elution are shown in Table 2. Full-scan mass spectrometry (m/z 350-1300) was acquired with a first mass resolution of 70K, and second resolution of 17.5K. The framsscan was applied with data-dependent acquisition (DDA), Top 20. Fragmentation was used for high-energy collision dissociation (HCD). The microscan was recorded using dynamic exclusion of 18 s.
ITRAQ data analysis
The MS/MS data of iTRAQ were analysed using Protein DiscovererTMSoftware 2.1 (Thermo Scientific) and searched in Uniprot-proteomes-homo-sapiens-70611.fasta. The database had 70611 entries, and date of download was July 15th, 2016. Result-filtered parameters were used to control peptide level false discovery rates (FDR) ≤1%. Protein quantification was performed by only unique peptides, and normalisation on protein medians was applied to rectify experimental deviation, which was accomplished by retrieval software. The ratios of the samples were weighted, and normalised by contrasting the negative control group (sample tagged as 113 and 117) to the denominator for protein quantitation. Regarding the quantitative changes, a≥1.2 or ≤0.83-fold change takeout and P-value (t-test) <0.05 were set for DEPs.
Gene Ontology (GO, http://www.geneontology.org/) and Kyoto Encyclopaedia of Genes and Genomes (KEGG, http: //www.enome. jp/kegg/) pathway were applied to annotate, and classify all authenticated proteins. DAVID 6.8 Functional Annotation Tool (http://david.abcc. ncifcrf. gov/) was arranged to process the DEPs for the enrichment analysis. Fisher Exact statistic methodology was used to filter results. The ClueGO of Cytoscape software (http://www.cytoscape. org/) was performed to assess GO biological network. String (Search Tool for the Retrieval of Interacting Genes) v10.0 (http://www.string-db.org/) was applied to analyse protein-protein interaction, and a high coefficient value of 0.7 was used as a cut-off. The expression patterns of DEPs (fold change≥ 1.2 or ≤ 0.83 and P<0.05) were identified by Cluster analysis using h-cluster (https://pypi.python.org/ pypi/hcluster/ 0.2.0) [9, 17].
Cells of siRNA-5 and NC groups (5×106 cells per cell group) were separately collected, and western blot analysis conducted [18-20]. Total proteins were extracted from cells with RIPA buffer (150 mM NaCl, 20 mM Tris, 0.1% SDS, pH 7.5, 1% deoxycholate and 1% Triton X‑100) containing protease inhibitors. Protein concentration was determined by a BCA Protein Assay kit (Appygen Technologies Inc., Beijing, China). Briefly, total protein (30µg) per lane was separated on a 10% SDS-polyacrylamide gel, and transferred onto polyvinylidene ﬂuoride membranes (Merck KGaA, Darmstadt, Germany). The membranes were blocked using Tris-buffered saline and Tween 20 (TBST) and 5% skim milk powder for 2hours, and incubated using primary antibodies against ILP-2 (rabbit IgG, 1:1,000; cat. no. Ab9664;Abcam, Cambridge, UK),MT1E(mouse IgG, 1:500; cat.no.MAD794Hu21; Cloud- CloneCorp, Wuhan, China), TDO2(rabbitIgG,1:500;cat.no. ab84926; Abcam, Cambridge,UK), AGA(rabbit IgG,1:500;cat.no. ab 231021; Abcam, Cambridge,UK), Tubulin (rabbit IgG, 1:1000;cat.no. 11224-I-AP; Proteintech, Shanghai, China) and GAPDH (mouse IgG,1:2500;cat.no. 60004-1-Ig; Proteintech, Shanghai, China) overnight at 4°C. On the second day, the membranes were washed using TBST, and incubated using a goat anti-mouseimmunoglobulin G-horseradish peroxidise (anti- mouse IgG, 1:5000; cat. no. SA001; Auragene Biotech,Hunan,China), and peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L) (anti-rabbit IgG,1:5000;cat. no. ZB-2301; ZSGB-BIO, Beijing, China) for 1 hour at room temperature. An enhanced chemiluminescence detection system (SuperECL-Plus, Applygen Technologies, Beijing, China) was usedto visualise immunoreactive proteins. The proteinwas visualised by chemical chemiluminescence imaging system (Beijing Sage Creation Science Inc., Beijing, China). The images were analysed with ImageJ. Tubulin,GAPDH was used as internal control.
To extract the total RNA, wash the cell sample twice with 1mL PBS in a cell culture dish, add 1mL RNA isolater Trizol (Vazyme, Nanjing, China) and pipette evenly, transfer it to a 1.5mL RNase free EP tube to fully lyse the cells. Let stand at room temperature for 10 min; add 200uL chloroform, shake vigorously for 15s, and stand at room temperature for 10 min; centrifuge at 4℃ 12000rpm for 15 min, collect the upper colorless liquid phase in a new 1.5mL RNase free EP tube; add 500 uL isopropanol, shake and mix well. After 10 minutes at room temperature, centrifuge at 4°C 12000rpm for 10 minutes, discard the supernatant; add 1mL 75% alcohol, and gently shake to wash the precipitate, centrifuge at 4°C 12000rpm for 5 min, discard the supernatant, and repeat twice; dry at room temperature for 10-15min; add 50uL of DEPC (diethyl pyrocarbonate ) water to dissolve the RNA. Measure the OD260/OD280 of RNA on the nucleic acid protein detector to detect the purity of total RNA, and run the RNA sample in 1% agarose gel electrophoresis to detect RNA integrity. Perform RNA reverse transcription according to the instructions of Bester qPCR RT Kit (DBI Bioscience, Germany). Denature the RNA by keeping it at 65°C for 5 minutes and put it on ice immediately for later use. Add the components to a 0.2ml RNase free EP tube in the following order to prepare a reverse transcription reaction solution, 2 μL 5×RT Buffer, 0.5 μL RT Enzyme Mix, 0.5 μL Primer Mix, 1 μg RNA, make the reaction solution up to 10μL by adding RNA-free Water. For reverse transcription reaction, put the above-mentioned mixture on a PCR machine immediately, perform reverse transcription reaction at 37°C for 15 minutes, and at 98°C for 5 minutes, and store the reversed transcribed RNA (the cDNA) at 4°C for immediate use or at -20°C for long-term use. For QPCR experiments, follow the instructions of Bestar SybrGreen qPCR Mastermix kit (DBI Bioscience, Germany). Prepare 20μL reaction solution by the following component accordingly, 10μL Bestar SybrGreen qPCR Mastermix, PCR Forward Primer (10μM) 0.5μL, PCR Reverse Primer (10μM) 0.5μL, DNA template 1μL, ddH2O 8μL. Run Real Time PCR amplification using the following thermal conditions, pre-denaturation at 95℃ for2min; PCR cycle (40 times) at 95℃ for 10s, 60℃ for 30s, and 72℃ for 30s; melting curve hold at 95℃ for 1min and 55℃ for 1min.
Histograms were constructed using GraphPad Prism 5. Statistical analysis was performed by conductingt-test withSPSS statistics 17.0(SPSS, Chicago, USA). A level of P<0.05 was considered statistically significant.