Cell culture
The 4T1 cell line was acquired from Cyagen Biosciences (Guangzhou, China). Cells were cultured in complete Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS, catalog number 10099141C, Invitrogen, USA) and a penicillin-streptomycin-glutamine mixture (catalog number 10378016, Invitrogen, USA). Cultures were maintained in a humidified atmosphere containing 5% CO2 at 37°C. The culture medium was refreshed every three days to ensure optimal growth conditions.
Lentivirus infection
Lentiviral vectors targeting miR-615-3p mimic and control lentiviral vectors were produced by GenePharma Co., Ltd. The 4T1 cells were infected with the lentivirus in the presence of polybrene (BL628A, Biosharp, China) for 12 hours. Forty-eight hours post-infection, the infected 4T1 cells underwent antibiotic selection to ensure the integration of the viral construct. The target sequence for the miR-615-3p mimic is as follows: miR-615-3p mimic, 5′-TCCGAGCCTGGGTCTCCCTCTT-3′.
Quantitative Real-Time PCR (qRT-PCR)
Quantitative Real-Time PCR (qRT-PCR): Total RNA was isolated using the Vazyme Total RNA Isolation Kit (R711-01, Vazyme, China) and cDNA was synthesized following the manufacturer's protocol using HiScript III RT SuperMix for qPCR (+ gDNA wiper) (R323-01, Vazyme, China). Real-time PCR was performed using the MagicSYBR Mixture (CW3008, CWBIO, China). For miRNA detection, reverse transcription polymerase chain reaction (RT-PCR) was conducted using the TaqMan MicroRNA Reverse Transcription kit (4366596, Invitrogen, USA). The TaqMan probe for hsa-miR-615 (Assay ID: 001960, Applied Biosystems, USA) was used to detect the specific microRNA, with U6 (Assay ID: 001973, Applied Biosystems) serving as the loading control. Quantitative real-time PCR (qPCR) was conducted on a BioRad CFX96 Real-Time PCR Detection System (BioRad, USA). Each reaction was performed in triplicate and the entire experimental process was repeated three times to ensure the reliability and consistency of the results. The relative expression levels were calculated using the 2-ΔΔCt method.
Protein extraction and digestion
SDT(4%SDS, 100mM Tris-HCl, pH7.6) buffer was used for sample lysis and protein extraction. The amount of protein was quantified with the BCA Protein Assay Kit (Bio-Rad, USA). 20 µg of protein for each sample were mixed with 5X loading buffer respectively and boiled for 5 min. The proteins were separated on 4%-20% SDS-PAGE gel (constant voltage 180V, 45 min). Protein bands were visualized by Coomassie Blue R-250 staining. Protein digestion by trypsin was performed according to filter-aided sample preparation (FASP) procedure described by Matthias Mann. The digest peptides of each sample were desalted on C18 Cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 ml, Sigma), concentrated by vacuum centrifugation and reconstituted in 40 µl of 0.1% (v/v) formic acid.
Western Blot
Total protein was extracted using RIPA buffer. After quantification, equal amounts of protein samples were loaded and separated by 10% SDS-PAGE, then transferred to a 0.45-µm PVDF membrane. The membrane was blocked with 5% non-fat milk for 2 hours, followed by overnight incubation at 4°C with primary antibodies. The primary antibodies used were anti-PICK1 (10983-2-AP, Proteintech, 1/1000 dilution) and anti-GAPDH (ab8245, abcam, 1/5000 dilution). Subsequently, the membrane was incubated with secondary antibodies at room temperature for 1 hour. Immunoblots were visualized using an ECL chemiluminescence detection kit (Beyotime, Shanghai) and observed with a Tanon 4600 system (Tanon Science and Technology Co., Ltd.).
Dual-Luciferase Reporter Assay
The coding sequences (CDS) of wild type (wt) or mutant (mut) PICK1 were cloned into the firefly luciferase expression vector pMIR-REPORT (Sangon Biotech, Shanghai, China). The PICK1-mut, PICK1-wt, and Vector or miR-615-3p mimic were co-transfected into the 4T1 cell line using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). The luciferase activity in the cells was normalized by co-transfecting with a plasmid containing the full-length Renilla luciferase gene (pTK-Renilla) as a reporter (Genomeditech), and quantified 48 hours after transfection using a Dual-Luciferase Reporter Assay System (Promega).
5-Ethynyl-2′-deoxyuridine (EdU) Assay
The EdU incorporation was detected using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, Shanghai, China). After washing with PBS, the cells were incubated with the EdU solution for 2 hours, followed by staining of the nuclei with DAPI solution. After washing, the samples were observed under an inverted microscope (Olympus).
Filter-aided sample preparation (FASP Digestion) procedure
The detergent DTT (with the final concentration of 10 mM) was added to each sample respectively and mixed at 600 rpm for 1.5 h (37℃).After the samples cooled to room temperature, IAA was added with the final concentration of 20 mM into the mixture to block reduced cysteine residues and the samples were incubated for 30 min in darkness. Next, the samples were transferred to the filters respectively. The filters were washed with 100 µl UA buffer three times and then 100 µl 25mM NH4HCO3 buffer twice. Finally, trypsin was added to the samples(the trypsin : protein (wt/wt) ratio was 1:50) and incubated at 37℃ for 15–18 h (overnight), and the resulting peptides were collected as a filtrate. The peptides of each sample were desalted on C18 Cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 ml, Sigma), concentrated by vacuum centrifugation and reconstituted in 40 µl of 0.1% (v/v) formic acid. The peptide content was estimated by UV light spectral density at 280 nm using an extinctions coefficient of 1.1 of 0.1% (g/l) solution that was calculated on the basis of the frequency of tryptophan and tyrosine in vertebrate proteins.
LC-MS/MS analysis
We conducted peptide analysis using an LC-MS/MS setup, specifically a Q Exactive mass spectrometer from Thermo Scientific, connected to an Easy nLC system from Proxeon Biosystems (now part of Thermo Fisher Scientific). The peptides were first introduced onto a reverse phase trap column, Thermo Scientific Acclaim PepMap100 (100 µm by 2 cm, nanoViper C18), and then transferred to a C18-reversed phase analytical column (Thermo Scientific Easy Column, 10 cm long, 75 µm inner diameter, with 3µm resin) using a buffer solution of 0.1% Formic acid in water. This setup separated the peptides with a gradient mixture of 84% acetonitrile and 0.1% Formic acid at a 300 nl/min flow rate. Operating in positive ion mode, the mass spectrometer targeted the most abundant precursor ions for higher-energy collisional dissociation (HCD) fragmentation, based on a data-dependent top20 method. We ensured precise measurement by setting the automatic gain control (AGC) target to 1e6, with a maximum injection time of 50 ms and a dynamic exclusion duration of 30.0 seconds. The system achieved a resolution of 60,000 at m/z 200 for survey scans and 15,000 at m/z 200 for HCD spectra, with an isolation width of 1.5 m/z. The normalized collision energy was set to 30 eV, and the underfill ratio was kept at 0.1%, ensuring that the system efficiently recognized and analyzed peptides.
Identification and quantitation of proteins
The MS raw data for each sample were combined and searched using the MaxQuant 1.6.14 software for identification and quantitation analysis.
Protein-protein interaction analysis
The protein–protein interaction (PPI) information of the studied proteins was retrieved from IntAct molecular interaction database (http://www.ebi.ac.uk/intact/) by their gene symbols or STRING software (http://string-db.org/). The results were downloaded in the XGMML format and imported into Cytoscape software (http://www.cytoscape.org/, version 3.2.1) to visualize and further analyze functional protein-protein interaction networks. Furthermore, the degree of each protein was calculated to evaluate the importance of the protein in the PPI network.
RNA extraction and RNA-seq
Total RNA was extracted from tumor of both VIM-KO and ctrl group. Nanodrop ND-2000(Thermo Scientific, USA) was used to detect the A260/A280 absorbance ratio of RNA samples. The Rins of RNA were determined by by an Agilent Bioanalyzer 4150 system (Agilent Technologies, CA. Only qualified RNA can be used for library construction. Prepare paired terminal libraries and purify mRNA, followed by synthesizing cDNA using mRNA fragments as templates. Afterwards, the synthesized double stranded cDNA fragments were adapter linked to prepare a paired end library and subjected to PCR amplification. Purification and evaluation of PCR products (AMPure XP system), sequencing and 150bp paired end reading on Illumina Novaseq 6000 (or MGISEQ-T7). Subsequently, quality control and related analysis will be carried out.
NGS data analysis
The quality of raw sequencing data was examined by FastQC (v0.11.7). Contaminated adapter and low-quality sequences were removed by using Trimmomatic (v0.38). Cleaned sequencing reads were then aligned to mm10 genome reference by STAR aligner (v2.5.2). Samtools (v1.3) was then used to remove unmapped reads and low-quality alignments. Htseq-count (v2.0.3) was used to calculate the raw counts for each transcript. The raw counts of different sample groups were compared by DESeq2 (v3.18) to identify the differentially expressed genes. For calling alternative polyadenylation (APA) events, DANPOS (v3.1.1) was used with default settings.
Functional enrichment analysis
To assess the biological functions, the computational web server g:Profiler(9) (https://biit.cs.ut.ee/gprofiler/gost) was utilized to conduct Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses for differentially expressed genes. The resulting GO and KEGG pathway terms were ranked according to the P value and visualized using R software (v4.1.2).