Cell lines and cell culture
Human cell lines SW48, LoVo and HEK293T were purchased from American Type Culture Collection (ATCC). Cells were cultured in DMEM high glucose medium (Gibco, USA) containing 10% FBS (Thermo Fisher Scientific, USA). All of the cells were cultured at 37°C in a 5% CO2 incubator.
Plasmid And Stable Cell Lines Construction
The expressing vector pcDNA3.1-Smad4-Flag was constructed by inserting the PCR product of HEK293T cells cDNA. The vector pCMV-HA-PRMT5 was a gift from Prof. GX Jin. pcDNA3.1-Smad4 R361K-Flag was generated using Mut Express II Fast Mutagenesis Kit V2 (Vazyme, China, based on the following sequences: 5′-CTTCTGGAGGAGATAAATTTTGTTTGGGTCAACTCTCCAA-3′ and 5′-TTTATCTCCTCCAGAAGGGTCCACG-3′). Smad4 knockout cell lines based on SW48, LoVo, and HEK293T were constructed by PX458-462-SpCas9 that were inserted with the guide sequence (5′-CACCGGACAGCACTACCACCTGGAC-3′ and 5′-AAACGTCCAGGTGGTAGTGCTGTCC-3′) into the sgRNA scaffold. We used pMD2G and psPAX lentivirus pack system to generate stable expression-silencing or over-expression cell lines. The pLKO.1 puro vectors expressing shPRMT5 (#1: 5’-CCGGCCCATCCTCTTCCCTATTAAGCTCGAGCTTAATAGGGAAGAGGATGGGTTTTTG-3’, #2: 5’-CCGGGGCTCAAGCCACCAATCTATGCTCGAGCATAGATTGGTGGCTTGAGCCTTTTTG-3’). pLVX-Smad4-Flag puro, pLVX-SMAD4 R361K-Flag puro, and pLVX-HA-PRMT5 puro plasmids were constructed by inserting the indicated DNAs into the pLVX puro vector. We used 1 µg/mL puromycin to select the infected cells for 48h. Routine analysis of all cell lines confirmed identity.
Antibodies And Reagents
Smad4 Rabbit mAb (A19116), Smad4 Rabbit pAb (A5657), PRMT5 Rabbit mAb (A19533), PRMT5 Rabbit pAb (A1520), Symmetric DiMethyl-Histone H4-R3 Rabbit pAb (A3159), Snail Rabbit pAb (A11794), Rhodamine Phalloidin (RM02835), Slug Rabbit pAb (A13352), were bought from Abclonal, China. Anti-PRMT5 (sc-376937) and anti-GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology, USA. Asymmetric Di-Methyl Arginine Motif [adme-R] MultiMab Rabbit mAb mix #13522, Symmetric Di-Methyl Arginine Motif [sdme-RG] MultiMab Rabbit mAb mix #13222, Vimentin (D21H3) XP Rabbit mAb #5741, DYKDDDDK Tag (D6W5B) Rabbit mAb, Rabbit (DA1E) mAb IgG XP Isotype Control #3900, were obtained from Cell Signaling Technology, USA. HA tag Polyclonal antibody (51064-2-AP) was purchased from Proteintech, China. Purified Mouse Anti-E-Cadherin (610182) was bought from BD Transduction Laboratories, USA. Cy3, Goat Anti-Rabbit IgG, (A22220, Abbkine); Cy3, Goat Anti-Mouse IgG (A22210, Abbkine); Dylight 488, Goat Anti-Rabbit IgG (A23220, Abbkine) and DyLight 488, Goat Anti-Mouse IgG (A23210, Abbkine) were used in the immunofluorescence assays. The following secondary antibodies were used in western blot assays: Goat anti-Rabbit IgG (H + L) Secondary Antibody, HRP (Thermo Fisher Scientific, # 31460, USA) and Goat anti-Mouse IgG (H + L) Secondary Antibody, HRP (Thermo Fisher Scientific, # 31430, USA) and HRP-conjugated AffiniPure Mouse Anti-Rabbit IgG Light Chain (AS061, Abclonal, China). The D-Luciferin, Potassium Salt D (40902ES08) was obtained from Yeasen, China. PRMT5 inhibitor GSK3329595 (HY-101563) was purchased from MCE, USA.
Western Blot, Dot Blot, And Co-ip Assays
For western blot assays, cells were washed with PBS and lysed with NP40 lysis buffer containing protease inhibitor cocktail (MCE, HY-K0010, USA). Bicinchoninic Acid Assay (BCA) assay kit (Thermo Fisher Scientific, USA) was used to measure the protein concentration. Cytoplasmic and nuclear protein fractions were extracted by subcellular structure nuclear and cytoplasmic protein extraction kit (Boster, AR0106, China). Protein samples were electrophoretically separated in Tris-HCl sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to PVDF membrane. For the dot blot assay, different quantified proteins were dropped on a PVDF membrane. The above antibodies and HRP-conjugated secondary antibodies were used to incubate with the membrane. The membrane bands were visualized with ECL reagents (Abbkine, China).
For Co-IP assays, corresponding antibodies were incubated with protein A/G magnetic beads (MCE, HY-K0202, USA), and the cell lysate was added to the reaction system for a further 2 h at room temperature. Magnetic beads were washed 4 times with NP40 lysis buffer and then boiled in SDS-loading buffer for 10 min. Protein binding to the beads was analyzed by Western blot subsequently.
Mass Spectrometric Analyses
The Smad4-Flag fusion protein was enriched by Co-IP assay. The protein sample was separated by SDS-PAGE. After Coomassie blue staining, the gel bands of interest were excised from the gel, reduced with 5 mM of DTT, alkylated with 11 mM iodoacetamide, and digested in the gel with α-lysate in 50 mM ammonium bicarbonate overnight at 37°C. The next day, the sample was quenched by adding 10% TFA to adjust the pH to below 2. Then the sample was digested with sequencing-grade modified chymotrypsin in 50 mM ammonium bicarbonate at 25 ℃ overnight. The peptides were extracted twice with 0.1% trifluoroacetic acid in 50% acetonitrile aqueous solution for 1 h and then dried in a speedVac. Peptides were redissolved in 25µL 0.1% trifluoroacetic acid and 6 µL of extracted peptides were analyzed by Orbitrap Fusion Lumos mass spectrometer.
For LC-MS/MS analysis, the peptides were separated by a 40 min gradient elution at a flow rate of 0.30 µL/min with a Thermo-Dionex Ultimate 3000 HPLC system, which was directly interfaced with an LTQ Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, Germany).
Immunofluorescence Assays
Cells were seeded on a cover slide in 24-well culture plates with different treatments. The cells on the cover slide were washed with pre-cool PBS and then fixed with 4% paraformaldehyde for 30 min at room temperature. Next, the non-specific antigens were blocked with 1% BSA-TBST solution for 2 h. Slides were incubated with the primary antibody overnight at 4°C. Then, the slides were embedded with fluorochrome-conjugated secondary antibodies for 2 h at room temperature in the dark and incubated with DAPI (Cat# 9542, Sigma, USA) for 10 min subsequently. We used a confocal scanning microscope (Olympus FLUOVIEW FV1000, Japan) or a fluorescence microscope (Nikon ECLIPSE Ti2, Japan) to obtain representative images.
In vitro kinase assays
His & Flag-tagged PRMT5 recombinant protein (Sino Biological, Cat: 11074-H18H, China), GSK-tagged fusion protein Smad4 (Proteintech, Cat No: Ag0299, USA), and MEP50 recombinant protein (Abnova, H00079084-P01, China) pure proteins were purchased. Each reaction system contained 1 µg His & Flag-tagged PRMT5 recombinant protein, 5 µL 5×protein arginine methyltransferase (PRMT) buffer (50 mM Tris-HCl, 2 mM DTT, 20 mM KCl, 5 mM MgCl2, 10% glycerol), 13 µM S-adenosyl-l-methionine (SAM), 1 µg MEP50 recombinant protein, and GSK-tagged fusion protein Smad4 as substrates, with the addition of ddH2O to 25 µL. The mixture was incubated at 37°C for 10 h followed by western blot analysis.
Tumor Specimens, Crc Tissue Microarrays, And Immunohistochemistry Staining
Clinical samples were obtained from CRC patients who underwent surgical resection at the department of gastrointestinal surgery, Wuhan Tongji Hospital, and informed consent was obtained. The human CRC tissue microarray of 80 primary tumors and adjacent normal tissues bought from Wuhan Baiqiandu Technology Co., Ltd. (Cat. #80A1) was used to evaluate the level of Smad4 R361me2s. For IHC staining, lesions or adjacent tissues were fixed with formalin solution followed by paraffin embedded. Slides were dewaxed, rehydrated, and heated in sodium citrate buffer for antigen retrieval. Endogenous peroxidase was inhibited with 3% hydrogen peroxide and 0.1% sodium azide for 30 mins at room temperature. Non-specific epitopes were blocked with incubation in 5% BSA for 2 h at room temperature. Next, the slides were incubated with indicated primary (diluted anti-PRMT5 (1:150) and diluted anti-Smad4 R361me2s (1:50)) antibodies at 4°C overnight and then with the secondary antibodies at room temperature for 1 h.
The level of Smad4 R361me2s was evaluated by the IHC score ranging from 0 to 12, which was calculated by multiplying the proportion and intensity score. Two experienced pathologists scored the immunostaining results independently. Staining intensity was scored as follows: 0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining. The proportion of positively stained tumor cells was scored as follows: 1 (< 10%), 2 (10%-50%), 3 (50%-75%), and 4 (> 75%).
Transwell Migration Assays
Transwell migration assays were carried out using 24 well 8 µm pore size Transwell chambers (Corning Incorporated, costar 3422, USA). Colorectal cancer cells (2 × 105) in a 600 µL volume of serum-free medium were seeded into the upper chambers and cultured at 37°C for 16 h for in vitro migration assay. The up surfaces of the chamber were wiped with cotton swabs. Cells across pores were fixed with 4% paraformaldehyde and then stained with 1% crystal violet solution. The migrating cell numbers were counted in 3 randomly selected microscope fields (200×).
Quantitative Real-time Pcr (Qpcr) Reactions
The total RNA of tumor cells was isolated with RNAiso reagent (Takara, Japan) and then reversely transcripted into cDNA with cDNA Synthesis Kit (gDNA digester plus) (Yeasen, 11121ES60, China). Reactions were carried out using Hieff UNICON Universal Blue qPCR SYBR Green Master Mix (Yeason, 11184ES03, China) on QuantStudio 3 System (AppliedBiosystem Thermo Fisher Scientific, USA). The results were calculated using the Comparative Ct method with GAPDH as a housekeeping control.
Nude Mice Crc Liver Metastasis Model And Imaging
BALB/c nude mice (6 w) were bought from Gempharmatech Co., Ltd and were kept in the Laboratory Animal Center of Wuhan Tongji Hospital. All mouse experiments were performed following a protocol approved by our institutional Animal Care and Use Committee. Mice were randomly assigned to groups. After administering appropriate anesthesia, 2 × 106 cells of each group suspended in 125 µL serum-free media were injected carefully into spleens. After 6 weeks, D-Luciferin, Potassium Salt D (150 mg/kg) was intraperitoneally injected, and imaging analysis was performed 10 min after injection. The mice were sacrificed and dissected subsequently. Then livers were removed, and tumors were stripped and weighed.
Statistical analysis
Data were statistically analyzed using one-way ANOVA or two-tailed Student’s t-tests for comparison between groups. Clinicopathological parameters associated with the level of R361 me2s were assessed by Chi-square tests and Fisher’s exact tests. A Spearman test was used to analyze the correlation between gene expressions. Survival curves were plotted by the Kaplan-Meier method. All statistical analysis was performed with GraphPad Prism 8.0 (GraphPad Software) and SPSS 22.0 (IBM). All data represent the mean ± SEM. Statistical significance was set at p < 0.05.