786-O, Caki-1 and ACHN cell lines were purchased from National Collection of Authenticated Cell Cultures, Chinese Academy of Sciences (Shanghai, China). The 769-P cell line was purchased form iCell Bioscience lnc. (Shanghai, China). 786-O and 769-P cells were cultured in RPMI-1640 medium (Invitrogen, Cat#11875-093) supplemented with 10% fetal bovine serum (FBS, Biological Industries, #04-001-1ACS; Beit Haemek, Israel) and 1% penicillin-streptomycin (Solarbio, Cat#P1400, Beijing, China). Caki-1 cells were cultured in McCoy’s 5A medium (Biological Industries, Cat# 01-075-1A) with 10% FBS and 1% penicillin-streptomycin. ACHN cells were maintained in MEM (iCell Bioscience lnc, Cat# iCell-0012; Shanghai, China) with 10% FBS and 1% penicillin-streptomycin. All cells were cultured in a humidified incubator at 37 °C and 5% CO2. All cell lines were verified to be free of mycoplasma contamination every 3 months. Individual cell line authentication was confirmed by Tsingke Biotech CO., Ltd. (Beijing, China).
Antibodies and reagents
Information on antibodies used in this study is provided in Supplementary Table 1. The following reagents were purchased from indicated companies: Doxycycline (Med Chem Express, Cat#HY-N0565B; New Jersey, USA), Cycloheximide (Med Chem Express, Cat#HY-12320), Protease Inhibitor Cocktail (Cell signaling technology, Cat#5871s), RNase Inhibitor (Beyotime, Cat#R0102-10kU, Shanghai, China), 4% paraformaldehyde (Servicebio, Cat#G1101; Wuhan, China), Opti-MEMTM Reduced Serum Medium (ThermoFisher Scientific, Cat#31985070), Protease K (Solarbio, Cat# P1120; Beijing, China), Mounting medium with DAPI (Abcam, Cat#ab104139).
Small interfering RNA and plasmid transfection
The small interfering RNAs (siRNAs) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China) and transfected using Lipofectamine™ RNAiMAX transfection reagent (ThermoFisher Scientific, Cat#13778030) according to manufacturer’s instructions. Plasmids pcDNA3.1-MILIP, pcDNA3.1-MILIP-DDFO, pEGFP-N1-YBX1(FL), pEGFP-N1-YBX1-DAP, pEGFP-N1-YBX1-DCSD, pEGFP-N1-YBX1-DCST, pGL3-MILIP promoter and pGL3-MILIP promoter-DBR were constructed by Tsingke Biotech CO., Ltd. (Beijing, China) and transfected using Lipofectamine 3000 transfection reagent (ThermoFisher Scientific, Cat#L3000001) according to manufacturer’s instructions. siRNA sequences are listed in Supplementary Table 2.
Inducible shRNA knockdown
Short hairpin RNA (ShRNA) oligos were constructed into the FH1-tUTG plasmid as previously described . Lentivirus particles were packaged in HEK293 cells through co-transfecting FH1-tUTG inserted with shRNA oligos (6 µg), pMDLg.pRRE (6 µg, Addgene, Cat#12251), pMD2.g (6 µg, Addgene, Cat#12259) and pRSV.Rev (3 µg, Addgene, Cat#12253) plasmids. The lentiviral particles were harvested after 48 hrs transfection and stored in -80℃. Caki-1 or ACHN cells were transduced with the lentiviral particles in 3.5 cm cell culture dishes to establish inducible knockdown cell sublines. The knockdown of MILIP was induced in response to doxycycline treatment. ShRNA oligos were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). ShRNA oligo sequences are listed in Supplementary Table 2.
Transwell migration assay
Cells with or without MILIP knockdown were seeded to the upper chambers of 24-well transwell inserts (8 µm; Corning, Cat#3422; 5×104 cells/well) and maintained in serum-free medium. The bottom chambers were filled with medium supplemented with 10% FBS. After 48 hrs of culture, cells grown in inserts were fixed with 4% paraformaldehyde (Servicebio, Cat#G1101; Wuhan, China) for 10 min and then stained using 1% crystal violet stain solution (Solarbio, Cat#G1062; Wuhan, China) for 15 min. The non-migrated cells on the upper surface of the membranes were removed by scrubbing with a cotton tipped swab. Cells that migrated to the lower surface of the membranes were further imaged using Macro Zoom Fluorescence Microscope System (Olympus, MVX10) prior to quantification with the ImageJ software.
Transwell invasion assay
Cell invasion was assayed using inserts pre-coated with 7% Matrigel (BD Biosciences, Cat#BD-356234). Cells with or without MILIP knockdown were loaded in the upper chambers of 24-well transwell inserts (1×105 cells/well) and maintained in serum-free medium. The bottom chambers were filled with medium supplemented with 10% FBS. After 24 hrs of culture, cells were fixed and stained as described in the migration assay. After removing the non-invaded cells on the upper surface of the membranes, the invasive cells were imaged and quantified following the methods in the migration assay.
Wound healing assay
Cells with or without MILIP knockdown were grown to 90% confluence, and then monolayers were scratched with 200 µl pipette tips across the center of wells. After being washed with PBS for three times, cells were then maintained in the serum-free medium and imaged over a 24 hrs period at 12 hrs intervals to monitor wound width. The wound width was quantified using the Vision Works software (Analytik Jena, Jena, Germany).
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA from ccRCC cells was extracted with Trizol reagent (Solarbio, Cat#R1100; Beijing, China) according to manufacturer’s instructions. cDNA was then synthesized from 1 µg of total RNA using HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Cat#R211; Nanjing, China). Of the resultant cDNA, 25 ng was used in the 20 μl qRT-PCR mix, containing 10 μl RealStar Green Power Mixture (GenStar, Cat#A313; Beijing, China) and 0.25 μM of each primer. Samples were amplified for 40 cycles using the Applied Biosystems® 7500 Real-Time PCR System (ThermoFisher Scientific). 2−ΔΔCT method was used to calculate the relative gene expression levels normalized against the housekeeping gene β-Actin. Primer sequences are listed in Supplementary Table 3.
Biotin RNA pulldown assay
The RNA pulldown assay was performed as previously described . In brief, Caki-1 and ACHN cell pellets were lysed in lysis buffer [50 mM Tris-HCl (PH 7.5), 150 mM NaCl, 2.5 mM MgCl2, 1 mM EDTA, 10% Glyceral, 0.5% Nonidet P-40/Igepal CA-630, 1 mM DTT, RNase Inhibitor and Protease Inhibitor], followed by sonication for 3 cycles with 10 sec on/30 sec off. Cell lysates were then rotated with streptavidin beads (ThermoFisher Scientific, Cat#20349) coated with 4 μg antisense/scramble biotin-labeled probes for 4 hrs at 4°C. Beads were then washed with lysis buffer for 6 times followed by RNA isolation and immunoblotting analysis. Information of antibodies and probes used in this study is shown in Supplementary Table 1 and Supplementary Table 4, respectively.
RNA immunoprecipitation (RIP) assay
The RIP assay was performed using the Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (Millipore, Cat#17-701) following instructions provided by the manufacturer. In brief, 2×107 cells were lysed with equal pellet volume of RIP lysis buffer. 100 μl cell lysate was incubated with PierceTM Protein A/G Agarose (ThermoFisher Scientific, 20422) pre-coated with YBX1 antibodies at 4°C overnight. Normal rabbit or mouse IgG were used as negative controls. For cells transfected with pEGFP-N1-YBX1(FL), pEGFP-N1-YBX1-DAP, pEGFP-N1-YBX1-DCSD or pEGFP-N1-YBX1-DCST plasmids, the lysates were incubated with anti-GFP mAb-Magnetic Agarose (MBL, Cat#D153-10; Nagoya, Japan). After being washed with RIP wash buffer, beads-bound immunocomplexes were prior to RNA isolation and immunoblotting analysis. Information of antibodies and primers used in this study is shown in Supplementary Table 1 and Supplementary Table 3, respectively.
Domain-specific chromatin isolation by RNA purification (dChIRP)
dChIRP assays were performed as previously described . Briefly, Caki-1 and ACHN cells were harvested and cross-linked in 1% glutaraldehyde for 10 min at room temperature. Cell pellets were then lysed in lysis buffer (50 mM Tris-HCl [pH 7.0], 10 mM EDTA, 1% SDS, PMSF, Superase-in) on ice for 30 min, prior to sonication for three cycles (10 sec on/30 sec off). Four micrograms antisense/scramble biotin-labelled probes against MILIP RNA was rotated with cell lysates at 37 °C for 4 hrs, followed by further incubating with 100 µl C-1 magnetic beads (Invitrogen, #65002) at 37 °C for 30 min. Beads were then washed in wash buffer for five times, followed by RNA isolation. For cell free system, dChIRP assays were performed using 1 µg in vitro-transcribed biotin-labeled MILIP RNA or MILIP RNA with its DFO deleted incubated with 1 µg Snai1 RNA or Snai1 RNA with MILIP-BR deleted. In vitro-transcribed MILIP RNA without biotin labeling was included as a negative control. Primer and probe sequences are shown in Supplementary Table 3 and Supplementary Table 4, respectively.
In situ hybridization (ISH)
The ISH assays were performed with RNAscope® 2.5 HD Detection Reagent-Brown kit (Advanced cell diagnostics, Cat#322310) according to manufacturer’s instructions. In brief, formalin-fixed paraffin-embedded (FFPE) ccRCC tissue microarray (Cat# HKid-CRC060CS-01) purchased from the Shanghai Outdo Biotech Co., Ltd (Shanghai, China) was deparaffinized, rehydrated, boiled in target retrieval solution, and treated with proteinase K. Sections were then incubated with probes (Advanced Cell Diagnostics, Cat#547581) for 4 hrs at 40°C. After being washed, sections were incubated with 3,3’-diaminobenzidine (DAB), and counterstaining was performed using hematoxylin. Quantification was performed by examining the percentage of positive cells ranged from 0 to 100%. The intensity of staining (intensity score) was judged on an arbitrary scale of 0–4: no staining (0), weakly positive staining (1), moderately positive staining (2), strongly positive staining (3) and very strong positive staining (4). A reactive score (RS) was derived by multiplying the percentage of positive cells with staining intensity divided by 10.
Subcellular fractionation was performed as previously described [19, 20]. 1×107 cells were harvested and lysed in 600 μl hypotonic buffer I (25 mM Tris–HCl, PH 7.4, 1 mM MgCl2, 5 mM KCl) for 5 min on ice. 600 μl hypotonic buffer II (25 mM Tris–HCl, PH 7.4, 1 mM MgCl2, 5 mM KCl, 1% NP-40) was then added to lysates and incubated for further 5 min on ice. After centrifugation at 5,000 × g for 15 min, supernatants were collected as cytoplasmic fractions. The pellets were washed with cold PBS followed by nuclear fractionation using nucleus resuspension buffer (20 mM HEPES, PH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF). Supernatants were harvested as nuclear fractions after centrifugation at 12,000 × g for 10 min.
In vitro transcription
The in vitro transcription assay was performed as previously described . DNA templates for in vitro transcription were generated by PCR amplification using forward primers containing the T7 RNA polymerase promoter sequence and reverse primers without the promoter sequence. In vitro transcription was then performed using TranscriptAid T7 High Yield Transcription Kit (Thermofisher Scientific, Cat#K0441) following the manufacturer’s instructions. Primer sequences are listed in Supplementary Table 5.
Chromatin immunoprecipitation assay (ChIP)
The ChIP assay was performed using Chromatin Immunoprecipitation (ChIP) Assay Kit (Beyotime, Cat#P2078; Shanghai, China) according to manufacturer’s instructions. In brief, cells crosslinked with 1% formaldehyde for 10 min at 37°C were lysed and sonicated. Cell lysates were then rotated with an anti-TFAP2C antibody or a corresponding anti-normal rabbit IgG antibody at 4 °C overnight. Then, 60 μl of protein A/G agarose beads was added to the antibody-lysate mixture and rotated for 1 hr at 4 °C. Beads were washed, and DNA fragments were eluted, purified and analyzed by RT-PCR with specific primers. Information of antibodies and primers used in this study is shown in Supplementary Table 1 and Supplementary Table 4, respectively.
Luciferase reporter assays
Luciferase reporter assays were performed using the Dual-Luciferase Reporter Assay System (Promega, Cat#E1910) according to the manufacturer’s instruction. In brief, cells were co-transfected with pGL3-MILIP promoter reporters or pGL3-MILIP promoter-DBR reporters, together with pGL4.73[hRluc/SV40] reporters expressing the renilla luciferase. Firefly and renilla luciferase activities were examined after 24 hrs transfection.
Mouse models of metastasis
1.5×106 ACHN.shMILIP.1 cells were injected via the tail vein into 5-week-old male NOD/SCID mice (Nanjing GemPharmatech Co., Ltd Company, China) (eight mice per group). Drinking water containing doxycycline (1 mg/ml, supplemented with 10 mg/ml sucrose) was used to induce MILIP knockdown, whereas drinking water containing PBS was included as the control. After 7 weeks of transplantation, mice were sacrificed, and lung tissues were excised. Studies on animals were conducted in accordance with relevant guidelines and regulations and were approved by the Animal Research Ethics Committee of Zhengzhou University (Zhengzhou, China). All mice were housed in a temperature-controlled room (21–23 °C) with 40–60% humidity and a light/dark cycle of 12 h/12 h.
All statistical analysis was performed using Graphpad Prism 8 and Microsoft Excel software to assess differences among experimental groups. Statistical differences were analyzed by two-tailed Student’s t-test or one-way ANOVA test followed by Tukey’s multiple comparisons. P values <0.05 were considered to be statistically significant.