Micropeptide MIAC inhibits the tumor progression by interacting with AQP2 and inhibiting EREG/EGFR signaling in renal cell carcinoma

TCGA database analysis

The RNA seq data and clinical pathologic information (Table S1) of 530 KIRC tumors and 72 normal renal tissues adjacent to tumors was obtained through the TCGA data portal (https://portal.gdc.cancer.gov/). The obtained reads (counts) were normalized to their library sizes and transcript length (RPKM normalization). To compare the percentage survival between different MIAC and AQP2 expression groups, Kaplan–Meier survival curves were generated.

Clinical specimens

All the clinical tumor specimens were obtained from Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School. The formalin-fixed and paraffin-embedded (FFPE) specimens from 7 patients and the frozen tumor tissues from 63 patients who underwent surgery. Tissues were obtained after patients’ written consent under a protocol approved by the institution’s Institutional Review Board.

Cell lines and cell culture

Human renal clear cell carcinoma 786-O and A498 cell lines, embryonic kidney (HEK) 293T cell line were obtained from the American Type Culture Collection. 786-O cells were cultured in RPMI-1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Bioind, USA). A498 cells were cultured in MEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS. HEK-293T cells were cultured in DMEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS.

Cell transfection and vector construction

Lentiviral vectors (pLenti-CMV-GFP-Puro, addgene) harbouring the cDNA sequence were co-transfected with psPAX2 (Addgene) and PMD2.G (Addgene) into HEK-293T cells. Cell transfections with plasmids or siRNAs were performed using Lipofectamine 3000 (Invitrogen) or GP-transfect-mate (GenePharma, Shanghai, China), respectively, following the manufacturer’s instructions. The stable cell lines were established by infecting cells with lentiviruses that expressed the target gene, followed by selection with puromycin. All the siRNAs were obtained from GenePharma and the sequences are listed in Table S2.

Quantitative real-time PCR (qRT-PCR)

Total RNA was isolated using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. And cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (G592, abm, Canada) following the manufacturer’s protocol. The mRNA levels were detected by SYBR Green real-time PCR Master Mix (G891, abm, Canada) and performed on ABI QuantStudio 3 Real-Time PCR System (Applied Biosystems). The mRNA expression was normalized by the expression of GAPDH and relative expression levels were calculated using the 2^-ΔΔCT method in cell and tissue lysates. Primers are shown in Supplementary Table S3.

Protein extracting and Western blotting analysis

Total protein was extracted from cell samples using RIPA lysis buffer (Beyotime Biotechnology) supplemented with Thermo Scientific Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Protein extracts were resolved by SDS-PAGE and then electrophoretically transferred onto a PVDF membrane (EMD Millipore). The membrane was incubated for 2 h in blocking buffer (1 × TBST containing 5% non-fat milk), and then was incubated at 4℃ overnight with following primary antibodies: MIAC (abmart, 1:1000), AQP2 (abcam, ab199975), EREG (Cell Signaling, 12,048), EGFR (Proteintech, 18,986–1-AP), Phospho-EGFR (Abmart, T55232), mTOR(Abmart, T55306), p-mTOR (Abmart, T56571), Akt (Abmart, T55561), Phospho-Akt (Abmart, T40067), ERK1/2 (Abmart, T40071), Phospho-ERK1/2 (Abmart, TP56192), GAPDH (Proteintech, 60,004–1-Ig), HA (Abmart, M20003S). The immunocomplexes were subsequently incubated with the HRP-conjugated secondary antibodies, and detected with the Western Blot Detection kit (Beyotime Biotechnology) and TANON-5200 system (Tanon, Shanghai, P.R. China). The densitometric ratio of protein bands was calculated by ImageJ program.

Co-Immunoprecipitation (Co-IP)

Cell extraction was performed using IP Lysis Buffer (Beyotime Biotechnology) supplemented with Thermo Scientific Halt Protease Inhibitor Cocktail, and then incubated with primary antibody overnight at 4℃. Next day, the antibody-bound protein of interest in lysis buffer was incubated with 40 μl of Protein A Agarose Beads (9863, Cell Signaling Technology). After three washes with Wash Buffer (0.5 M Tris–HCl pH 7.4, 1.5 M NaCl), protein-bound beads were mixed with 5 × loading buffer (Yeasen, Shanghai, P.R. China) and boiled for 10 min at 95℃. The samples were then stored at -20℃ or ready for western blotting analysis.

Cell proliferation assay

Cell proliferation assays were performed using Cell Counting Kit-8 (Hanbio). Briefly, cells were seeded in 96 well plate with 100 μl of complete medium per well. CCK-8 reagent was added into each well at 4 h prior to measurement. Absorbance at 450 nm was measured by a Multiskan Plate Reader (Thermo Fisher Scientific) at indicated time points.

CellTrace Violet Cell Proliferation kit used for in vitro labeling of different cells to trace multiple generations using dye dilution by Flow cytometry (BECKMAN COULTER, CytoFlex). Specifically, the indicated cells were suspended at a density of 1 × 106 cells mL−1 in PBS/0.1% bovine serum albumin (BSA) in preparation for cell labeling. CellTrace Violet (Molecular Probes, Thermo Fisher Scientific, C34557) was prepared at twice the final concentration in PBS/0.1% BSA, and a volume equivalent to each cell suspension was added to each tube immediately prior to incubation in a 37℃ water bath for 20 min. The staining was stopped with completed medium, washed twice for 5 min, and cells were detected by FACS analysis and placed into culture.

Transwell migration assays

The RCC cells were harvested and seeded with serum-free medium into the upper chambers at 4 × 104 cells/well, and the bottom chambers containing culture media and 10% FBS as a chemoattractant, and then incubated for 48 h at 37℃, successfully translocated cells were fixed by ice-cold methanol and stained with 2% crystal violet, and imaged using an inverted microscope (Olympus, IX53P1F).

Cell apoptosis and cell cycle detection

Apoptosis assay in cells was performed using an Annexin V-PE/7-AAD Apoptosis Assay Kit (KeyGEN BioTECH) according to manufacturer’s instructions. Briefly, A498 cells were harvested and incubated with Annexin V-PE and 7-AAD for 15 min. Apoptotic cells populations were analyzed by FACS system. The cell cycle phase distribution were determined by propidium iodide (KeyGEN BioTECH) staining. Briefly, A498 cells were harvested and and fixed with 70% ice-cold ethanol for at least 2 h. After centrifugation at 1000 rpm for 5 min, the cells were washed and resuspended with 0.5 mL PI/RNase buffer for 30 min in the dark. Subsequently, the cell cycle distribution was tested by FACS system.

In vivo animal models

Male BALB/c-nude mice (6–8 weeks old) were maintained under pathogen-free conditions in the Laboratory Animal Center of China Pharmaceutical University and subsequent procedures were performed in accordance with the institutional ethical guidelines for animal experiments. Briefly, to detect the effect of MIAC on the tumourigenicity of RCC cells, 5 × 106 A498 cells stably with MIAC overexpression or mock cells were subcutaneously injected into the mice, respectively. The length and width of the mice tumors were measured every three days and the tumor volume was calculated using the following formula 0.5 × length × width2.

In the in vivo metastasis model, 3 × 106 A498 cells stably with MIAC overexpression or mock cells were injected into the tail vein of mice, tumor progression was monitored twice a week through IVIS Spectrum living imaging system (Perkin Elmer) for four weeks. At the end point of the experiment, mice were humanely euthanized.

For efficacy of chemosynthetic MIAC peptide studies, 5 × 106 A498 cells were subcutaneously injected into the mice according to the above protocol. When the volume of xenograft reached 100 mm3, mice were randomly assigned into seven groups and intravenous (IV) injections with different dosage of MIAC peptides (5 mg/kg, 10 mg/kg, 15 mg/kg) or saline every day for four weeks, and intragastric injections with sunitinib (Selleck, 20 mg/kg, 40 mg/kg) or Axitinib (Selleck, 30 mg/kg) every day for four weeks. The length and width of the mice tumors were measured every three days and the tumor volume was calculated using the above formula.

Molecular Docking

The 3D structures of the peptide MIAC was generated in PEP-FOLD3 (https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/). PEP-FOLD is a de novo approach aimed at predicting peptide structures from amino acid sequences [16]. This method, based on structural alphabet SA letters to describe the conformations of four consecutive residues, couples the predicted series of SA letters to a greedy algorithm and a coarse-grained force field. The 3D structure of the protein AQP2 were downloaded from RCSB Protein Data Bank (PDB ID: 4OJ2). Protein–protein docking in HDOCK [17] was used to predict the binding affinity with MIAC and AQP2. ClusPro [18] is a web-server that based on a hybrid algorithm of template-based modeling and ab initio free docking. The docked structures and interface residues were analyzed using MOE 2019.1.

Streptavidin–biotin pull-down assay

HEK293T cells transfected with plasmids including recombinant HA-AQP2 and different AQP2 mutation for 72 h, cells were lysed in ice-cold lysis buffer as the above protein extracting protocol. The soluble fractions from cell lysates were incubated with synthesized biotin-conjugated peptide MIAC (GL Biochem, Shanghai) overnight at 4℃. Then, Streptavidin beads were added the mix and rotated for 2 h at 4℃, followed by washing with PBST buffer three times. The binding components were eluted by boiling with 1 × Loading Buffer and were analysed by western blot for HA.

Microscale thermophoresis (MST) assay

MST assay was performed to detect binding interactions of peptide MIAC and AQP2 protein as previously described [19]. Chemosynthetic peptide MIAC (NANJING ANJI BIOTECHNOLOGY CO. LTD) was labeled by CY5 fluorescence (Xi’an ruixi Biological Technology Co., Ltd) via the sulfhydryl group on the cysteine. HEK293 cells were used to express the membrane protein Flag-Mcherry-AQP2. With the existence of the TEV cleavage site, the Flag-Mcherry tag was excised by a TEV protease. The obtained AQP2 protein was identified by liquid phase and mass spectrometry. The labeled MIAC with AQP2 protein were incubated 5 min at room temperature, and reaction mixtures were enclosed in premium-coated glass capillaries and loaded into the instrument (Monolith NT.115, NanoTemper, Germany). Kd values were determined using the NanoTemper MO. Affinity Analysis tool as described.

SPR (Surface Plasmon Resonance) assay

A BIAcore T200 instrument (GE Healthcare) was used to detect binding interactions using a direct binding assay format [20]. Prior to activation, the research grade CM5 chip surface was preconditioned using 100 μL injections of HBS-EP buffer at a flow rate of 30 μL/min. AQP2 (10 μg/mL) was immobilized on the sensor surface using standard amine coupling with 10 mM NaAc, pH 4.0 buffer. Peptide were injected over the active protein and reference surface with at least 30 s association and dissociation times. Surface regeneration was achieved using dissociation for a time period allowing the response to return to baseline. Control injections of different concentration of MIAC peptide to allow monitoring of the functionality of the protein surface. SPR equilibrium binding data, consisting of Req values from 5 points concentration series, were analyzed by fitting a simple 1:1 binding to yield Rmax and Kd values using Biacore T200 Evaluation Software.

Statistical analysis

Experimental data were statistically analyzed using Prism 9.0 software program (GraphPad Software Inc., San Diego, CA, USA). The statistical signifcance of diferences was evaluated by two-tailed Student’s t test or two-way ANOVA, derived from triplicate samples of at least three independent experiments. For Kaplan–Meier analysis the log rank (Mantel-Cox) test was performed. *P < 0.05 was considered statistically signifcant.

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