piRNA-1742 promotes renal cell carcinoma malignancy by regulating USP8 stability through binding to hnRNPU and thereby inhibiting MUC12 ubiquitination

Accumulating studies have confirmed that PIWI-interacting RNAs (piRNAs) are considered epigenetic effectors in cancer. We performed piRNA microarray expression analysis on renal cell carcinoma (RCC) tumor tissues and paired normal tissues and performed a series of in vivo and in vitro experiments to explore piRNAs associated with RCC progression and investigate their functional mechanisms. We found that piR-1742 was highly expressed in RCC tumors and that patients with high piR-1742 expression had a poor prognosis. Inhibition of piR-1742 significantly reduced tumor growth in RCC xenograft and organoid models. Mechanistically, piRNA-1742 regulates the stability of USP8 mRNA by binding directly to hnRNPU, which acts as a deubiquitinating enzyme that inhibits the ubiquitination of MUC12 and promotes the development of malignant RCC. Subsequently, nanotherapeutic systems loaded with piRNA-1742 inhibitors were found to effectively inhibit the metastasis and growth of RCC in vivo. Therefore, this study highlights the functional importance of piRNA-related ubiquitination in RCC and demonstrates the development of a related nanotherapeutic system, possibly contributing to the development of therapeutic approaches for RCC.

3 strand cDNA using the stem-loop method by a specific stem-loop reverse transcription kit (Vazyme Biotech Co., Ltd, China). SYBR qPCR kit (Vazyme, China) and ABI Prism 7500 sequence detection system (Applied Biosystems, CA) were used for qRT-PCR.
piR-1742 primer designed and synthesized by RiboBio (PQPSCM001-2, RiboBio Biotechnology, Guangzhou, China). The qPCR parameters are as follows: 40 cycles of 30 seconds at 95°C, then 10 seconds at 95°C, and 30 seconds at 60°C. U6/GAPDH was used as an endogenous control. The relative fold change is analyzed by the 2 -ΔΔC(t) method.

Fractionation of nuclear and cytoplasmic RNA
To detect the subcellular localization of piRNAs, a PARIS kit (Thermo Fisher, MA, USA, Cat#AM1921) was used for the extraction of nuclear and cytoplasmic RNAs following the manufacturer's protocol. The qPCR was used to detect RNA abundance in different cell fractions. U6 and GAPDH were used as internal controls for the nucleus and cytoplasm, respectively.
Immunofluorescence RCC cells were seeded onto cell slides. Cells were fixed in 4% formaldehyde for 15 min and permeabilized with Triton X-100 (0.1%) for 20 min. After blocking with goat serum for 30 minutes, the primary antibody was added and incubated overnight at 4°C. The next day, cells were incubated with a secondary antibody for 1 hour at room temperature, and DAPI was used for nuclei staining. A confocal microscope (Zeiss, Pleasanton, CA, USA) was used to capture the images.

Northern Blot
Total RNA was extracted, and 15% urea denaturing PAGE gel was used for total RNA electrophoresis. RNA was immobilized with Stratagene UV cross-linker after about 1h at a constant voltage of 60V. According to the instructions of the Northern Blot Assay Kit, the piR-1742 RNA probe modified with locked nucleic acid and digoxigenin-labeled was 4 used for hybridization reaction with the membrane, and the U6 probe was used as the internal reference. Bands are exposed and analyzed after using HRP Anti-Digoxigenin.

RNA immunoprecipitation
The RIP assays were performed using a RIP kit (BersinBio, Guangzhou, China).
According to the manufacturer's instructions. 1×10 7 RCC cells were immunoprecipitated with RIP buffer containing hnRNPU antibody-coupled magnetic beads, and RNA was extracted from RNA-protein complexes. Then, the expression of piR-1742 was verified by qPCR. IgG was used as a negative control.

RNA pull-down
The piR-1742-binding proteins were examined using an RNA pull-down kit (BersinBio, Guangzhou, China). Biotin-modified piR-1742 and NC probes were incubated with protein extract overnight, followed by precipitation with streptavidin magnetic beads. The retrieved protein was eluted from the RNA-protein complex. Afterward, western blot or mass spectrometry was performed (Oebiotech, shanghai, China).

EdU assay
3×10 4 cells were seeded on cell slides. The next day, the EdU reagent (Yeasen Biotechnology, shanghai, China) was added to the cells and incubated for 1 hour. Cells were fixed in 4% paraformaldehyde for 15 minutes and then permeabilized by adding Triton X-100 (0.3%) for 15 minutes. Add 500 μl of EdU reaction mix (containing Azide 555) to cells and incubate in the dark for 30 min. Hoechst was used for nuclear staining.
Images were captured under a fluorescence microscope.

IHC
Human RCC tissue or mouse tumor tissue was fixed in 4% paraformaldehyde, followed by paraffin embedding and sectioning. For IHC, tissue sections were deparaffinized, dehydrated, antigen retrieved, and blocked. After blocking, the tissue was incubated with antibodies overnight at 4°C. The next day, tissues were incubated with biotinylated goat anti-rabbit IgG for 20 minutes at room temperature, followed by streptavidin-horseradish peroxidase for 30 minutes. Finally, staining was performed using diaminobenzidine-H2O2 and hematoxylin.

Dual-luciferase reporter assays
The dual-luciferase reporter assays were performed to detect the epigenetic regulation of PiR-1742 and USP8. Bioinformatics analysis was performed using the miRanda algorithm.
The wild-type (WT) and mutant (Mut) pGL3 plasmids were constructed and transfected to 293T. After 48 hours, cells were lysed to detect firefly and Renilla fluorescence by a dual-luciferase reporter assay system, and statistical analysis was performed.

ISH)
RCC cells were seeded on cell slides, after which cells were fixed in 4% paraformaldehyde and 0.5% Triton X-100. Subsequently, cy3-labeled piR-1742 and NC probes (Zima, Shanghai, China) were added and hybridized overnight at 37°C. DAPI was used for nuclei staining and images were captured by confocal microscopy (Carl Zeiss AG, Germany) for further analysis. The double (5' and 3')-digoxigenin (DIG)-labeled probe targeting piR-1742 was designed and synthesized by RNAscope®. Expression of piR-1742 was detected using the RNAscope 2.0 detection kit (Red, Cat No. 324500) according to the manufacturer's instructions. The piR-1742 expression was calculated as follows: H-score=Σpi(i+1), pi represents the percentage of the number of positive cells; i represents the intensity of staining.

Nude mouse xenograft assay
1×10 7 RCC cells were injected subcutaneously into the right armpit of a 5-week-old female BALB/c nude mouse. 5-week-old female BALB/c nude mice were subcutaneously 6 injected with 1×10 7 RCC cells. When tumors were~100 mm 3 (10 days), the mice were randomly divided into two groups. One was intraperitoneally injected with antagomir piR-1742 (16 mg/kg), and the other groups were injected with PBS. After 4 weeks, the mouse was sacrificed, and the tumors were removed for IHC, IF, or western blot. The mouse weight and tumor diameter/volume were monitored every 3 days according to the formula (tumor volume = π/6×length×width 2 ).

Tail vein metastasis in nude mouse
1×10 6 RCC cells in 100 μL of PBS were injected into the tail vein of the mouse to observe metastasis in the nude mouse. After 4 weeks, tumor metastasis in nude mice was detected by in vivo imaging system (IVIS) every 3 days. Finally, the nude mouse was sacrificed, and the lung tissues were removed to detect tumor metastasis by IVIS. Lung tissue was used for HE and IHC experiments.

TCGA database and Gene Set Enrichment Analysis (GSEA)
GSEA was used to obtain different biological pathways between groups

Synthesis of piR-1742-inhibitor@PDA@MUC12 nanoparticles
The nano-delivery systems are designed for the targeted therapy of RCC. We developed piR-1742-inhibitor@PDA@MUC12 nanoparticles (pPM-NPs) through a simple and green method. Firstly, 40 nM piR-1742-inhibitor was dissolved in the commercial liposomes (100 μL; Yeasen, China) through a 20s vortex. And the obtained solution was subsequently added to the tris-HCl (pH 8.8; 10 mM) with a final volume of 5 ml.
Meanwhile, 5 mg dopamine hydrochloride was incorporated in the above solution to form polydopamine (PDA) modified liposomes under stirring for 3 h. The PDA-modified liposomes were concentrated by 8,000 rpm centrifugation (15 min) and washed with RNase-free water. Next, the resulting mixture was dissolved in 1 ml streptavidin solution (2 mg/mL; dissolved in PBS with the pH 8~9) and shaken for 12 h in the dark under 4℃ for the preparation of piR-1742-inhibitor@PDA@str(pP). Finally, the prepared pP was added to 1 ml biotinylated MUC12 antibody solution (50 μg/mL; Bioss, China) and shaken for 1 h in the dark under 4℃ to generate the pPM-NPs. The obtained pPM-NPs suspension was washed with distilled water and stored in a PBS buffer under 4℃.

Characterization of nanoparticles
Transmission electron microscope (TEM) imaging was established to characterize SPM-NPs. The samples were immersed in water and dropped on the carbon-cover copper TEM grids. After preparation, the sample was photographed by TEM (JEOL, Tokyo, Japan). Ultraviolet-visible (UV-vis) absorption spectra of pPM-NPs were examined by UV-vis spectrophotometer. Fourier Transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were conducted to evaluate the synthesis of PDA by FTIR-8300 series spectrometer (Shimadzu, Japan) and ESCALAB 250 Xi Mg (Thermo Scientific, Japan) X-ray resource. Meanwhile, the Raman spectroscopy of pPM was examined by using a WITEC Spectra Pro 2300I spectrometer. Additionally, the scanning transmission electron microscopy (STEM) of pPM-NPs was also measured, which was accompanied by energy dispersive X-ray spectrometry (EDS) element mapping examination. The diameters of pP and pPM-NPs were measured using a particle size potentiometer (Nano ZS90, Worcestershire, UK); and the Zeta potential was also simultaneously examined.
Encapsulation capability of siRNA 8 The gel electrophoresis was conducted to examine the encapsulation capability of siRNA possessed by pPM-NPs. There were three subgroups including siRNA as control, pP, and pPM. 0.5 g agarose was added into 40 ml 1×TAE buffer to form the gels through the boil and cool process. And the RNA or NPs were mixed with 2 μl 6×DNA loading buffer and 1 μl SYBR Green I nucleic acid gel stain solution to gain the 12 μl final mixture. Then the mixture was added to the dented pores with 80 V electrophoresis voltage for 30 min. After the electrophoresis, the gel was imaged using the gel imaging system and photographed by the Tanon Gel image system (Shanghai, China).

MUC12 incorporation measurement
Western blot analysis was conducted to examine the incorporation of MUC12 antibody with the contribution of coomassie blue staining. The experimental subgroups were divided into PDA, pP, and pPM. 16 μl samples were mixed with 4 μl 5× protein loading buffer and heated at 100℃ for 30 mins. And the mixtures were separately added in the dented pores of the 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) for electrophoresis with 120 V. After 1.5 h electrophoresis, the gel was dyed by coomassie blue staining buffer for 3 h and washed by distilled water subsequently. The final gel was photographed the next day.