RCC tissues and correspondingly matched adjacent normal tissues were obtained from 90 patients who underwent nephrectomy between January 2014 to December 2019 at the Department of Urology, Shanghai Tenth People's Hospital of Tongji University (Shanghai, China), Zhongda Hospital of Southeast University (Nanjing, China) and Beijing Chao-Yang Hospital of Capital Medical University (Beijing, China). Histopathology of all the cases of RCC was confirmed by senior pathologists and staged according to the 8th edition of the American Joint Committee on Cancer (AJCC). None of the patients received radiotherapy or other treatment modalities prior to surgery. Demographics and clinicopathological information of these patients are listed in Table S1. The protocol for the collection of tissue samples was evaluated and approved by the Ethics Committee of the Shanghai Tenth People's Hospital (SHSY-IEC-BG/02.04/04.0–81,602,469). All patients provided written informed consent.
Cell lines and cell culture
Human RCC cell lines, 786-O (RRID: CVCL_1051), A498 (RRID: CVCL_1056), OSRC-2 (RRID: CVCL_1626), 769-P (RRID: CVCL_1050), Caki-1 (RRID: CVCL_0234), Caki-2 (RRID: CVCL_0235), ACHN (RRID: CVCL_1067), normal renal tubular epithelial cells (HK-2) (RRID: CVCL_0302), and human umbilical vein endothelial cells (HUVECs) (RRID: CVCL_2959) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The culture medium used for maintaining each cell line and the culture conditions were followed as described previously [3, 27]. The RCC cell lines were stored at -80 °C using CELLSAVING reagent (NCM, Suzhou, China).
Tube formation assay
Fifty μL Matrigel (BD Biosciences, USA) was added to each well of a 96-well plate (Corning, USA) separately and allowed to incubate at 37 °C for 1 h. Next, 5000 HUVECs were seeded per well and incubated for 6 h in media containing the supernatant of the pre-treated cells. Photographs were captured and cells were counted under a microscope (Leica Microsystems, Mannheim, Germany).
Sphere formation assay
Transfected cells were seeded into ultra-low attachment 96-well plates (Corning, USA) and cultured in DMEM-F12 media (Gibco, USA) supplemented with serum-free media containing 5 μg/mL insulin (Sigma, USA), 20 ng/mL fibroblast growth factor (Sigma, USA), and 20 ng/mL epidermal growth factor (Sigma, USA). The spheres thus formed were imaged after two weeks.
Fluorescence in situ hybridization (FISH)
In situ hybridization assay was performed using specific probes for SLERCC and UPF1 to observe the localization of SLERCC and UPF1 in RCC cells. Briefly, 786-O and ACHN cells were grown on crawl sheets according to the manufacturer's instructions (C10910, RiboBio, Guangzhou, China). Following fixation and permeabilization, the cells were probed with cy3-labeled UPF1 and fluorescein isothiocyanate (FITC)-labeled SLERCC probes overnight at 37 °C; the nuclei were stained with DAPI.
5′-azacytidine (5-AZA) treatment
ACHN and 786-O cells were seeded into 12-well plates (Corning, USA) and allowed to adhere for 16 h. The cells were then treated with 5 μmol/L 5-AZA for three days, following which the media was discarded. RNA and DNA were extracted as described previously .
Bisulfite sequencing PCR (BSP)
BSP was performed according to the protocol specified in the DNA methylation kit (Qiagen, cat# 59,824). Assessment of the methylation status of the SLERCC promoter region by BSP was performed by IBSBIO (Shanghai, China). The methylation patterns were evaluated using the BiQ Analyzer software.
RNA immunoprecipitation (RIP) assay
RIP experiments were performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA) following the manufacturer's protocol. Briefly, the correspondingly treated cells were harvested and lysed using RIP lysis buffer. Samples were immunoprecipitated using magnetic beads conjugated with anti-UPF1 antibody (Abcam, MA, USA) or rabbit anti-IgG as the negative control. The purified total RNA was subsequently analyzed by qRT-PCR.
Analysis of the TCGA-RCC dataset
The information on TCGA-RCC patients was extracted from The Cancer Genome Atlas (TCGA) database, which included data on gender, age, TNM stage, histological grade, pathological stage, survival status, survival time, lncRNAs, and transcriptome profile (Fragments Per Kilobase Million [FPKM] value). Kaplan–Meier survival curves, univariate and multifactorial Cox regression analyses were used to assess the effects of SLERCC, MUC12, and DNMT3A on OS and DFS.
We used the online software starbase (https://starbase.sysu.edu.cn/) to predict the proteins that could potentially bind directly to SLERCC. Next, we used the catRAPID (http://service.tartaglialab.com/page/catrapid_group) website to evaluate the Z-score, interaction strength, and RNA binding domain instances for each protein, and RPISeq (http://pridb.gdcb.iastate.edu/RPISeq/) to assess the random forest (RF) and support vector machine (SVM) classifiers.
si-DNMT3A, a negative control (si-NC), and three small interfering RNA oligos (si-SLERCC#1, si-SLERCC#2, and si-SLERCC#3) specifically targeting SLERCC, were purchased from IBSBIO (Shanghai, China). The full-length cDNA of human SLERCC was synthesized by Invitrogen and cloned into the pCDNA3.1 expression mini vector. Control plasmids and plasmid-mediated SLERCC overexpression, as well as knockdown vector constructs (sh-SLERCC) and plasmid-mediated DNMT3A overexpression, were obtained from IBSBIO (Shanghai, China). For functional in vitro assays in RCC cells, transient transfection was performed using lipofectamine-3000 (Thermo Fisher Scientific, USA) at cell confluency of 30-50%. For lentiviral transduction, packaging plasmids along with sh-NC, SLERCC, or sh-SLERCC vectors were co-transfected into HEK-293 T cells and incubated for 48 h. Virus-containing supernatants were collected and added to the target cells, and finally, the infected cells were screened by puromycin selection (Gibco, USA).
To identify RCC-associated lncRNAs, we analyzed three pairs of human RCC tissues and matched paracancerous normal tissue gene arrays. RNA sequencing was performed following the procedure described previously . The screening criteria for the differential genes expression were absolute fold change ≥ 1 and false discovery rate (FDR) < 0.05.To elucidate the molecular mechanism underlying SLERCC involvement in RCC progression, we also performed an RNA sequencing of the ACHN and Caki-1 cells transfected with sh-SLERCC and sh-NC lentiviral constructs.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells or tissue specimens stored in liquid nitrogen using the Trizol reagent (TaKaRa, China). RNA was isolated from the nuclear and cytoplasmic fractions using the NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo Fisher Scientific, Waltham, USA). cDNA was obtained by reverse transcription using a cDNA kit (R312, Vazyme Biotech, Nanjing, China), following which qRT-PCR was performed using the SYBR Green PCR kit (Q141, Vazyme Biotech, Nanjing, China). Subsequently, the CT values of the samples were determined on the ABI Prism 7500 sequence detection system (Applied Biosystems, USA). The relative expressions of SLERCC, UPF1 and DNMT3A were calculated using the 2-ΔΔCt method, and the expression of GAPDH was used as the internal reference. All primer sequences are listed in Table S2.
Cell Counting Kit-8 (CCK-8) assay
Pre-treated or transfected cells were inoculated in 96-well plates (Corning, USA) at a density of 2000 cells per well. After seeding cells and incubating them for 12 h, 24 h, 48 h, 72 h, and 96 h, the media were discarded and 10 µl CCK8 solution (Yeasen, Shanghai, China) in 100 µl serum-free medium was added to each well. The cells were incubated in the dark for 2 h at 37 °C, and the optical density (OD) values were measured at 450 nm using a microplate spectrophotometer (BioTek, Winooski, USA).
5-Ethynyl-2′-deoxyuridine (EdU) assay
Pre-treated or transfected cells were seeded in 6-well plates (Corning, USA) and cultured overnight. After incubation with 10 μM EdU reagent for 2 h, the cells were fixed with paraformaldehyde (4%), permeated with 0.5% Triton X-100 in PBS buffer, followed by washing with PBS buffers. Next, the cells were incubated with AlexaFluor-488 in the dark; nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI), and finally, images were captured using an Olympus microscope (Tokyo, Japan).
Wound healing assay
Transfected cells were seeded into 6-well plates (Corning, USA). When the cells attained 80% confluency, they were scratched using a 200 μL pipette tip. The debris was subsequently washed out using PBS buffer, and media supplemented with 2% fetal bovine serum was added to each well. Photographs were taken at 0 h and 24 h of wounding using an Olympus microscope (Tokyo, Japan).
Assays to assess cellular migration and invasion were performed. The upper chamber was pre-coated with Matrigel (BD Biosciences, USA) for cellular invasion experiments. Specifically, pre-treated or transfected cells were inoculated into the upper chamber, and 600 μL of 10% media was added to the lower chamber. After 12–24 h of incubation, cells in the upper chamber were removed using cotton swabs, while those on the surface of the lower chamber were fixed using anhydrous ethanol, stained with 0.1% crystalline violet (Vicmed, China), photographed, and counted using a microscope (Leica Microsystems, Mannheim, Germany).
Western blotting, immunohistochemistry (IHC), and RNA pull‐down assay
Western blotting and IHC were performed as described previously [28, 29], and information on the antibodies used is listed in Table S3. SLERCC biotin-labeled and NC biotin-labeled probes were added to the cell lysate products, accordingly, and the complexes were subsequently incubated with 50 μl of streptavidin magnetic beads (Thermo Fisher Scientific, Inc.) at room temperature. The products were subjected to RNA extraction and purification protocols, and finally, the PCR products were analyzed by agarose gel electrophoresis and Western blotting.
Synthesis of Plasmid-SLERCC@PDA@MUC12 nanoparticles (PSPM-NPs)
The Plasmid-SLERCC@PDA@MUC12 nanoparticles (PSPM-NPs) were synthesized by a rapid and green method. Briefly, 50 nM SLERCC plasmid dissolved in RNase free water was loaded into the commercial liposomes (50 μL; Yeasen, China) by vortexing for 30 s to form Plasmid-SLERCC@lipsome (PS). The resulting suspension was dispersed in 5 mL Tris–HCl (pH 8.8; 10 mM) solution with the subsequent addition of dopamine hydrochloride (5 mg) resulting in the formation of polydopamine (PDA) (Adamas-beta Inc. China) modified liposomes after stirring for 3 h. Next, the PDA-modified liposomes (Plasmid-SLERCC@PDA, PSP) were collected by centrifugation at 8,000 rpm for 10 min and washed using distilled water. Next, the obtained mixtures were dispersed in 1 ml streptavidin solution (2 mg/mL; dissolved in PBS with the pH 8 ~ 9) and shaken thoroughly for 24 h in dark at 4 ℃ resulting in the synthesis of Plasmid-SLERCC@PDA@Str (PSPs). For PSPM-NPs synthesis, the obtained PSPs was mixed with 1 ml biotinylated MUC12 antibody solution (50 μg/mL; Bioss, China) and incubated for 1 h. The obtained suspension was washed using PBS buffer and stored at 4 ℃ till further use.
Characterization of NPs
Imaging by transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) with energy dispersive X-ray spectrometry (EDS) element mapping was performed to characterize PSPM-NPs. The sample was placed on the carbon cover copper TEM grids and photographed by TEM (JEOL, Tokyo, Japan).
Determination of particle size potential of NPs
Separately, 20 μL of PSP and PSPM-NPs were dissolved in 1 mL distilled water. The zeta potentials and sizes of the samples were determined using a particle size potentiometer (Nano ZS90, Worcestershire, UK).
Encapsulation capability of SLERCC plasmid
The encapsulation of SLERCC plasmid was examined by gel electrophoresis. Using empty plasmid as a negative control, PSP and PSPM were separately examined to determine their RNA encapsulation capability. The gels were prepared by mixing 0.6 g agarose in 30 ml 1 × TAE buffer. After mixing with 2 μL 6 × DNA loading buffer and 1 μL SYBR Green I nucleic acid gel staining solution, 9 μL samples were loaded in the dented pores. Electrophoresis voltage was set at 120 V for 20 min. Subsequently, the gel was imaged and analyzed on the Tanon Gel image system (Shanghai, China).
Measuring MUC12 incorporation
The incorporation of MUC12 was measured by western blot analysis. After adding 4 μL 5 × protein loading buffer, 16 μL samples were heat-denatured by boiling for 30 min at 100℃. Next, PSP and PSPM-NPs samples were loaded into the 10% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) for electrophoresis with PDA as the negative control. After electrophoresis for 2 h, the gels were dyed using Coomassie blue staining buffer for 3 h and washed by distilled water, and stored overnight. On the next day, the gels were photographed; the MUC12 band was observed at ~ 62 kDa.
Cytophagy of PSPM-NPs
For successful plasmid delivery, the cytophagy of PSPM-NPs was examined by bio-TEM imaging. ACHN cells were cultured in a 6-well plate for 16 h till they reached 70 ~ 80% confluency. The cells were incubated for 6 h with 40 μL SPM diluted in 2 mL DMEM media and washed using distilled water. Next, the cells were trypsinized and collected by centrifugation. The cells were fixed with glutaraldehyde fixative (2.5%) at stored overnight at 4℃. The prepared samples were washed and dehydrated before polymerization using Spurr’s low-viscosity solution at 60℃ for two days. Finally, the samples were sliced and stained with lead citrate before bio-TEM imaging.
A total of 60 male BALB/c-nu mice, aged 4–6 weeks, were purchased from Slac Laboratory Animal (Shanghai, China). All mice were housed in a pathogen-free environment, and all the animal experiments were performed in accordance with the protocol approved by the Animal Research Ethics Committee of the Shanghai Tenth People's Hospital. The length and width of the tumors in mice were measured weekly, and the tumor volume was calculated using the following formula: volume (mm3) = 0.5 × width2 × length. After sacrificing, the weights of each tumor from all mice were recorded.
Subcutaneous xenograft model
a) 100 μl of 5 × 107 ACHN cells were mixed with 100 μl of Matrigel (BD, USA) and injected subcutaneously into mice (4 groups, n = 3 in each group). 3 weeks later, PBS, Sunitinib, PSPM-NPs or PSPM-NPs + Sunitinib (both 10 nmol) were injected intravenously three times a week (200 μl) for two weeks. b) 100 μl of 5 × 107 sh-NC and sh-SLERCC#3 stably transfected ACHN cell lines were mixed with 100 μl of Matrigel (BD, USA) and injected subcutaneously into mice (2 groups, n = 4 in each group). Tumor size and tumor changes were observed.
Tail vein lung metastasis model
Two hundred μl of 1 × 106 ACHN cells were injected into the tail vein of each mouse (4 groups, n = 6 in each group). 3 weeks later, PBS, Sunitinib, PSPM-NPs, or PSPM-NPs + Sunitinib (both 10 nmol) were injected intravenously three times a week (200 μl) for two weeks, respectively. Tumor progression was observed on an IVIS imaging system (Calipers, Hopkinton, USA).
Orthotopic xenograft model
1 × 106 sh-NC and sh-SLERCC#3 stably transfected ACHN cell lines were orthotopically implanted into mice (2 groups, n = 5 in each group) in the subrenal positions of both kidneys. Tumor progression was observed on an IVIS imaging system (Calipers, Hopkinton, USA).
In vivo biocompatibility of PSPM-NPs
Six male BALB/c mice were randomly divided into two groups (2 groups, n = 3 in each group). The control group and PSPM-NPs group were injected with 200 μl of PBS or PSPM-NPs (both 10 nmol), respectively. Mice were sacrificed at 15 days, and lung, liver, spleen, kidney, heart and colon were harvested for subsequent HE staining and IHC staining.
R-Studio (Boston, USA), SPSS 20.0 (RRID:SCR_002865, Inc., Chicago, USA), and GraphPad Prism 8.3 software (San Diego, USA) was used for statistical analyses. Two-tailed Student's t-test or χ2 test was used to assess differences between components. Survival analysis was performed using the Kaplan–Meier method and significance was confirmed by a log-rank test. Univariate and multivariate Cox proportional hazards models were used for survival analyses. P-values < 0.05 were considered statistically significant.