Cell lines and culture
CNE-1 and SUNE-1 cells were purchased from China Center for Type Culture Collection (Wuhan, China). CNE-2 and NP69 were obtained from the Chinese Academy of Science Cell Bank (Shanghai, China). 6-10B was a kindly gift of Prof. Yiwen You from Affiliated Hospital of Nantong University. Four NPC cell lines (CNE1, CNE2, SUNE-1 and 6-10B) were cultured in RPMI medium 1640 (NCS, Hyclone, Invitrogen) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). The immortalized normal nasopharyngeal epithelial cell line NP69 was cultured in Keratinocyte-SFM medium supplemented with epidermal growth factor. Cultures were maintained at 37 °C under 5% CO2.
Patients
Samples from 129 NPC patients and 20 healthy volunteers were collected between January 2007 and December 2009 at Fujian Cancer Hospital. All patients were newly diagnosed and histologically proven with NPC. The TNM stages of NPC were classified according to the International Union Against Cancer/American Joint Committee on Cancer (UICC/AJCC). This study was approved by the ethics committee of Fujian Cancer Hospital (Approved number, SQ2020–056-01).
Quantitative real-time PCR
Total RNA was extracted using Trizol (Invitrogen) according to the manufacturer’s protocol. To examine the expression of RNF38 mRNA (forward primer: 5′-GCACGCAACAGAAGAAGTCC-3′; reverse primer: 5′-TGGATGAGCAGCAGGATGTAG-3′), complementary DNA (cDNA) was synthesized with the Transcriptor First strand cDNA synthesis Kit (Roche), PCR was performed with LightCycler 480 SYBR green I master (Roche). Data were normalized to GAPDH expression. The fold changes were calculated through the relative quantification method (2−ΔΔCt). All reactions were run in triplicate times.
Lentivirus production and infection
The RNF38 lentiviral vector and control vector were purchased by GeneChem, Shanghai, China. CNE2 cell lines and SUNE1cell lines were stably transfected with RNF38 lentiviral vector (GSV138) or control vector and named RNF38 or NC, respectively. 6-10B cell lines were stably transfected with shRNF38 lentiviral vector (GV248) or control vector and named in-RNF38 or in-NC, respectively. The sequence of shRNF38 was as follows, CTTCCTTCTTATCGGTTCAAT. Enhanced infection solution and polybrene were used to assist the process of lentivirus infecting NPC cells. Puromycin was used to select NPC cells that stably overexpressing and downregulating RNF38. All experiments were carried out on mycoplasma-free cells.
Western blot analyses
Western blot analyses were performed according to the standard methods. Primary antibodies were listed as follows: anti-RNF38 (1:250, Abcam), anti-ACTN4 (1:1000, Cell Signaling Technology), anti-p-Erk1/2 (1:1000, Cell Signaling Technology), anti-p-p65 (1:1000, Cell Signaling Technology), anti- catenin (1:1000, Cell Signaling Technology), anti-MMP2 (1:200, Cell Signaling Technology), anti-MMP9 (1:250, Abcam). Briefly, the blots were incubated with the following primary antibodies at 4 °C overnight, followed by incubation with the Horseradish Peroxidase-Conjugated secondary anti-rabbit or anti-mouse (1:3000, Santa Cruz) for 1 hour at room temperature. GAPDH (1:10000, Abcam) serves as the endogenous loading control.
Immunohistochemistry (IHC) examination
Two independent pathologists (Lihua Zhong and Wenhui Liu), who were unaware of the patient characteristics, evaluated the specimens. Expression of RNF38, Ki67, E-cadherin and Vimentin were examined using immunohistochemistry assays by two independent pathologists. The indirect streptavidin-peroxidase method was used according to the manufacturer’s protocol. Anti-RNF38 (1:50, Abcam), Anti-Vimentin (1:200, Cell Signaling Technology), Anti-E-Cadherin (1:400, Cell Signaling Technology), and Anti-Ki67 (1:400, Cell Signaling Technology) were used as primary antibodies. The expression of RNF38 was accessed by the staining intensity and the proportion of positive cells. Testis tissues were used as positive control and NPC tissue stained with the same standards apart from using anti-RNF38 antibody as a negative control. RNF38 was primarily found in the cell nucleus. The staining intensity was graded as 0 (absent), 1 (weak), 2 (mediate), and 3 (strong). The percentage of positive cells was graded as 0 (0%), 1 (< 25%), 2 (25–50%), 3 (50–75%) and 4 (> 75%). The scores were calculated by multiplying the intensity score and percentage score to figure out a semiquantitative H-score. The mean H-score from all patients was used as the cutoff value to determine RNF38 positivity, which was described in our previous article [27].
Cell proliferation, migration, and invasion assays
Transfected cell lines were seeded in plates at a density of 2000 cells/well and were cultured for 1, 2, and 3 Days. Cell index was calculated by applications named xCELLigence RTCA S16(Real-Time Cell Analyzer, AECA, Biosciences, Inc). The clone assays, cell migration and invasion assays were conducted as described in our previous study [20].
Apoptosis test
NPC Cells were cultured in six-well plates and treated with 0.01% DMSO, cisplatin (10 um or 20um) or etoposide (80 um) for 48 h. Then, cells were washed twice with cold PBS and resuspended in 1 X Binding Buffer. 100ul of the solution (1× 10^5 cells) was transferred to a new tube, followed by adding 5ul of PE Annexin V and 5ul 7-AAD (PE Annexin V Apoptosis Detection Kit I, BD PharmingenTM), and then incubated for 15 min at room temperature in the dark. Cells were analyzed by Beckman-Coulter system.
Nude mouse tumor cell xenograft growth and lung metastasis model
Female BALB/c nude mice (4–6 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). All animal studies were performed according to protocols approved by the Institutional Animal Care and Use Committee. Briefly, for the tumor xenograft model, SUNE-1 cells were subcutaneously injected into mouse (2 × 107 cells in 100 μl PBS). After 5 weeks, tumors were removed, photographed, and weighed. Xenografts were monitored daily, and tumor size was measured every four days. For the lung metastasis model, SUNE-1 cells were (1.0 × 106 cells in 100 ul PBS) were intravenously injected through the tail vein. All mice were subjected to fluorescent imaging on ANLT-9MACIMSYSPLUS whole-body imaging system (Lighttools Research; Encinitas, CA, USA) after six weeks. Then mice were sacrificed by CO2 asphyxiation, and pulmonary metastatic lesions were sampled and quantified. Samples of tumor xenografts and lung metastasis model were fixed and paraffin-embedded, and serial 4um sections were conducted for IHC analysis.
Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)
NPC cells with overexpressed Flag-tagged RNF38 was prepared and then anti-Flag antibody was added to pull-down the proteins that may interact with RNF38. Then, 1 mL cold RIPA lysis buffer containing protease inhibitor (Roche) was added to cultured cells to prepare the samples. Next, the mixture was centrifuged 15 min at 4 °C with top speed, and the supernatant was transferred to a new ep and kept on ice. Next, the lysates were incubated with EZview Red ANTI-FLAG M2 Affinity Gel (Sigma-ALDRICH) overnight at 4 °C. Centrifuge for 30 seconds at 8200×g and remove the supernatant carefully. Wash the bead pellet by adding 500 μl of TBS. Vortex, incubate and gentle mixing at 2–8 °C for 5 minutes. Centrifuge for 30 seconds at 8200×g and remove the supernatant. Repeat washing the bead two more times and remove the supernatant. Then, add 50 μL 1% TFA to Dynabeads, incubate 10 min at 37 °C with high-speed shaking to elute binding proteins. Finally, Tryptic digestion and Peptide desalting were conducted for further LC-MS/MS. Raw MS files were processed with MaxQuant (Version 1.5.6.0). This database and its reverse decoy were then searched against by MaxQuant software. Both peptide and protein FDR should be less than 0.01. Only unique & razor peptides were used for quantification.
Co-immunoprecipitation, ubiquitination assay and CHX assay
For Co-immunoprecipitation assay, cell lysate of SUNE-1-NC and SUNE-1-RNF38 cells were incubated with primary antibody RNF38, ACTN4 or IgG at 4 °C overnight. The mixture was added to protein A/G magnetic beads (Thermo Scientific™ Pierce™), then placed on a low-speed rotating shaker for 1 hour at room temperature. After washed by RIPA buffer three times, proteins were eluted with separated by SDS-PAGE, and immunoblotted with antibodies against RNF38, ACTN4 and GAPDH, respectively. As to ubiquitination assay, SUNE-1-NC and SUNE-1-RNF38 cells were treated with MG132, followed by immunoblotting (IB) analysis to detect ubiquitin. CHX chase assay was used to examine the half-life period of ACTN4, SUNE-1-NC and SUNE-1-RNF38 cells were treated with CHX for 0, 2 and 4 hours, then IB was performed.
Statistical analysis
Data are analyzed using software SPSS (v24.0; Inc.; Chicago, IL, USA) and Graph Pad Prism 8 (GraphPad; La Jolla, CA, USA). Two sets of data are analyzed using Student’s t-test. The relationship between RNF38 expression and various Characteristics is analyzed by the chi-square test. Kaplan-Meier method and the log-rank test are used to evaluate the survival probability or to determine statistical significance according to RNF38 expression, respectively. Multivariate analysis is performed using the Cox regression model. The correlation between RNF38 and ACTN4 mRNA expression was evaluated with Spearman’s correlation analysis. All statistical tests are two-sided. A P value of < 0.05 is considered statistically significant.