Cell culture
The human NSCLC cell lines A549 and SK-MES-1 were purchased from the Cell Bank of the Chinese Academy of Sciences (CBCAS, Shanghai, China) and maintained in RPMI-1640 medium (Thermo Fisher, CA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher, CA, USA). All cells were passaged by 0.25% trypsin digestion (Thermo Fisher, CA, USA) and cultured in an atmosphere of 5% CO2 at 37°C in a humidified incubator (MCO-175, SANYO, Japan).
Collection and immunohistochemical staining of clinical NSCLC samples
Twenty sets of NSCLC and adjacent normal tissues were obtained from the Shanghai Pulmonary Hospital between May 2017 and April 2018 (Table 1). The patients gave written informed consent, and the study was approved by the Ethics Committee of Shanghai Pulmonary Hospital. None of the patients had received chemotherapy before sample collection. Each sample was divided into the two parts, one for western blotting of UbcH10, KIAA0101, BubR1, Mad2 and CyclinB proteins, and one for immunohistochemistry of UbcH10 and KIAA0101. For western blotting, separate samples which were used for extracting total protein were rinsed with saline and transferred to 2 ml microtubes that were labeled and subsequently stored in liquid nitrogen. For immunohistochemistry, separate samples were placed in tissue clips immersed in 10% neutral formaldehyde. Then, paraffin embedding and pathological sectioning were performed successively. The embedded sections were cut into 4 μm slices and placed on a glass slide before immunohistochemical staining to examine UbcH10 and KIAA0101. The following antibodies were used: anti-UbcH10 (1:400) and anti-KIAA0101 (1:450) and HRP-labeled goat anti-rabbit (1:1200) (Abcam, Cambridge, UK).
Cell cycle arrest and release
SK-MES-1 cells in the logarithmic growth phase were seeded in 6-well plates at 5×105 cells/well with RPMI-1640 medium containing 10% FBS and cultured overnight under normal conditions. Then, nocodazole (Sigma, CA, USA) at a final concentration of 1 µg/ml was added to the medium, and the cells were cultured for 18 hours. Next, cycloheximide (CHX, Sigma, CA, USA) at a final concentration of 10 µg/mL was added to the medium, and the cells were cultured for another 5 hours; some cells were collected every hour. UbcH10 and KIAA0101 expression in the cells harvested at different time points was detected by western blotting.
Immunofluorescence assay
SK-MES-1 cells in the logarithmic growth phase were fixed with 4% formaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.4% Triton X-100 in PBS, incubated with blocking buffer (10% donkey serum in PBS), and stained overnight at 4°C with primary antibody. Then, the cells were incubated for 1 hour at room temperature with secondary antibodies. Hoechst 33342 (Thermo Fisher, CA, USA) was used to stain nuclear DNA. The following antibodies were used: anti-UbcH10 (1:200) and anti-KIAA0101 (1:350) , the TITC or Alexa Fluor-labeled goat anti-rabbit were used as secondary antibodies (1:1200,Abcam, Cambridge, UK).
Genetic intervention using a lentiviral approach
SK-MES-1 and A549 cells in the logarithmic growth phase were seeded into 6-well plates at a density of 5×105 cells/well. On the following day, the cells were infected with virus encoding UbcH10 (Lv-shRNA-UbcH10 contains a siRNA sequence 5’-GACCTGAGGTATAAGCTCT-3’) or silencing KIAA0101(Lv-shRNA-KIAA0101 contains a siRNA sequence 5'- GTGGCAAAAAGGAATTGGAG-3’) or control (Lv-NC contains a mistranslated sequence 5’- GAAGCCAGATCCAGCTTCC-3’) at a multiplicity of infection (MOI) of 10. The infection efficiency was assessed by observing and analyzing green fluorescent protein (GFP) fluorescence 72 hours after infection with an inverted fluorescence microscope (IX71-F22, Olympus, Japan). The infection rate was estimated by dividing the number of cells expressing GFP by the total number of cells in each view. Total RNA and protein were isolated from the cells and subjected to real-time quantitative PCR (RT-qPCR) and western blotting to determine UbcH10 and KIAA0101 mRNA and protein expression.
Cell cycle analysis
SK-MES-1 and A549 cells infected with recombinant lentiviruses(Lv-NC contains a mistranslated sequence 5’- GAAGCCAGATCCAGCTTCC-3’ or Lv-shRNA-UbcH10 contains a siRNA sequence 5’-GACCTGAGGTATAAGCTCT-3’or Lv-shRNA-KIAA0101 contains a siRNA sequence 5’-GGAGGACAAATACGCAATG-3’ or Lv-shRNA-UbcH10 combined Lv-shRNA-KIAA0101) for 72 hours were trypsinized, washed twice with PBS, and fixed with 70% ethanol at 4°C overnight. The fixed cells were washed twice with PBS, resuspended in 100 μl propidium iodide (50 μg/ml and 100 μg/ml ribonuclease A in PBS), and incubated at room temperature for 30 min. The cell suspensions were detected by a FACSCalibur flow cytometer (BD Biosciences, NJ, USA).
Cellular proliferation assay
SK-MES-1 and A549 cells infected with recombinant lentiviruses for 72 hours were trypsinized and seeded into 96-well plates at a density of 1×105 cells per well. The cells were cultured under normal conditions, and cell viability was examined using a Cell Counting Kit-8 (CCK-8) assay at 24, 48, and 72 hours. Briefly, 10 µl CCK-8 solution (Dojindo, Japan) was added, and the cells were cultured under normal conditions for an additional 4 hours before the absorbance at 450 nm was measured.
Coimmunoprecipitation analysis
The coding sequence of KIAA0101 was amplified from human cDNA and cloned into the pcDNA3.1-HA expression vector (Invitrogen) to construct the wild-type (wt) KEN box vector pcDNA-HA-wt-KIAA0101 (wt KEN box: 5’-AGAAAGGTGCTT-3). Then, the mutant KEN box vector, pcDNA-HA-mt-KIAA0101 (mutant KEN box: 5’-GAAAAGGTTGCT-3’), was constructed through point mutation. SK-MES-1 cells transfected for 48 hours with pcDNA-HA-wt-KIAA0101 or pcDNA-HA-mt-KIAA0101 by using Lipofectamine 2000 (Thermo Fisher, CA, USA) were lysed at 4°C in ice-cold immunoprecipitation assay lysis buffer (Pierce, USA) for 10 min, and the resulting cell lysates were centrifuged for 3 min at 12,000 g. A negative control (NC) group was established that contained lysates from normal cells that were incubated with IgG. Before coimmunoprecipitation, samples containing equal amounts of protein were precleared with protein A or G agarose/Sepharose beads (Abcam,CA,USA) at 4°C for 3 hours and subsequently incubated with irrelevant IgG or anti-HA antibody (Santa Cruz, 2 μg/ml) in the presence of protein A or G agarose/Sepharose beads (Abcam, CA,USA) for 2 hours at room temperature or overnight at 4°C with gentle shaking. After incubation, the agarose/Sepharose beads were extensively washed with PBS, and the proteins were eluted by boiling in 2×SDS sample buffer before separation by SDS-PAGE. Subsequently, the target protein was identified by western blotting.
Animal xenografts
Animal studies were performed in accordance with ARRIVE guidelines and all processes were approved by Shanghai General Hospital Institutional Review Board (Permit Number: 2018KY201). Sixty female athymic nude mice (Shanghai Slake Laboratory Animal Co., Ltd.; age, 10 weeks; average weight, 18±2g) were housed at 23°C in a humidified atmosphere and a 12-h-light/dark cycle, with standard rodent chow and water ad libitum at the Second Military Medical University Animal Experiment Center, where the implantation experiment was performed. SK-MES-1 cells (1×105) were suspended in 200 μl medium and injected subcutaneously into the flanks of mice. Two weeks after inoculation, subcutaneous tumors were visible, and these tumors were approximately 2.5 mm in diameter. All animals were randomly divided into 5 groups (12 mice per group): the model group, NC group, UbcH10-silence group, KIAA0101-silence group, and co-silence group. In the intervention groups, each animal received 50 μl recombinant lentivirus (5×107 IFU) twice a week (on Monday and Thursday) starting at the second week after inoculation and continuing for 4 weeks, while the model group received the same volume of saline. The diameter of each tumor was measured weekly, and the data were used to plot tumor growth curves. At the end of the gene intervention (4 weeks), the subcutaneous tumors were stripped and used to detect the protein levels of UbcH10, KIAA0101, BubR1, Mad2 and CyclinB by western blotting. The mice were put to death by dislocation after experiments and all operations were performed under sodium pentobarbital anesthesia with every efforts made to minimize mice suffering.
RT-qPCR
Total RNA was isolated with TRIzol Reagent (Thermo Fisher, CA, USA) according to the manufacturer’s instructions and was reverse transcribed into cDNA using M-MLV Reverse Transcriptase and the Random9 primer (Takara). The following specific primers were used for quantitative PCR: UbcH10-forward 5’-GGCTACCCTTACAATGCGCCC-3’ and UbcH10-reverse 5’-CCTGACATCATACAGGGC-3’; KIAA0101-forward 5’-AACATAGCGTAAACCCTATC-3’ and KIAA0101-reverse 5’-CCTTGTTAGGCAGGATGGTCTC-3’; and β-actin-forward 5’-CCTGTACGCCAACACAGTGC-3’ and β-actin-reverse 5’-ATACTCCTGCTTGCTGATCC-3’. Real-time PCR was performed using the SYBR Premix Ex Taq kit and ABI7500 System (Thermo Fisher Scientific, CA, USA). One microliter of cDNA was used as the template. The mRNA levels of target genes were normalized to those of the endogenous housekeeping gene β-actin using the 2-ΔΔCt method.
Western blotting
Total protein was extracted from the cells using the M-PER mammalian protein extraction reagent or from tissues using the T-PER tissue protein extraction reagent (Pierce, IL, USA). Equal amounts of protein (12 μg per lane) as estimated by a bicinchoninic acid protein assay kit (Pierce) were separated by 11% SDS-PAGE and then transferred onto nitrocellulose membranes. The blots were probed with rabbit monoclonal antibodies against UbcH10 (1:200), KIAA0101 (1:500), BubR1 (1:600), Mad2 (1:250), CyclinB (1:350) and β-actin (1:1000) (Abcam, CA, USA), followed by incubation with secondary HRP-conjugated goat anti-rabbit antibody (Abcam, CA, USA). After a washing step, the bands were detected by chemiluminescence and imaged on X-ray films. β-Actin was used as an endogenous reference for normalization.
Statistical analysis
All statistical analyses were performed using SPSS 20.0 (SPSS, Chicago, IL, USA). The data are expressed as the mean ± standard error of the mean (SEM). Differences between groups and the control were analyzed by Tukey’s post-hoc test. P < 0.05 was considered to indicate a significant difference. All data were obtained from three independent experiments.