UBE2O targets Mxi1 for ubiquitination and degradation to promote lung cancer progression and radioresistance

UBE2O targets Mxi1 for ubiquitination and degradation to promote lung cancer progression and radioresistance

Cell culture and plasmids

The human bronchial epithelioid cell line HBE, HEK293T, SMCC-7721, HeLa, and all human lung cancer cell lines including A549, H1299, H292, HCC827, and H1975 cells were purchased from American Type Culture Collection and cultured in RPMI1640 or DMEM with 10% FBS in incubator at 37 °C with 5% CO2. All plasmids were subcloned into entry vector and then transferred to destination vector with indicated Myc, HA, SFB, or GST tag for the expression using Gateway Technology (Invitrogen). K46R mutation was generated by the KOD Hot Start DNA Polymerase (Novagen) and validated by DNA sequencing.

Antibodies and reagents

Anti-UBE2O antibody (GTX119315) for immunohistochemical (IHC) analysis was purchased from GeneTex. Anti-UBE2O antibody (A301-873A) for immunoblotting was purchased from Bethyl Laboratories. Anti-Mxi1 antibody (HPA035319), anti-Flag antibody (F1804), and Cycloheximide (01810) were purchased from Sigma-Aldrich. Anti-Myc (sc-40) and anti-Mxi1 (sc-1042) antibodies were obtained from Santa Cruz Biotechnology. Anti-GAPDH (60004-1-Ig) antibody was obtained from Proteintech. Anti-HA (#3724) and anti-GST (#2624) antibodies were obtained from Cell Signaling Technology. Anti-ATM (A19650) and anti-phospho-ATM-Ser1981 (AP0008) antibodies were obtained from ABclonal. The proteasome inhibitor MG132 (474790) was purchased from Millipore. Arsenic trioxide (ATO) was a clinically available drug which was obtained from Beijing ShuangLu Pharmaceutical Co., Ltd., China (approval number: H20080664).

RNA interference

Lipofectamine RNAiMAX reagent (Invitrogen) was used for siRNA transfection. After 48 h of transfection, cells were collected and analyzed by western blotting. The siRNAs ON-TARGETplus SMARTpool for Mxi1 were purchased from Dharmacon. The siRNAs targeting UBE2O and β-Trcp were as follows:

si-UBE2O#1: 5-GGUUGUAGAGUUGAAAGUUTT-3;

si-UBE2O#2: 5-CCACCCAGUGUGAAACCAATT-3;

si-β-Trcp: 5-AAGUGGAAUUUGUGGAACAUC-3 [20, 21].

GST pull-down assay

This assay was carried out as described previously [22, 23]. In brief, SFB-UBE2O plasmid was transfected into HEK293T cells for 24 h, and then the cell lysates were incubated with purified GST-only or GST-Mxi1 fusion proteins plus GST beads (GE Healthcare) overnight. The samples were centrifuged and washed five times with NETN buffer and subjected to SDS-PAGE.

Western blotting and co-immunoprecipitation assay

Cells were lysed in NETN buffer (100 mM of NaCl, 1 mM of EDTA, 20 mM of Tris HCl, pH 8.0, and 0.5% Nonidet P-40), and then cell lysates were separated by SDS-PAGE. For the exogenous co-immunoprecipitation, cell extracts were incubated with Myc beads (Santa Cruz Biotechnology), S beads (Novagen), or HA-beads (Millipore) overnight. For the endogenous binding, cell lysates were incubated with protein A/G agarose (Santa Cruz Biotechnology) plus IgG or anti-UBE2O/Mxi1 antibody overnight. After being centrifuged and washed with NETN buffer five times, the precipitates were analyzed by immunoblotting.

In vivo ubiquitination assay

The indicated plasmids and siRNAs were transfected into cells and proteasome inhibitor MG132 was added for 4 h before harvested. Cell lysates were incubated with S protein beads for co-immunoprecipitation, and ubiquitinated Mxi1 was detected by immunoblotting with anti-HA antibody.

Identification of Mxi1 ubiquitination modification site

SFB-Mxi1, Myc-UBE2O, and HA-ubiquitin plasmids were co-transfected in HEK293T cells for 24 h. Cells were then harvested and lysed with NETN buffer. SFB-Mxi1 protein was enriched by S beads co-immunoprecipitation. The ubiquitination-modified lysine (K) sites were identified by mass spectrometry (MS) analysis.

Viral packaging and infection

This method was carried out as previously described [23,24,25]. In brief, HEK293T cells were co-transfected with viral packaging plasmids pSPAX2 and pMD2. G as well as control shRNAs or shRNAs against UBE2O, respectively. After 48 h of transfection, the lentivirus supernatants were harvested and filtered. H1299 cells were infected with virus particles in addition with 10 μg/mL Polybrene. Stable cells were screened by puromycin (2 μg/mL) and confirmed by immunoblotting with anti-UBE2O antibody. The shRNAs against UBE2O were as follows:

sh-UBE2O#1: 5-CGATGATTCCTATGGCTTCTA-3;

sh-UBE2O#2: 5-CGGGTCTCTTCTTCGATGATT-3 [26].

EdU assay

The Cell-Light EdU Apollo567 in vitro kit was purchased from RIBOBIO, and the EdU assay was conducted as per the instruction manual. Briefly, cells transfected with indicated siRNAs or treated with ATO were suspended and seeded in a 96-well plate. After 2 h incubation with 50 μM EdU, cells were washed and fixed with 4 % formaldehyde for 30 min. EdU-Apollo was added for 30 min and cells were treated with 0.5% Triton × 100/PBS for 10 min. Cells were counterstained with DAPI and then detected by fluorescent microscope.

Flow cytometry analysis

This assay was performed as described previously [25]. Cells transfected with the indicated siRNAs for 24 h were irradiated and collected after another 24 h. The samples were analyzed by flow cytometry.

CFSE assay

Cells were plated in six-well plates for 24 h and incubated with CFSE (carboxy fluorescein succinimidyl ester) for 10 min. The CFSE labeling was stopped by the treatment with 40% cold bovine serum. Flow cytometry analysis was used to measure cell proliferation.

Clonogenic cell survival assay

Cells were plated in six-well plates and exposed to irradiation with indicated doses. After being cultured for 14 days, cell clones were fixed with 4% formaldehyde and treated with crystal violet solution for 30 min. Colonies with more than 50 cells were evaluated.

Neutral Comet assay

The reagent kit (4250-050-K) for neutral comet assay was purchased from Trevigen, and this assay was carried out as per manufacturer’s instructions. Briefly, 4 h after irradiation, cells were collected and then combined with molten LMA agarose and immediately pipetted onto a slide. Subsequently, the slides were immersed and placed in 1× Neutral Electrophoresis Buffer at 21 V for 45 min and then immersed in DNA precipitation solution for 30 min. Samples were stained with SYBR for 10 min and viewed by a fluorescent microscope. The olive tail moment of at least 100 cells was calculated.

Immunofluorescence staining

Cells were transfected with indicated siRNAs or treated with ATO and then irradiated. After 4 h of irradiation, cells were fixed with paraformaldehyde and then blocked with 5% bovine serum albumin. After being incubated with Rad51 antibody and secondary antibody, samples were washed with PBS buffer for three times. Rad51 foci was detected under a laser confocal microscope. Cells with more than 10 foci were considered positive cells.

Mouse xenograft models

Experiments in xenograft mouse models were approved by the Medical Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. This assay was carried out as previously described [23]. Briefly, BALB/c nude mice (5 weeks old) were randomly grouped and injected subcutaneously with 5 × 106 sh-Control, sh-UBE2O#1 or sh-UBE2O#2 H1299 cells. For irradiation treatment, mice were irradiated with 10 Gy when the tumor volume reached 100  mm3. For ATO treatment, ATO (2.5 or 5 mg/kg, i.p.) were administered to mice every day when the tumor volume reached 130 mm3. Tumor sizes and weights were monitored every 2 or 3 days.

Immunohistochemical (IHC) staining

The lung adenocarcinoma tissue microarray, which contained 82 paired carcinoma tissues and adjacent tissues, was purchased from Shanghai Outdo Biotech (Shanghai, China). IHC analysis was performed as previously described [23, 27, 28]. Briefly, the tissue section was dewaxed and hydrated, and the endogenous peroxidase was blocked by 3% hydrogen peroxide. The section was blocked with 5% skimmed milk for 1 h and then incubated with the anti-UBE2O antibody (GTX119315) at 4 °C overnight. After that, biotinylated secondary antibody was added for 1 h. For immunohistochemistry scoring, the intensity of staining (0 = negative, 1 = weak, 2 = moderate, 3 = strong) and the percentage of positively stained tumor cells (1 = 0–25%, 2 = 26–50%, 3 = 51–75%, 4 = 75–100%) were used for the quantification. The total IHC score equals the product of the intensity of staining and the percentage of positively stained tumor cells. The total IHC scores ≤6 were defined as low expression, and >6 were defined as high expression.

Statistical analysis

Each experiment was performed at least three times independently, and the data were shown as mean ± SD unless stated otherwise. The statistical significance between two independent groups was analyzed by unpaired two-tailed Student’s t test. Correlations of UBE2O and Mxi1 expression in tumor tissues was analyzed using the Pearson chi-square test. Overall survival analysis was evaluated by the Kaplan–Meier method. P value of <0.05 was considered significant.

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