Animal care and use
All mice were raised in a specific pathogen-free environment in the Jinan University and all experimental protocols were approved by the Jinan University. All animal work is carried out in accordance with the approved protocols. In the single-dose virus infusion experiment, the siblings of double transgenic mice were randomly divided into two groups (3–4 in each group). TetO-KrasG12D/CC10rtTA (designated KC) double transgenic mice were fed with doxycycline (Dox)-containing diet for 2 months to induce lung Adenocarcinoma. Retrovirus for overexpressing ZNF24 (KC-Z) or Control (KC-C) was inhaled through the nostrils into lungs. After recovery for a week, all the mice were fed with diet containing Dox until the day of the sacrifice. The lung tissues were stained with hematoxylin eosin. Computed Tomography (CT, PINGSENG Healthcare) was used to quantify tumor burdens. To quantify the tumor burden, we calculated the total size (mm3) of all tumor regions in H&E sections under the microscope.
Lsl- KrasG12D Mice of 6–8 weeks of age were treated with recombinant lenti-virus co-expressing Cre and CRISPR/Cas9 via nasal inhalation. Tumor burdens were monitored through CT in mice with ZNF24 knockout (K-sgZNF24 mice) and with Td-Tomato knockout (K-sgTD mice for control) six months after infection.
Mouse treatment
The sgRNA targeting ZNF24 was cloned into pSECC plasmid. The resultant plasmid was sequenced to verify before used to prepare recombinant viruses. These viruses were dripped into Lsl-KrasG12D mice through nostrils. Tumor burden was documented by computed tomography (CT, PINGSENG Healthcare) six months after lung cancer induction. Mice were treated with NF-κB inhibitor BAY11-7082 (selleckchem, 20 mg/kg/day), BI3406 (selleckem, 25 mg/kg/day) and anti-PD-1 (BioXcell, BP0033-2, 5 mg/kg). Tumor burden were monitored by computed tomography.
In vivo xenograft model
8-week-old male BALB/C Nude mice were used for xenotransplantation study (Guangdong Medical Lab Animal Center). Briefly, 2 × 106 A549i cells were inoculated subcutaneously on the right back of mice in 100 μL Matrix (Gel356237, CORNING). Mice were randomly grouped to give doxycycline or normal diet when tumor volume reached about 100 mm3. Body weight and tumor volume (D × d2/2 (mm3)) were measured every 2 days (D was the longest and d was the shortest diameter). At the end of treatment, tumors were harvested for photographing and weighing.
Cell culture and generation of engineered cell lines
HEK293T, NCI-H446, EKVX, NCI-H322, NCI-H460, NCI-H1975, PC9, HCC827, Hop62, NCI-H3255, Hop92 and A549 were purchased from ATCC as part of NCI-60 (American Typical Culture Collection, Manassas, VA, USA). BEAS-2B, and HEK293T cells were provided by Dr. Kwok-Kin Wong (The Helen and Martin Kimmel Center for Stem Cell Biology, NYU). All cell lines were held in a standard tissue incubator at 37° C with 5% CO2. BEAS-2B, HEK293 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin /glutamine. (FBS, Gibco, Life Technologies, Carlsbad, CA, USA). The remaining lung cancer cells were cultured in RPMI-1640. For preparing recombinant lenti-virus, HEK293 cells were transfected with two packaging plasmids (psPAX2 and pMD2.G) together with a pLVX-TetOne-Puro vector control or the same vector constructs for expressing ZNF24, RelA respectively. Cells were changed with fresh medium after 6 h. The recombinant virus-containing medium was filtered with 0.22 µm and used to infect cells in the presence of polybrene (8 µg/mL), followed by selection with 1 μg/mL puromycin (J593, Amresco) for 1 week. For shRNA knockdown, cells were infected lenti-virus packaged from pLKO.1-shGFP-Puro vector control or the same vector constructs encoding shZNF24, shNR3C2, shCST4, shARHGDIG, shCRYBB3. Cells were selected the cells with 1 μg/mL of puromycin for 1 week.
Reagents and antibodies
Following reagents were used in current study: Doxycycline hyclate (Dox, D9891, Sigma, St. Louis, MO, USA), Protease and phosphatase inhibitor cocktail (CatNO:C0001, C0002, TargetMol), trizol (15596018, ambion), Lipofectamine3000 (L3000015, Invitrogen), Western blotting substrate (WBKLS0500, Millipore), Cell Counting Kit-8 (TN623, DojindoLaboratories). Cell Signaling Senescence β-Galactosidase Staining Kit (#9860, CST), dual-specific luciferase assay kit (Promega), anti-ZNF24 (HPA024062, Sigma Aldrich), anti-ZNF24 (11219–1-AP, Proteinch), anti-Caspase1 (22915–1-AP, Proteinch), anti-P-MLKL (phospho S358, D6H3V, CST), anti-LaminB1 (ab16048, Abcam), anti-CDK2, Cyclin B1, Cyclin D1 (ab228528, Abcam), anti-Cyclin E1 (ab33911, Abcam), anti-Caspase9 (#9504, CST), anti-Caspase3 (#9661, CST), anti-Caspase8 (#8592, CST), anti-FLAG (#14793, CST), anti-β-actin (A5316, Sigma), anti-P65 (ab16502, Abcam), anti-P50 (ab32360, Abcam), anti-P-P65 (phospho S536, ab86299, Abcam), anti-IκBα (ab32518, Abcam), anti-p-IκBα ( phospho S36, ab133462, Abcam), anti-GAPDH (ab8245, Abcam).
The antibody in flow cytometry as follows: anti-mouse-CD45 (559664, BioLegend, APC), anti-mouse-CD8a (45–0081-82, eBioscience, PerCP-Cyanine5.5), anti-mouse-CD4 (48–0041-82, eBioscience, BV421), anti-mouse-TIM-3 (119703, BioLegend, PE), anti-mouse-TNF-α (506305, Biolegend PE), anti-Human-Calreticulin (D3E6, CST), anti-Human-HSP70 (ab2787, Abcam), anti-HMGB1 (10829–1-AP, Proteinch), anti-mouse-IL-2 (12–7021-82, eBioscience, PE), anti-mouse- IFNγ (12–7311-81, eBioscience, PE).
Constructs
Plasmids, pLVX-TetOne-Puro and pLVX-TetOne-zeocin were purchased from Clontech, and psPAX2, pMD2.G and pLKO.1-puro from Addgene. plasmids encoding shRNAs were constructed by standard molecular biology techniques. shRNA target sequences were as follow:
shCST4: 5ʹ- CTTTCGAGATCTACGAAGTTC-3ʹ.
shARHGDIG: 5ʹ-CCCAGGAGTATGAGTTTGTGA-3ʹ.
shCRYBB3: 5ʹ- CCCAGGAGTATGAGTTTGTGA-3ʹ.
shZNF24: 5ʹ-GAGGATTTGGAGAGTGAACTT-3ʹ.
shNR3C2: 5ʹ-CCTTGCCTTCAGCAAGACAAT-3ʹ.
The constructs, pCDH-NF-κB reporter, FLAG-tagged ZNF24 and pGL3-P65-Luciferase were cloned by standard molecular biology techniques and verified through sequencing.
Cell proliferation assay
Cell proliferation test: 1 × 103 cells (A549i, Hop62i) were inoculated into each well of 96-well plates and cultured overnight. 1 μg/mL of Dox was added for 5 days in 1640 culture medium containing 10% FBS. The proliferative activity was then determined using the CCK8 following the manufacturer's protocols.
Colony formation assay
1000 cells were seeded in 6-well plates containing medium supplemented with 10% FBS. The cells were cultured without or with Dox incubation for 14 days. Fixed in methyl alcohol and stained with 0.5% crystal violet.
Soft-agar colony formation assay
The soft-agar colony formation test was carried out in soft agar (0.6% lower gel and 0.35% upper gel). Add 1 mL of 0.5% base gel to each well of the six-well plates and let stand for 3 h. 1 × 104 A549i and Hop62i cells were mixed with the upper gel and Dox (1 μg/mL), and then seeded in 6-well plates. 0.5 mL 1 × fluid media containing Dox (1 μg/mL) was added weekly to the upper gel surface. The cells were cultured around 3–4 weeks with or without Dox treatments before imaging and colonies counting.
Senescence-associated β-Galactosidase (SA-β-Gal) staining assay
SA-gal staining was performed with cell signal transduction aging galactosidase Staining Kit (CST, #9860). 200,000–300,000 cells were seeded in each well of the six well plates and cultured until staining time. A549i and Hop62i cells were treated with Dox (1 μg/mL) for 2 days, and then SA-gal staining was performed to observe the effect on senescence.
Cell cycle analysis
A549i and Hop62i cells were treated with or without 1 μg/mL of Dox for 48 h, and fixed with 70% ethanol at − 20 °C overnight. After fixation, the cells were subsequently centrifuged at 3000 × g for 5 min and washed with PBS. Cell cycle was performed with the cell cycle kit (Beyotime, C1052) following the manufacturer’s instructions. Cell cycle distribution was analyzed using BD Accuri™ C6 flow cytometer.
RNA extraction and real-time RT-PCR
Total RNA was extracted with Trizol reagent and analyzed by real-time PCR to detect the mRNA level of the gene. 2 μg of total RNA was reverse-transcribed to cDNA through AccuRT Genomic DNA Removal Kit (G492, Applied Biological Materials). Real-time PCR was performed with Bio-Rad Real-Time PCR System and SYBR Green qPCR Mix (110344, Monad). The data shown is the relative abundance of the referred mRNA normalized to Actin. The sequence of gene-specific Primers is as follows:
CDK2-Forward: 5′- ATGGATGCCTCTGCTCTCACTG-3′,
CDK2-Reverse: 5′- CCCGATGAGAATGGCAGAAAGC-3′,
Cyclin E1-Forward: 5′- TGTGTCCTGGATGTTGACTGCC-3′,
Cyclin E1-Reverse: 5′- CTCTATGTCGCACCACTGATACC-3′,
Cyclin D1-Forward: 5′- TCTACACCGACAACTCCATCCG-3′,
Cyclin D1-Reverse: 5′- TCTGGCATTTTGGAGAGGAAGTG-3′,
P65-Forward: 5′- TGAACCGAAACTCTGGCAGCTG-3′,
P65-Reverse: 5′- CATCAGCTTGCGAAAAGGAGCC-3′,
RelB-Forward: 5′- TGTGGTGAGGATCTGCTTCCAG-3′,
RelB-Reverse: 5′- TCGGCAAATCCGCAGCTCTGAT-3′,
C-Rel-Forward: 5′- AGTTGCGGAGACCTTCTGACCA-3′,
C-Rel- Reverse: 5′- CGTGATCCTGGCACAGTTTCTG-3′,
P105-Forward: 5′- GCAGCACTACTTCTTGACCACC-3′,
P105- Reverse: 5′- TCTGCTCCTGAGCATTGACGTC-3′,
Actin-Forward: 5′-ACGTGGACATCCGCAAAG-3′,
Actin- Reverse: 5′- GACTCGTCATACTCCTGCTTG-3′,
ZNF24-Forward: 5′- GTGACAGTGCTGGAGGATTTGG-3′,
ZNF24- Reverse 5′- GGTTCTCCACAGCATCAAGCTC-3′,
IL-6-Forward: 5′- AGACAGCCACTCACCTCTTCAG-3′
IL-6- Reverse: 5′-TTCTGCCAGTGCCTCTTTGCTG-3′.
IL-1β-Forward: 5′- CCACAGACCTTCCAGGAGAATG-3′,
IL-1β-Reverse: 5′- GTGCAGTTCAGTGATCGTACAGG-3′,
TNF-Forward: 5′- CTCTTCTGCCTGCTGCACTTTG-3′,
TNF-Reverse: 5′- ATGGGCTACAGGCTTGTCACTC-3′,
Protein extraction and immune-blotting
Whole cell lysates were extracted with RIPA lysis buffer (P0013K, Beyotime) supplemented with protease and phosphatase inhibitor cocktail. Protein concentrations were determined by the Bradford assay. Soluble Protein (30–40 μg) was analyzed by SDS-PAGE followed by immunoblot analysis.
Virus packaging and concentration
The lenti-virus plasmid (psPAX2 and pMD2.G) were co-transfected into HEK293T cells with VigoFect (Vigorous Biotechnology, Beijing, China). The cells were washed with fresh medium 6 h after transfection and cultured in fresh medium. The supernatant containing virus was collected after 24 h. All the retrovirus used in the mouse experiments were concentrated by centrifuging the viral medium at 27,500 rpm/min for 2 h, carefully removing the supernatant, and re-suspending the virus particles in a 100 μL of opti-mem medium. Shake gently at 4° C overnight.
pGL3-P65-Luciferase reporter assay
500 ng of pGL3-P65-luciferase plasmid was mixed with 200 ng of renilla plasmid. The mixture was transfected into A549i cells seeded in 12-well plates via Lip3000. After 6 h, changed with fresh medium containing Dox for inducing ZNF24 expression for 12, 24 and 48 h. Cells were then digested with Trypsin and equal number of cells were then measured for luciferase activity.
Chromatin immunoprecipitation (ChIP)-seq
The A549i cells were inoculated into eight 150-mm plates, and Dox was added to the final concentration of 1 μg/mL when the cell concentration reached 70–80%. After 2 days of culture, the cells were cross-linked with 1% formaldehyde (final concentration) and sonicated with the following parameters: 30 s on, 30 s off, 25% set power for 15 cycles. The total Lysis was divided into two bisected samples, one mixed with 1 mg/mL of Flag antibody and 40 μL of protein agarose bead. The other was mixed with 1 mg/mL of normal mice IgG (#F2416, Santa Cruz Biotechnology) and 40 μL of agarose beads. After overnight incubation, the beads were washed with a ChIP cleaning buffer. Chromatin was eluted, cross-linked DNA was reversed and DNA was extracted with phenol/chloroform. Finally, the eluted DNA was resolved in ddH2O and then sent to BGI (https://www.bgi.com/bgi-online) for sequencing.
RNA sequencing
A549i cell was treated with or without 1 μg/mL of Dox for 48 h. The cells were digested with Trypsin. Total RNA was extracted with Trizol and sent to BGI for library construction and sequencing.
Flow cytometry
To detect cell surface proteins (CD45, CD4, CD8, TIM-3), grind lung tissues separate from mice on grid 200. Collect cell suspension and use lysis solution (#NH4CL2009, TBD) to disrupt red blood cells. Then filter the cells to prepare a single cell suspension and staining antibody. For IL-2, IFNγ and TNF-α detection, firstly, stain the membrane proteins with antibodies against CD45, CD8 or CD4, wash once with PBS and use BD fixation and permeabilization kit (00–5523-00, eBioscience) overnight, then wash once with PBS, and stain with the corresponding antibody (IL-2, IFNγ, TNF-α). For CRT, HSP70 detection, EKVX-shZNF24 cells were treated with BAY11-7082, BI3406, or combination for 24 h. Cells were incubated with primary anti-CRT antibodies (1:100), or anti-HSP70 antibodies (1:100) for 1 h. The cells were washed and incubated with the FITC-conjugated monoclonal antibody for 30 min. Cells were analyzed by flow cytometry. For HMGB1 detection, EKVX-shZNF24 cells were treated with BAY11-7082, BI3406, or combination for 24 h. Cells were resuspended and fixed with the BD fixation and permeabilization kit (000-5523-00, eBioscience) overnight. These cells were then washed with PBS, stained with anti-HMGB1 antibodies for 1 h. The cells were washed and incubated with the FITC-conjugated monoclonal antibody for 30 min. Cells were analyzed by flow cytometry.
CRISPR/Cas9 screen
We first infect EKVX cells with virus for encoding Cas9, and selected with blasticidin (3513–03-9, Solarbio) for two weeks. Monoclone was picked to generate a stable cell line (EKVX-Cas9). EKVX-Cas9 was further infected with the sgRNA library (addgene, #73178). After 24 h infection, a portion of the cells were collected as a control. The cells were selected with puromycin (1 μg/mL) for a week to eliminate uninfected cells. The infected cells were inoculated at 2 × 106 cells subcutaneously into nude mice. After 14 days, the tumor was harvested to prepare genomic DNA. DNA fragment containing CRISPR sequence were PCR amplified and sent for sequencing.
Histopathological analysis
Lung tissues were fixed with 10% neutral buffered formaldehyde (HT501320; Sigma-Aldrich) overnight, and dehydrated, embedded in paraffin. Then the tissues were sectioned for staining with hematoxylin and eosin (H&E) for histopathology.
TCGA data analysis
We compared ZNF24 expression between normal tissue and primary tumor tissues was performed with UCSC Xena (http://xena.ucsc.edu/compare-tissue/). For correlation analysis between ZNF24 and RELA, RELB, CCNE1, CDKN1B, CDKN1A in lung adenocarcinoma patients, we obtained the gene expression data from TCGA by UCSC Xena and the gene expression correlation was analyzed by GraphPad Prism 7.04. For the expression analysis of the ZNF24 with KRAS, EGFR mutant in lung adenocarcinoma patients, patients were divided into high and low expression of ZNF24 according to the median expression of ZNF24.
Survival curve analysis
The survival curve analysis of ZNF24 expression in lung cancer patients was usingKaplan-Meier-Plotter(https://kmplot.com/analysis/index.php?p=service&cancer=lung).
Statistical analysis
Using GraphPad Prism 7.04 for statistics. Student’s t-test was used to compare differences between two experimental groups. Data are presented as means ± SEM and error bars denote SEM; n = 3; *P < 0.05; **P < 0.01; ***P < 0.001.