Tissue Samples
Fresh tissue samples (138 in total) including lung adenocarcinoma (LUAD) (n = 69) and paired adjacent normal lung (n = 69, > 5 cm from the tumor edge) tissues, were collected from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. All patients had received no chemotherapy or radiotherapy before surgery and signed a written informed consent before sample collection. Clinical data of the patients are provided in Additional Table 1. The studies were approved by the Human Assurance Committee of Tongji Hospital (IRB: TJ-IRB20160601).
Data Mining From Tcga And Human Protein Atlas Database
We assessed the association between DDB2 expression and melanoma using the cohorts from TCGA (The Cancer Genome Atlas) database (TCGA-melanoma cohort). The survival curves for MBD2 and DDB2 in LUAD and melanoma patients were evaluated in the data from the Human Protein Atlas database (https://www.proteinatlas.org/). The UCSC Xena browser (https://xenabrowser.net) was employed to access and analyze the data.
Animals
The C57BL/6 mice (8-week old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animal experiments were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China).
Cell Culture
The A549 cells, NCI-H1975 and murine melanoma B16F10 cells were purchased from the American Type Culture Collection (Rockville, MD, USA). Those cells were cultured at 37°C in a 5% CO2 incubator. To stimulate EMT, each type of cells was induced by TGF-β1 (10 ng/ml, 24 h) after transfected with MBD2 siRNA or a scramble siRNA for 48 h. The scramble siRNA transfected cells without TGF-β1 were served as the controls.
Reagents
Recombinant human TGF-β1 (Cat: 100 − 21) was purchased from the PeproTech Corporation (Cranbury, NJ, USA). Antibodies against E-cadherin (Cat: Sc-7870), N-cadherin (Cat: Sc-8424), Vimentin (sc-58899) and GAPDH(Sc-47724) were obtained from the Santa Cruz Biotechnology (Dallas, TX, USA). The crystal violet staining solution (Cat: C0121) and the ChIP assay kit (Cat: P2078) were purchased from the Beyotime Biotechnology (Shanghai, China). The lipidoid C12-200 was purchased from the Xinjiahecheng Medical Chemistry Corporation (Wuhan, Hubei, China). The 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (mPEG-DMG) was ordered from the NOF Corporation (Tokyo, Japan). The CCK-8 assay kit (Cat: 40203ES60) was originated from Yeasen Biotechnology (Wuhan, China), and the Lipofectamine 3000 (Cat: L3000015) was obtained from the Invitrogen Corporation (Shanghai, China). All other reagents were obtained from Sigma (St. Louis, MO, USA).
Rna Interference And Preparation Of Sirna-loaded Liposomes
The MBD2 siRNAs and a corresponding scramble siRNA were synthesized by the RiboBio (Guangzhou, China), and were then transfected into A549 cells, B16F10 cells and H1975 cells using the Lipofectamine 3000 according to the manufacturer’s protocol or a liposome-based siRNA transfection method as previously described [14]. The following two siRNAs were used for MBD2: siRNA1 5’-GCA AGA GCG AUG UCU ACU A-3’, and siRNA2 5’-GCG AAA CGA UCC UCU CAA U-3’. The siRNA-loaded nanoparticles were prepared as reported [15].
Mbd2 Overexpression
A549 cells and B16F10 cells were seeded on 12-well plates and transduced with Adenovirus-MBD2 and a Vector (Abcam, MA, USA) at the multiplicity of infection (MOI) 1:4 for 48 h. The transduced cells were then stimulated with TGF-β1 (10 ng/ml) for 24 h and analyzed by Western blotting.
Western Blot Analysis
Total proteins were extracted from tissues and cells using the Lysis buffer (Beyotime, Shanghai, China), and then subjected to Western blot analysis using the established techniques [16]. The primary antibodies against MBD2; E-cadherein, N-cadherein; Vimentin.were employed for the analyses, and GAPDH band was used as the internal control.
Immunofluorescence Assay
Cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. The samples were washed with PBS and blocked with 5% BSA in PBS for 1 h. Subsequently, the cells were incubated firstly with indicated primary antibodies overnight at 4°C, and then with an Alexa Fluor 488-conjugated antibody or an Alexa Fluor 594-conjugated antibody (Abbkine, Redlands, CA, USA, 1:200) for 1 h at room temperature, respectively. Nuclei were counterstained with DAPI for 8 min. Immunofluorescence images were acquired under a microscope (Olympus, Tokyo, Japan) in a blinded manner.
Quantitative Real-time Pcr
Quantitative Real-Time PCR was conducted using the SYBR Premix Ex Taq (TaKaRa, Tokyo, Japan) as previously reported [17]. The primer sequences were provided in Additional Table 2.
Cell Migration And Invasion Assays
The cell migration and invasion were evaluated by using a Transwell assays (Corning, MA, USA) using the established techniques [18].
Cell Counting Kit-8 Assay
The cell viability was examined by a Cell Counting Kit-8 kit based on the manufacturer’s protocol. The absorbance was measured at 450 nm using a microplate reader (ELx800, BioTek Instruments, Winooski, VT, USA).
Apoptosis Assay
Apoptosis assay was conducted using an Annexin V/PI detection kit (Beyotime, Shanghai, China) and analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA) as reported previously [18].
Rna Deep Sequencing (Rna-seq)
RNA from A549 cells activated by TGF-β1 was extracted using an RNA isolation kit (TaKaRa, Tokyo, Japan). The prepared mRNA libraries were sequenced on an Illumina HISEQ 2500 platform, and the HISAT2 v2.0.4 software (Center for Computational Biology, Baltimore, Maryland) was used to map the clean reads to the mm10 reference genome. Fragments per kilobase of exon per million mapped fragments (FPKM) values were calculated using the CuffNorm version 2.2.1 software (University of Washington, Seattle, Washington, USA). The genes with a calculated normalized FPKM value greater than 5.0 were considered to be expressed. Significantly differentially expressed genes were defined as those with a | log2 (fold change) | ≥ 0 and a P value ≤ 0.05. Heatmaps were generated using the R package heatmap.
Go Term And Kegg Pathway Enrichment Analysis
The biological significance of the differentially expressed genes (DEGs) was explored by the Gene Ontology (GO) term enrichment analysis covering 3 functional groups (biological process, cellular component and molecular function), and the Genomes (KEGG) pathway enrichment analysis of the DEGs was performed with the Bioconductor package “GeneAnswers” to identify vital pathways related to EMT in A549 cells.
Global Dna Methylation Assay And Bisulfite Dna Sequencing
Global DNA methylation was evaluated by a MethylFlashTM Methylated DNA Quantification Kit (Epigentek, NY, USA) according to the instructions and bisulfite DNA sequencing was carried by Sequenom MassARRAY platform (BGI.write, Beijing, China) as previously reported [19].
Chromatin Immunoprecipitation (Chip) Assay And Ddb2 Promoter Reporter Assay
ChIP assays were conducted using a ChIP assay kit (Beyotime, Shanghai, China) as reported [20]. The primers for DDB2 used in the ChIP PCR were F: 5' ACT CCC CAA CTA CAC CCT GT 3' and R: 5' CCG GCT AAT TTC TCT CTC TCT 3'. A dual luciferase reporter system (Promega, Madison, WI) was used for DNA methylation dependent DDB2 promoter luciferase reporter assays, in which the MBD2 binding sites within the DDB2 promoter were disrupted using the established techniques [21].
Flow Cytometric Analysis
A549 and B16F10 cells were transfected with DiI-labeled liposomes for 2 or 4 h. After washing, the cells were analyzed using a FACSCanto II (BD Biosciences, San Jose, CA, USA) as reported [22]. All data were analyzed using the FACSExpress V3 software (De Novo Software) based on the manufacturer’s protocol.
Anticancer Activity In The B16f10 Lung Metastasis Model
The melanoma tumor-bearing mouse model was established through the subcutaneous injection of B16F10 melanoma cells into the right flanks of C57BL/6 mice (6×105 cells/mouse). Once the tumor volume reached approximately 80 mm3, the mice were intratumorally injected with PBS, L-scramble siRNA, empty liposomes or L-MBD2 siRNA (1 mg/kg) for a total of three times with a three-day of interval. Tumor progression was assessed by monitoring the tumor volume with the following equation: V = (length×width2)/2. Two days after the last injection, the mice were sacrificed, and the tumor tissues were collected and weighed. The tumors were cryosectioned into pieces. The sections were co-stained with a FITC-labeled anti-N-cadherin antibody and an Alexa Fluor 594-labeled anti-E-cadherin antibody at 37°C for 30 min. After rinsing the sections with PBS. the distribution of N-cadherin and E-cadherin was observed by confocal microscopy, and the relative fluorescence intensity was determined using the ImageJ software.
To further investigate the inhibitory capability of L-MBD2 siRNA in tumor metastasis, B16F10 melanoma cells were incubated with PBS, empty liposomes, 50 nM L-scramble siRNA or 50 nM L-MBD2 siRNA for 6 h, respectively. The pretreated B16-F10 tumor cells were then intravenously injected into the C57BL/6 mice (2×105 cells/mouse). After 20 days of injection, metastatic tumor-bearing lung tissues were collected from sacrificed mice, and the pulmonary metastatic nodules were counted. Tumor-bearing lung sections were also prepared for H&E staining.
Statistical analysis
All experiments were conducted with at least three independent replicates, and statistical analyses were performed using the GraphPad Prism 5.0 software (San Diego, CA, USA). Data are presented as the mean ± SEM, and an independent Student’s t test was applied to analyze the statistical significance of differences between two groups. As for comparisons among multiple groups, one-way ANOVA followed by Tukey’s post hoc test was performed. In all cases p < 0.05 were considered with statistical significance.