All experimental protocols were approved by our local quality committee, and were carried out in accordance with our institutional guidelines.
Human lung cancer and corresponding non-cancer tissues were collected at the time of resection from 64 patients with NSCLC from May 2017 to June 2018 at the department of thoracic surgery in HuaDong hospital affiliated to FuDan University. Tissues were frozen in liquid nitrogen and stored at -80℃ refrigerator immediately. Patients who relapsed within 3 months after the first course of chemotherapy were defined as chemoresistance; ≥3 months was sensitive.
The signed informed consent was obtained from all patients and the clinical research ethics committee of FuDan university approved the present study.
Cell Culture And Regents
The NSCLC cell lines (SW900, SW900-Taxol) were purchased from the Cell Resource Center, Shanghai Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences. SW900-Taxol cell line refers to paclitaxel-resistant SW900 cell line. Both cells were maintained at 37°C in humidified air atmosphere containing 5% carbon dioxide in RPMI 1640 supplemented with 10% FBS. Rapamycin (R0395) and chloroquine (C6628) were purchased from Sigma; vorinostat (SAHA) was purchased from Selleck (Houston, TX, USA) and dissolved in dimethylsulfoxide (DMSO) as 10 mmol/L stock solutions.
RNA extraction and quantitative real-time PCR
Total RNA was extracted from cultured cells using the Isolation Kit (Ambion; Life Technologies), RNA was extracted from formalin-fixed/paraffin-embed normal and lung cancer specimens using the Recover All Total Nucleic Acid Isolation Kit (Ambion; Life Technologies). cDNA was synthesized from total RNA with specific stem-loop primers and the TaqMan Reverse Transcription Kit (Applied Biosystems; Life Technologies).
The sequences of the primers were as follows:
SIRT6, forward, 5'-CCCACGGAGTCTGGACCAT -3′;
Reverse, 5′- CTCTGCCAGTTTGTCCCTG -3′;
TGF-β, forward: 5'- GGCCAGATCCTTCCAAGC -3',
Reverse: 5'-GTGGGTTTCCACCATTAGCAC -3';
GAPDH was used as an internal control and amplified with forward primer: 5′-GGAGCGAGATCCCTCCAAAAT-3′,
Reverse primer: 5′-GGCTGTTGTCATACTTCTCATGG-3′.
Western Blot Analysis
According to the standard procedure, proteins were separated by 8% SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad). After blocked in 5% non-fat milk, the membranes were incubated with the primary antibodies: rabbit anti-SIRT6 and anti-TGF-β monoclonal antibody (mAb; 1:1000; CST), rabbit anti-β-Actin mAb (1: 1000; CST). The proteins were visualized with enhanced electrochemiluminescence (ECL) agents (Pierce).
Migration And Invasion Assay
For transwell migration assays, 2.5×104 to 5×104 cells were plated in the top chamber with the non-coated membrane (24-well insert; pore size, 8 um; BD Biosciences). For invasion assays, 1.25×105 cells were plated in the top chamber with matrigel coated membrane (24-well insert; pore size, 8 um; BD Biosciences). In both assays, cells were plated in medium without serum or growth factors, and medium which supplemented with growth factors or serum was used as a chemo-attractant in the lower chamber. The cells were incubated for 24 h and cells that did not migrate or invade through the pores were removed by a cotton swab. Cells on the lower surface of the membrane were stained with the Crystal Violet (GuangZhou Xilong Chemical industry co.) and counted.
The resected tissues were fixed with 10% formaldehyde, embedded in paraffin blocks. All sections were incubated at 63°C for 60 mins, deparaffinized in xylene, rehydrated, and incubated with fresh 0.3% hydrogen peroxide in 100% methanol for 30 mins at 37°C to block endogenous peroxidase activity. After rehydration through a graded ethanol series, antigen retrieval was carried out in 10 mM citrate buffer (pH6.4) at 98°C–100°C for 20 mins.The sections were passively cooled to 30°C. After rinsing the sections in 0.1M phosphate - buffered saline (pH7.4), non-specific binding sites were blocked by incubating with 10% normal goat serum for 30 mins. The sections were incubated overnight at 4°C or 37°C for 30 mins with anti-SIRT6 antibodies (CST) at a dilution of 1:100 in PBST. The sections were washed using PBST, incubated with biotinylated anti-mouse IgG, A, M solution (NichireiCo., Tokyo, Japan) for 30 mins at 37°C, and finally incubated in S-ABC solution (Nichirei Co.) for 30 mins. The chromogen, 3,3’-diaminobenzidine tetrahydrochloride, was applied as a 0.02% solution containing 0.005% hydrogen peroxide in a 50 mM ammonium acetate-citrate acid buffer (pH6.0).
The sections were counterstained in Mayer’s hematoxylin and mounted on glass slides. The expression levels were defined as follows:(1) low expression: no staining, weak staining or strong complete cytoplasm/ nuclear staining in <20% of tumor cells; (2) high expression/strong complete cytoplasm/nuclear staining in ≥20% of tumor cells. The expression levels were evaluated by two independent investigators who reached a consensus for all samples.
Cells were fixed with 4% formaldehyde, permeabilized with 0.4% Triton, and blocked with 2% BSA. The cells were stained with anti-SIRT6 (1:100), anti-LAMP2 (1:100), anti-LC3 (1:100) at 4°C. In the next day, the cells were incubated with FITC-conjugated secondary antibody for 1 h, and observed under a fluorescence microscope. To label the nuclei, cells were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI, Invitrogen) and were visualized using a confocal microscope.
Cells were plated at low conଂuence in 12-well plates (5*104 cells/well). On the 2 day, cells were exposed to serum starvation (0% FBS), normal medium (10% FBS), Rapamycin (30 umol/L), chloroquine (50 mmol/L) and SAHA for 24 hours. Medium was removed, cells were washed with PBS and treated with 4% paraformalde hyde/PBS for 20 minutes at room temperature, washed, and permeabilized with 0.1% Triton X-100 for 10 mins. Cells were blocked with 5% normal goat serum (CST) containing 0.4% Triton X-100 in PBS for 60 mins. Diluted primary antibody, anti-mouse LC3 A/B (CST), was applied in blocking buffer overnight at 4℃. Alexa Fluor-555 secondary antibodies diluted in 1% normal goat serum in PBS were added for 1 hour at ambient temperature. Cells were ଁxed using Vectashield hard set mounting medium containing DAPI dye (Vector Laboratories). Images were acquired using confocal microscopy (Olympus FV-1000) and overlaid using ImageJ.
Cell Viability Assays
Cells were seeded in 96-well plates (1*103–3*103 cells per well depending on the cell type) and incubated overnight before treatment. Cell viability was measured using the CellTiter 96® AQueous MTS Reagent (Promega, Madison, WI). For Giemsa staining, cells were washed with PBS and fixed with formaldehyde and washed again before incubation with Giemsa staining solution (Sigma, St Louis, MO). After 30 mins, cells were washed and allowed to dry. Each subgroup has three holes.
Flow cytometry after annexin-V/propidium iodide (PI) staining
The collected cells were centrifuged at 2000 rpm/min for 5 min, and the centrifuged cells were washed twice with PBS. The washed cells were centrifuged and collected, and the treated cells (1*106) were transferred into an Ependorff (EP) tube and 500 µL of bidding buffer was added into EP tube. PI (5 µL) and Annexin V (5 µL) (KeyGEN BioTECH, Nanjing, China) were added into each EP tube. After the dyes were added, the cells were incubated in the dark for 10-15 mins. The incubated cell suspension was filtered through 400 mesh cell screen to filter off the cell clumps. After filtration, the cells were placed on ice and analyzed within 30 mins using flow cytometer. Annexin V+PI− indicated early apoptosis, Annexin V+ PI+ indicated late apoptosis. Apoptotic rate = early + late apoptosis rates.
Data were shown as mean±SD, the student-t test was used for statistical analysis; all statistical analyses were performed with the SPSS 22.0 software. The Cox regression method was adopted to estimate overall survival (OS) and progression free survival (PFS), and the log-rank test was used to compare survival time. The independent prognostic factors were performed in multivariate analysis using the cox hazard model. P value <0.05 were considered to be significant. Graphical displays were prepared using Graph Pad Software (Graph Pad Software, Inc, La Jolla, CA) to show the distributions of expression.