Patients

Tissues were gathered from 60 patients with advanced LAD and 20 patients with other lung diseases (control group) in the Nanjing General Hospital of Nanjing Military Command between March 2005 and January 2010. All 60 patients with LAD met the previously described criteria [20]. Tumor response to chemotherapy was assessed via computed tomography (CT) every two or three cycles of chemotherapy, and defined according to the Response Evaluation Criteria in Solid Tumors as follows: progressive disease (PD), stable disease (SD), complete response (CR), or partial response (PR) [20]. PD and SD were regarded as “insensitive”, whereas CR and PR were considered as “sensitive” [20]. Tissues were rapidly frozen with liquid nitrogen and then stored at − 80 °C. Tissue acquisition was permitted by the Review Board of Hospital Ethics Committee of Nanjing General Hospital of Nanjing Military Command (Nanjing General Hospital of Nanjing Military Command, Nanjing University, China). All patients or authorized persons provided written informed consents.

Cell culture and treatment

Human LAD H1299 cells were obtained from the ATCC Cell Bank (Manassas, VA, USA), and human LAD SPC-A1 cells were from Procell life Science & Technology Co., Ltd. (Wuhan, China). Cells were cultured in RPMI-1640 medium (Gibco, Rockville, MD, USA) supplemented with 1% penicillin/streptavidin (Gibco) and 10% FBS (Gibco). DTX-resistant H1299 and SPC-A1 cells, termed H1299/DTX and SPC-A1/DTX, respectively, were established in our laboratory as described previously [14, 21]. H1299/DTX and SPC-A1/DTX cells were maintained in 50 μg/L of DTX (Selleck Chemicals, Houston, TX) and routinely grown as described in a previous work [22]. All cells were maintained in a humidified incubator with 5% CO2 at 37 °C.

Quantitative real-time polymerase chain reaction (RT-qPCR)

Total RNA extraction was performed with TRIzol reagent (Takara, Shiga, Japan), and then cDNA was synthesized by using PrimeScript RT Reagent Kit (Takara) as per the manufacturer’s instruction. Quantitative analyses were performed using SYBR PrimeScript RT-qPCR Kit (Takara). Relative gene expression was detected based on 2-ΔΔCt method by normalizing to GAPDH mRNA or U6 snRNA.

Subcellular fractionation

PARIS™ Kit (Ambion, Austin, TX) was used for the nuclear/cytoplasmic fractionation in line with the manufacturer’s instruction. In brief, cells (1 × 107) were collected and suspended in cell fractionation buffer and then lysed in cell disruption buffer. The MARCKSL1–2 content of both cell fractions was examined by RT-qPCR.

Fluorescent in situ hybridization (FISH)

RNA FISH probe for MARCKSL1–2 was designed by Ribobio Company (Guangzhou, China). After culturing with probes in hybridization solution, cell samples were counter-stained in DAPI solution and then visualized using a fluorescence microscope (Olympus Corp., Tokyo, Japan).

Cell transfection

The specific shRNAs targeting MARCKSL1–2 (shMARCKSL1–2#1 and shMARCKSL1–2#2), HDAC1 (sh/HDAC1), or SUZ12 (shSUZ12#1 and shSUZ12#2), as well as relative control shRNAs (sh/Ctrl), were procured from GenePharma (Shanghai, China). Then, the above shRNAs were cloned into pLKO.1 shRNA lentiviral vector (Addgene, Shanghai, China), followed by co-transfection of psPAX2 lentiviral packaging vector (Addgene) into HEK-293 T cells. Next, the collected lentiviruses particles containing the above respective shRNAs were used to infect LAD cells, and the stable cells were selected by puromycin treatment. For the overexpression of MARCKSL1–2, HDAC1, or SUZ12, the respective cDNA sequences were separately sub-cloned into pcDNA3.1 vectors (Invitrogen, Carlsbad, CA, USA) to construct pcDNA/MARCKSL1–2, pcDNA/HDAC1, and pcDNA/SUZ12 vectors. The empty vector (pcDNA3.1) was used as the negative control. Besides, cells were transfected with miR-200b mimics/inhibitor (Ribobio) to mimic or inhibit miR-200b, with NC mimics/inhibitor (Ribobio) as the negative control, respectively. The transfection of indicated pcDNA3.1 plasmid and miR mimics/inhibitor into LAD cells were achieved by using Lipofectamine 3000 (Invitrogen).

Colony formation assay

Cells were seeded into 6-well plates and cultured for 2 weeks. Then, cells were fixed by formaldehyde for 30 min and stained by 0.5% crystal violet. Subsequently, the plates were imaged and the colonies with over 50 cells were counted manually.

5-Ethynyl-2′-deoxyuridine (EdU) assay

EdU assay was undertaken with BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, Shanghai, China). After washing in PBS, EdU solution was used to incubate cells for 2 h. Cell nuclei were then stained by DAPI solution. After washing, samples were observed with an inverted microscope (Olympus).

Flow cytometry analysis

Cell apoptosis was assessed with flow cytometry (BD Biosciences, Franklin Lakes, NJ) as instructed. The Annexin V-FITC/PI Apoptosis Detection Kit (Elabscience, Wuhan, China) was utilized to stain cells for 15 min. Next, cell samples were collected from 6-well plates by centrifugation and the apoptotic cells were analyzed by flow cytometry.

Terminal-deoxynucleoitidyl transferase mediated Nick end labeling (TUNEL) assay

Cells were rinsed using PBS and then fixed with ethanol. The apoptotic cells were stained by using TUNEL reagents (Merck KGaA, Darmstadt, Germany) in accordance with the standard method. The apoptotic cells were captured by an optical microscope (Olympus).

Luciferase reporter assay

The promoter sequence of miR-200b or HDAC1 was separately inserted into the pGL3-basic vector (Promega, Madison, WI, USA) for luciferase reporter assays. Then, LAD cells (H1299 and SPC-A1) or DTX-resistant LAD cells (H1299/DTX and SPC-A1/DTX) were separately co-transfected with MARCKSL1–2 silencing or overexpression plasmids along with the recombinant pGL3 vectors for 48 h. The relative luciferase intensity was detected via luciferase reporter assay system (Promega).

Chemosensitivity assay

Drug sensitivity was evaluated by cell counting kit-8 (CCK-8) assay. The parental LAD cells (H1299 and SPC-A1) and DTX-resistant LAD cells (H1299/DTX and SPC-A1/DTX) were treated with different doses of DTX and fixed in 96-well plates. Then, CCK-8 solution (Dojindo, Osaka, Japan) was added. OD value was measured at the absorbance of 450 nm. The 50% inhibitory concentration (IC50) value of indicated LAD cells to DTX treatment was defined as the DTX dose which induces 50% cell death.

Chromatin immunoprecipitation (ChIP)

ChIP assay was undertaken by using the EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore, Bedford, MA, USA) according to the manufacturer’s direction. In short, cells were fixed with 1% formaldehyde for 15 min and subjected to ultrasonic treatment for shearing DNA into fragments (500 bp). The immunoprecipitation was implemented by mixing DNA fragments with 30 μl of magnetic beads conjugating with HDAC1 (#34589, 1/50 dilution, Cell signaling technology, Boston, MA, USA), SUZ12 (#3737, 1/100 dilution, Cell signaling technology), acetyl-histone H3 (#8173, 1/100 dilution, Cell signaling technology) or tri-methyl-histone H3 (lys27) (H3K27me3, #9733, 1/50 dilution, Cell signaling technology), and those anti-IgG antibodies (Millipore) acted as the negative control. Finally, the precipitated DNA fragments were analyzed via qPCR and agarose gel electrophoresis (AGE).

RNA pull-down assay

The RNA pull-down assay was conducted by using the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA, USA). In short, total protein extracts, obtained from cells via RIPA lysis buffer, were mixed with biotinylated probe for MARCKSL1–2 or MARCKSL1–2 antisense (AS). The pulled-down mixture was processed with SDS-PAGE and sliver staining, and the proteins were analyzed via mass spectrometry. Finally, the existence of interested protein in the pull-down complex was detected through Western bloting.

RNA immunoprecipitation (RIP)

Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) was used for RIP assay in accordance with the instructions. The processed cells were conjugated with anti-SUZ12 antibody (#3737, 1/100 dilution, Cell signaling technology) and control IgG antibody (Millipore) on magnetic beads. After digestion, the precipitated RNAs were extracted for RT-qPCR analysis.

Western blot

Cells were lysed in RIPA lysis buffer. Total proteins were separated on 12% SDS-PAGE. Then, they were transferred onto PVDF membranes and blocked with 5% skimmed milk. The primary antibodies against loading control GAPDH (ab181602, 1/10000 dilution), HDAC1 (ab109411, 1/2000 dilution) and SUZ12 (ab12073, 1/1000 dilution) were used. Then, incubation with the HRP-labelled secondary antibodies (ab216773, 1/10000 dilution) was conducted. All antibodies were bought from Abcam (Cambridge, MA, USA) and used as recommended. All protein bands were finally measured after adding the ECL reagent (Bio-Rad, Hercules, CA, USA). The experiment was independently repeated three times, and the representative blots of three independent experiments were provided in the Figures.

In vivo experiments

Nude mice (4–6 weeks old) used for in vivo experiments were obtained from the Nanjing General Hospital of Nanjing Military Command. The animal study was approved by the Review Board of Hospital Ethics Committee of Nanjing General Hospital of Nanjing Military Command (Nanjing General Hospital of Nanjing Military Command, Nanjing University, China). Animal models were established by subcutaneously injecting 2 × 106 H1299/DTX cells transfected with pcDNA3.1 and pcDNA3.1/MARCKSL1–2 (n = 6 each group). Two weeks later, three mice from the above two groups were randomly selected and treated with DTX (1 mg/kg) through intraperitoneal injections. After 4 weeks, all mice were sacrificed. The primary tumors were resected and weighed, followed by embedded with paraffin and fixed with formalin.

Statistical analysis

All experimental data were obtained from three bio-repeated experiments and shown as the mean ± standard deviation (S.D.). GraphPad Prism 7 (La Jolla, CA) was used for statistical analyses. Comparison among multiple groups was analyzed by one-way or two-way ANOVA, and that between two groups was analyzed using Student’s t test. Results were considered statistically significant when a p-value< 0.05.