Cell culture Normal airways were obtained from 1st through 4st order bronchi from pneumonectomy and lobectomy specimens for primary lung cancer. Informed consent was obtained from all surgical participants as part of an approved ongoing research protocol by the ethics committee of Zhejiang University school of Medicine. Human bronchial epithelial cells (HBEC) were isolated with protease treatment and characterized as previously described. HBEC were serially passaged and used for experiments until passage 3. The majority of experiments were performed with HBEC from non-COPD patients. HBEC were cultured in DMEM (Beyotime, MA0212) with 10% fetal calf serum (Beyotime,C0227) and penicillin-streptomycin (Beyotime, C0222).
Antibodies and reagents Antibodies used were rabbit anti-p21(Abcam,ab109520), rabbit anti-PINK1(Abcam,ab216144), rabbit anti-MAP1LC3B/LC3B (Abcam, ab247327),rabbit anti-MAP1LC3B/LC3B(Novus, 600–1384),rabbit anti-TOMM20 (Abcam,ab186735), Mouse anti-ACTB/β-actin(Abcam, ab8226), The following reagents were used: bafilomycin A1 (BafA1; Sigma-Aldrich, B1793), MG-132 (Enzo Life Sciences, BML-P102), CM-H2DCFDA (Life Technologies, C6827), Hoechst 33258 (Sigma-Aldrich, B2883), MitoSOX Red (Molecular Probes/Life Technologies, M36008).
Small interfering and transfection pcDNA-SAL-RNA1 were constructed by inserting SAL-RNA1 cDNA. pcDNA was used as a negative control. For SAL-RNA1 konckdown, small interfering RNA targeting SAL-RNA1(siRNA-SAL-RNA1)were synthesized by Ribobio Co.,Ltd.(Guangzhou, China).siRNA was used as a negative control. Transfection experiments were conducted using Lipofectamine 2000 reagent (Invitrogen).
Preparation of cigarette smoke extract (CSE) Cigarette smoke extract (CSE) was prepared as previously described with minor modifications. The research reference cigarettes 3R4F were used for experiments (Louisville, KY, USA). Forty milliliters of cigarette smoke were drawn into a syringe and slowly bubbled into sterile serum-free cell culture media in 15-ml BD falcon tubes. One cigarette was used for the preparation of 10 mL of solution. CSE solution was filtered (0.22 µm; Merck Millipore, SLGS033SS) to remove insoluble particles and was designated as 100% CSE solution.
qRT-PCR Total RNAs were extracted from glomeruli and podocytes using Trizol reagent (Beyotime Biotechnology) according to the manufacturer’s instructions. RNAs were transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA). PCR mixtures (25 µl) containing 2.5 µM of primer pairs, cDNA template, and SYBR Green Real-Time PCR Master Mixes (AppliedBiosystems) were subjected to PCR amplification using QuantStudio 5 Real-Time PCR System (Applied Biosystems). The relative SAL-RNA1 and SIRT1 expressions were calculated using the 2−ΔΔCT method. GAPDH was used as an internal reference.
Measurement of ROS production HBEC were grown on 8-well culture slides. HBEC transfected with pcDNA,pcDNA SAL-RNA1 were treated with CSE at 48 h post transfection or SRT1720 administration. HBEC were transfected with control siRNA, SAL-RNA1 siRNA, and were treated with CSE at 48 h post-transfection or selisistat. HBEC were fixed after 24-h treatment with CSE. Mitochondrial ROS production was analyzed by MitoSOX Red staining according to the manufacturer’s instructions, which was evaluated by fluorescence microscopy (Olympus, Tokyo, Japan and Keyence, BZ-X700).
RNA pull-down Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific) was used to determine the interaction between SAL-RNA1 and SIRT1. HBEC were transfected with biotinylated SAL-RNA1 and negative control (NC). The lysate was incubated with streptavidin magnetic beads. Then, the SAL-RNA1-SIRT1 complex bound to the beads were eluted and detected by western blot analysis.
RNA immunoprecipitation (RIP) RIP assay was performed to confirm the interaction between SAL-RNA1 and SIRT1. Briefly, HBEC were lysed with RIP lysis buffer.The lysate was incubated with an anti-SIRT1 rabbit polyclonal antibody at 4°C overnight with Magnetic Beads.protein A/G. The beads were washed with cold RIP wash buffer for six times. Then, beads were resuspended in 150 µl Proteinase K buffer and incubated at 55°C for 30 min. RNA was extracted and purified. qRT-PCR was then conducted with the purified RNA. Total RNAs (input controls) and isotype controls (normal mouse IgG) were simultaneously determined.
Ubiquitination assay To examine whether SNHG17 affects Mst1 ubiquitination, HA-Ub, FLAG-SIRT1, siRNA-control, siRNA-SAL-RNA1 were transfected into HBEC using transfection reagent for 12 h. After transfecting, HBEC were treated with MG132 (10 µM) for 4 h and then collected and lysed. Western blot analysis of cell lysates was used to confirm Mst1 protein expression. Then, cell lysates were immunoprecipitated with an anti-FLAG antibody at 4°C followed by immunoblotting with an anti-HA antibody.
SA–β-gal activity assay. SA–β-gal activity was quantitatively measured by the rate of conversion of 4-methylumbelliferyl-β-d-galactopyranoside (MUG) to the fluorescent hydrolysis product 4-methylumbelliferone (4-MU) at pH 6.0, as described previously. Briefly, lung tissues were homogenized in the lysis buffer (5 mM CHAPS, 40 mM citric acid, 40 mM sodium phosphate, 0.5 mM benzamidine, and 0.25 mM PMSF, pH 6.0), and kept on ice for 1 hour. The lysates were centrifuged for 5 minutes at 12,000g, and the supernatant was mixed with 2× reaction buffer (40 mM citric acid, 40 mM sodium phosphate, 300 mM NaCl, 10 mM β-mercapto-ethanol, and 4 mM MgCl2 [pH 6.0] with 1.7 mM MUG), which was placed into a 37°C water bath for 3 hours. Finally, 50 µl of the reaction mix was added to 500 µl of 400 mM sodium carbonate stop solution (pH 11.0), which was read at 150 µl/well in a 96-well plate using a SpectrumMax M5 plate reader (Molecular Devices) with excitation at 360 nm, emission at 465 nm, 40 µs integration, and gain held constant at 46. Normalized SA–β-gal activity was expressed as observed fluorescence (rate of conversion of 4-methylumbelliferyl-β-d-galactopyranoside to 4-MU) divided by milligram total protein in the assay.
Mouse model and CS exposure protocol C57BL/6J (CLEA Japan INC, Tokyo, Japan) and B6.129-SAL-RNA1−/− (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) mice were purchased, and were maintained in the animal facility at the Zhejiang University School of Medicine. All experimental procedures were approved by the Zhejiang University School of Medicine Animal Care Committee. 6 to 8-week-old mice were exposed using a whole-body exposure system (SCIREQ ‘InExpose’) within a barrier facility. Mice were exposed at a total suspended particulates of 200 mg/m3 using 3R4F cigarettes for 5 days a week. After 6 months, immediately following sacrifice, the right lung was inflated with 10% buffered formalin at a pressure of 20 cm H2O. Fixed lungs were embedded in paraffin and cut into 4-µm sections for tissue staining. The sections stained with hemaoxylin & eosin (H-E) and Picro-Sirius Red Stain (PSR) according to conventional protocols for histopathological evaluation. Immunohistochemistry was performed on the paraffin-embedded lung tissues as previously described with minor modifications. The left lungs were removed and homogenized for protein evaluation or the frozen sections stained with SA-β- gal staining and immunofluorescence staining. Tracheotomy was performed to insert a tracheal tube for collecting bronchoalveolar lavage fluid (BALF) sample with PBS (Wako, 041-20211; total 3 ml). The cell numbers in BALF were counted using a hemocytometer. Differential cell counts in BALF were analyzed on 300 cells stained with Diff-Quick (Sysmex, 16920).
Immunofluorescence staining HBEC were grown on 8-well culture slides. HBEC transfected with pcDNA SAL-RNA1 were treated with CSE at 48 h post-transfection or selisistat. HBEC were transfected with control siRNA, SAL-RNA1 siRNA and were treated with CSE at 48 h post-transfection or SRT1720 administration. HBEC were treated with BafA1 6 h before collection. HBEC were fixed with 100% methanol (Wako, 137–01823). After blocking with 1% BSA (Sigma-Aldrich, A2153) for 60 min, the primary and secondary antibodies were applied according to the manufacturers’ instructions. Confocal laser scanning microscopy analysis of BEAS-2B cells was performed using mouse anti-TOMM20 and rabbit anti- MAP1LC3B (Cell Signaling Technology, 3868), and evaluated by fluorescence microscopy (Carl Zeiss LSM880, Tokyo, Japan). Quantitative measure of immunofluorescence staining to evaluate mitophagic activity was analyzed by dividing the yellow intensity of mitophagy areas by the red intensity of mitochondrial areas. Fluorescence microscopy analysis of phospho-histone H2AFX and Hoechst 33258 staining were performed in lung frozen sections and HBEC, and evaluated by fluorescence microscopy (Olympus, BX60, Tokyo, Japan and Keyence, BZ-X700, Tokyo, Japan).
Lung morphometry. Mouse lungs (which had not been lavaged) were inflated with 1% low-melt agarose at a pressure of 25 cm H2O, then fixed with 4% neutral buffered PFA. Fixed lung was dehydrated, embedded in paraffin, and sectioned into 4-µm sections using a rotary microtome (MICROM International GmbH). H&E staining was performed on the lung midsagittal sections to determine Lm of airspace using MetaMorph software (Molecular Devices). Ten randomly selected ×100 fields per slide were photographed in a blinded manner, and the images were manually thresholded. The airway and vascular structures were eliminated from the analysis.
Measurement of lung mechanics. Lung mechanical properties, including lung compliance and RL, were determined as described previously. Briefly, the mouse was weighed, deeply anesthetized by i.p. injection of pentobarbital (90 mg/kg BW) and pancuronium (0.5 mg/kg BW), and tracheostomized. The trachea was cannulated, and the cannula was connected to a computer-controlled small animal ventilator (FlexiVent; SCIREQ). Estimated lung compliance, RL, and Rn at 3 cmH2O positive end expiratory pressure were obtained by fitting a model to each impedance spectrum. The calibration procedure removed the impedance of the equipment and tracheal tube within this system.
Electron microscopy Electron microscopy was performed as previously described. Pieces from mouse lung were fixed with 2% glutaraldehyde, 0.1 M phosphate buffer (pH 7.4) and after 48 h of incubation were dehydrated with a graded series of ethanol. Fixed lung pieces were then embedded in epoxy resin (Epok812; Oken, 02–1001). Ultrathin sections were stained with uranyl acetate and lead citrate and observed with the Hitachi H-7500 transmission electron microscope (Hitachi, Tokyo, Japan). For quantitative evaluation of mitochondria in airway epithelial cells, 10 airway epithelial cells imaged at 10,000X magnification were selected for each sample and mitochondria were counted.
Statistics Data are shown as the average (± SEM) taken from at least 3 independent experiments. Comparisons between 2 different groups were determined by Student t test for parametric data or Mann-Whitney test for nonparametric data. One-way analysis of variance was used for multiple comparisons and Tukey or Bonferroni post-hoc tests used to test for statistical significance. Significance was defined as P < 0.05. Statistical software used was Prism v.5 (GraphPad Software, Inc., San Diego, CA).