Cell culture and reagents
Human SCLC cell line NCI-H1688 and human lung bronchial epithelial cell line BEAS-2B were obtained and authenticated from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). NCI-H82, NCI-H526 and 16HBE were purchased and authenticated from the ATCC. All cell lines were maintained in either RPMI-1640 medium or DMEM (Thermo Fisher Scientific, MA, USA) medium supplemented with 10% fetal bovine serum. Cells were cultured at 37°C in a humidified atmosphere with 5% CO2. The natural compound library containing 640 natural compounds and panobinostat(T2383), THZ1(T3664), Gemcitabine(T0251), (+)-JQ-1(T2110) were purchased from Target Mol (Shanghai, China).
Cell proliferation and clone formation assay
Cell proliferation was determined by using Cell Counting Kit-8 (CCK-8) kit (Dojindo Molecular Technologies, Inc) according to the manufacturer’s protocol. For the formation assay, a total of 1000 cells were seeded into six-well plates and cultured in RPMI-1640 medium containing various concentrations of Sg (0, 0.5, 1, 1.5 µM). After 24h treatment, cells were changed to fresh complete medium and cultured for 2 weeks. The colonies were then fixed with methanol and stained with a 0.1% crystal violet solution.
Cell cycle and apoptosis assay
Cells were collected after 24h treatment with different concentrations of Sg. For cell cycle analysis, the collected cells were fixed in 70% ethanol at 4°C overnight and stained with Propidium Iodide (P4170, Sigma). Apoptosis was determined using FITC Annexin-V apoptosis detection kit (556547, BD Biosciences) according to the manufacturer’s protocol. Both cell cycle and apoptosis were analyzed by flow cytometry (Accuri C6, BD Biosciences).
Wound healing assay
3×105 cells were seeded in six-well plates and grown to 90% confluency in an incubator, followed by 48h of starvation in serum-free medium. The culture medium was removed and the monolayers were scratched using a 200µl pipette to create a uniform cell-free wound area. Debris was removed by gently washing with PBS. Cell migration into the wounded area was monitored and photographed at each designated time point under a microscope.
Transwell cell migration and invasion assay
Cell migration assay was carried out in a 24-well plate containing a polycarbonate membrane with a pore size of 8mm. For cell invasion assay, the top side of the membrane was coated with Matrigel and was placed in the upper chamber. Cells were seeded into the upper chambers. The lower chambers were filled with complete medium. After 24h, the chambers were removed, and inner side was wiped with cotton swaps. The cells were fixed with 4% formaldehyde, stained with 0.1% crystal violet and counted under light microscope.
Plasmids and cell transfections
Short hairpin RNA (shRNA) sequences were cloned into psiF-copGFP vectors (System Biosciences, Mountain View, CA). The shRNA sequences for CDKN1A were as follows: 5′-GACACCACTGGAGGGTGACTT-3′, 5′-GACAGCAGAGGAAGACCATGT-3′, 5′-ACAGATTTCTACCACTCCAA-3′, 5′-CCGCGACTGTGATGCGCTAAT-3′. The sequence for negative control(shRNA-control) was 5′-GGTGTGCAGTTGGAATGTA-3′.
To establish stably transfected cell lines, lentivirus was produced by HEK-293T cells with the second-generation packaging system pMD2.G and psPAX2, and harvested at 48h after transfection. SCLC cell lines were transduced with virus in the presence of 8µg/mL polybrene and screened them with puromycin for 7 days.
Western blot
Cells were lysed with RIPA buffer (Cell Signaling). Western blot analysis was then carried out using a standard protocol. Antibodies for CDKN1A (A19094), CDKN1B (A19095), CDKN1C (A2060) and p53 (A0263) were purchased from ABclonal Technology. β-actin antibody (H11459) was purchased from Sigma as a loading control. The immunoreactive proteins were visualized with SuperSignal West Dura Chemiluminescent (Thermo Fisher).
Quantitative real-time PCR (qPCR)
Total RNA was isolated by using Trizol reagent and reverse transcribed with a StarScript II First-strand cDNA Synthesis Kit (GenStar, A212-05) in accordance with a standard protocol. QPCR was performed using the SYBR Green Supermix (Bio-Rad, CA, USA) and CFX96 real-time PCR detection system (Bio-Rad). The mRNA expression of genes was evaluated by using the 2−△△Ct method. The primers for CDKN1A were: 5’-GGAAGGGACACACAAGAAGAAG-3’ and 5’-AGCCTCTACTGCCACCATCTTA-3’; the primers for CDKN1B were: 5’-GCTTCTTGGGCGTCTGCTC-3’ and 5’-TAACCCGGGACTTGGAGAAG-3’; the primers for CDKN1C were: 5’-GCGGCGATCAAGAAGCTGT-3’ and 5’-ATCGCCCGACGACTTCTCA-3’; the primers for β-actin were: 5’-TCGTGCGTGACATTAAGGAG-3’ and 5’-ATGCCAGGGTACATGGTGGT-3’.
Xenograft model
Animal experiments were performed according to protocols approved by the ethical committee of Guangzhou University of Chinese Medicine Institutional Animal Care and Use Committee. Five-week-old BALB/C nude mice were used for in vivo anti-SCLC effect of Sg. 2×106 NCI-H1688 cells were injected subcutaneously into athymic nude mice. Tumor volume was measured as [(length × width × height)/2] every 3 days. When the tumor volume reached ~ 30 mm3, treatments were carried out via intraperitoneal injection. After treatment for 21 days, the mice were sacrificed by cervical dislocation method, and tumor tissues were collected for immunohistochemical staining (IHC) and Western blot.
Immunohistochemistry
Tumors were collected at the indicated times and fixed with 10% neutral buffered formalin. After fixation for 24h, the tissues were paraffin-embedded, sectioned (4µm). For immunohistochemistry staining, deparaffinized and rehydrated sections were boiled in Na-citrate buffer (10mM, pH 6.0) for 30min for antigen retrieval. The sections were incubated with primary antibodies and developed by using the Ultra Vision Detection System (Thermo Fisher Scientific). Images were acquired using an Olympus IX51 microscope and processed using cellSens Dimension software.
RNA-sequencing and bioinformatics analysis
NCI-H1688 cells were treated with Sg at 1µM. After 24h, total RNA was extracted for RNA-sequencing (RNA-Seq) by using the TRI Reagent and Direct-zol RNA kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s instructions. The sequencing was performed by a sequencing service company (Novogene, Beijing, China) using the Illumina sequencing platform. Log2|Fold change| ≥ 1 and P < 0.05 were used as a cut-off to define overexpression or downregulation. Ingenuity Pathway Analysis (IPA) was used to identify enriched molecular pathways, gene networks, canonical pathways, and cellular biological consequences. Protein-protein interaction(PPI) analysis of genes was processed by STRING (https://string-db.org/) and visualized by Cytoscape. The hub genes in the network defined as possessing a connective degree > 10.
Gene expression analysis from the public database
Data from the Gene Expression Omnibus (GEO, www.ncbi.nlm.nih.gov/geo) was used to analyze CDKN1A mRNA expression in SCLC, GSE6044[20] and GSE40275[21], a total of 19 normal lung and 17 SCLC samples were included in the present study. Patient data from George J [22] was used to conduct OS analysis, in brief, patients were grouped into the higher (CDKN1A-H) and lower (CDKN1A-L) group based on their relative expression of CDKN1A.
Statistical analysis
Data are presented as the means ± SD. All statistical analyses were carried out using SPSS software (version 22). An unpaired two-tailed Student’s t-test was performed for two-group comparisons and one-way ANOVA analysis was performed for multiple group comparisons. The statistical significance was set at a * p < 0.05, ** p < 0.01, ***p < 0.001.