Patients and samples
We collected lung cancer tissues from the Chaohu Hospital affiliated to Anhui Medical University. All patients signed the informed consent of the using samples. Inclusion criteria were primary lung cancer patients receiving surgery as initial treatment and all clinicopathologic data were collected. Smoking patients have been smoking for at least 20 years and smoked 20 cigarettes a day before surgical operation. The human ethics and research ethics committees of Anhui Medical University approved the study (approval no. 202104001).
Cells culture and transfection
Lung cancer cell lines of A549, HCC827 were obtained from Cell Culture Center, Chinese Academy of Medical Sciences (Beijing, China). We identified the characteristics of A549 and HCC827 cell lines by STR analysis in 2020 and 2021 respectively. We stated that all experiments were performed with
mycoplasma-free cells. A549 and HCC827 cell lines were cultured in RPMI 1640 (Invitrogen) with 10% fetal calf serum (Hyclone, Logan, UT), streptomycin(100 μg/mL) and penicillin(100 U/mL). Cells were transiently transfected with plasmid or siRNA oligonucleotide (Invitrogen). Nicotine was gotten from Sigma-Aldrich (Code N3876). Nicotine was added to lung cancer cells with the concentration 5 μg/ml or 10μg/ml.
RNA isolation and quantitative reverse-transcription polymerase chain reaction (qRT-PCR)
We identified SNHG5 by qRT-PCR assay. Total cells or tissues RNAs were extracted with TRIzol (Invitrogen, Carlsbad, CA). QPCR assay was conducted using QuantiTect SYBR Green PCR Kit (Takara Bio Inc., Otsu, Japan) on Stepone Real-Time PCR System (Applied Biosystems, Carlsbad, CA). All primers were designed by Primer Premier 5.0. The primers were gotten from Sangon Biotech (Shanghai, China) (Supplementary Table 1). RNA expression was normalized to GAPDH or U6. RNA relative expression levels were calculated by using the 2-ΔΔCt method (Biorad CFX manager software 3.1).
SNHG5 expression vector construction
According to the instructions of manufacturer total RNA in the cells was extracted by using Rneasy Mini Kit (Invitrogen). The first-strand complementary DNA (cDNA) of SNHG5 was obtained by using Maxima TM First Strand cDNA Synthesis Kit (Fermentas) and DNA was amplified using the primers (supplement table 1). PCR products and pcDNA3.1 vector were digested. The full length sequence of SNHG5 was inserted into pcDNA3.1 vector to get pcDNA3.1-SNHG5.
Cellular fractionation
Norgen’s Cytoplasmic and nuclear RNA purification kit (Norgen BioTek, Canada) were used to extract nuclear and cytoplasmic RNA.
Apoptosis detection
We washed the cells three times using phosphate-buffered saline (PBS). The cells were stained using the Annexin V/fluorescein isothiocyanate (FITC). The single cell suspension was first incubated with 500 μL of 1×buffer, 5 μL of Annexin V and 5 μL of Propidium Iodide (PI) at 37°C for 35 min in dark place. The above cells were detected by FACSAriaTM flow cytometry and analyzed by Win MDI. 2.9 software (TSRI Flow Cytometry Core Facility, La Jolla, CA).
Western blot
Using RIPA Lysis Buffer (RIPA; Beyotime, Shanghai, China) cells total protein was extracted. Using bicinchoninic acid (BCA) method (Pierce, Waltham, MA, USA) the protein was quantified and loaded for electrophoresis. The protein was transferred on a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Then we blocked the membrane with 5% skim milk for about 2 hours and the membrane was soaked in primary antibodies at 4°C for 12h and secondary antibodies for 2 h. Bands were exposed by enhanced chemiluminescence (ECL) and analyzed by Image J Software (NIH, Bethesda, MD, USA).
RNA pull-down assay
SNHG5 and SNHG5-antisense were transcribed using the templates of pcDNA3.1-SNHG5 and pcDNA3.1-SNHG5-antisense plasmids. The plasmids were amplified. And the primers were showed in the supplementary table 1. Using Biotin-RNA Labeling Mix (Roche, Indianapolis, IN, USA) biotin-labeled RNAs were transcribed in vitro. Then biotin-labeled RNAs were treated with RNase-free DNase I (TaKaRa, Kyoto, Japan) and purified with an RNeasy Mini Kit (Roche). RNA structure buffer [10 m M Tris (pH 7), 0.1 M KCl, and 10 mM MgCl 2 ] included biotinylated RNA was in 98 °C for about 2 min, on ice for 5 min, and then stayed at room temperature for approximately 35 min. Total protein lysates of A549 cells were mixed with biotinylated RNA and incubated at room temperature. Streptavidin agarose beads (GE Heathcare, Little Chalfont, UK) were added to each binding reaction and further incubated at room temperature with rotation. The RNA-protein-beads compound were washed three times and dissolved into free nuclear water. Then the compound was boiled with sodium dodecyl sulfate polyacrylamide gel electrophoresis-loading buffer for about 15 min. The captures were given electrophoresis with 12% sodium dodecyl sulfate polyacrylamide gel and silver stained. Mass spectrometry (LC-MS/MS, A TripleTOF, ABsciex, Concord, ON, USA) identified the protein bands after in-gel trypsin digestion.
RIP assay
Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore, Billerica, MA, USA) was used to performe RIP according to the manufacturer’s instructions. Antibody against XIAP obtained from Cell Signaling Technology (Beverly, MA). The coprecipitated RNAs were adsorbed with magnetic beads and detected by reverse transcription-PCR.
UV-RIP
Cells applied 100 mM 4-thiouridine (4-SU) were cultured for about 12 hours. Then cells were cross-linked by 365 nm UV light at a dose of 400 mJ/cm2 in UV-RIP assay. The other UV-RIP protocols were same as RIP.
Statistical analysis
We made the statistical analysis with SPSS software (version 15.0). The differences between the two groups were analyzed by t-test. A p<0.05 showed the significant difference.