Ethics statement
Approved by the ethics committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, this study was conducted following Declaration of Helsinki. An signed consent was provided by each participant. Animal treatment was humanely performed.
Sample collection
Lung cancer tissues and normal tissues were harvested from 80 patients (not received radiotherapy or chemotherapy) in Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from April 2018 to March 2019. All tissues were frozen in liquid nitrogen. The diagnosis was conducted by the pathologists in Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology in compliance with the principles of the latest classification regulations of the World Health Organization [17].
Cell culture
Human lung cancer cell lines (A549, H1299 and H460) and normal human lung epithelial cell line (BEAS-2B) were supplied by the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and maintained in 10% fetal bovine serum (FBS)-Dulbecco's modified Eagle medium (DMEM) (both from HyClone, Los Angeles, USA) [18].
Cell transfection
Based on the known miR-449a and KDM3A sequences in NCBI, plasmids were constructed by Sangon Biotech (Shanghai, China). Cells of passage 3 were trypsinized and cultured into a monolayer in a 24-well plate at 1 × 103 cells/well. Cells at 75% confluence were transfected with miR-449a-mimic/inhibitor, sh-KDM3A, overexpression (oe)-KDM3A, miR-449a-mimic + oe-HIF-1α, miR-449a-mimic + oe-KDM3A, or their corresponding negative control (NC) through Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, USA) [19].
Cell counting kit (CCK)-8 assay
Cells in a 96-well plate (1 × 103 cells/well) were cultured for 0, 24, 48, and 72 h, respectively. At each time point, each well was supplemented with 10 μL CCK-8 reagent (Dojindo, Kumamoto, Japan). Cells that were continuously cultured for further 3 h were detected by a microplate reader (Infinite M200 PRO, TECAN, Switzerland) to measure the optical density (OD450 nm) [18].
Transwell assay
Cells (6 × 104 cells) were resuspended in serum-free DMEM (200 μL), and added in the chamber. An attractant was set with 10% FBS-DMEM (600 μL) in the lower chamber. Cells were incubated for 24 h in migration assay and for 48 h in invasion assay. Cells that migrated or invaded were fixed with 4% paraformaldehyde, followed by staining with 0.1% crystal violet and photography by a microscope (TE2000; Nikon, Tokyo, Japan) in at least five random fields of view [20].
Flow cytometry
A549 cells transfected for 48 h were pre-cooled with 70% frozen ethanol and treated with propidium iodide (PI) and RNase (BD Company, New Jersey, USA). Cell cycle distribution was analyzed with a flow cytometer (BD Biosciences, New Jersey, USA) [21]. For cell apoptosis, cells were detached with 0.25% trypsin, added with Roswell Park Memorial Institute-1640 medium containing 10% FBS, centrifuged at 1000 r/min, fixed with 70% ethanol and adjusted to 1 × 106 cells/mL. The cell suspension was added with 10 mL Annexin V-fluorescein isothiocyanate (FITC)/PI (556,547, Shanghai Shuojia Biotechnology Co., Ltd., China) and cell apoptosis was detected in a flow cytometer (XL type, Conlter, USA). Excitation wavelength was 480 nm, FITC was detected at 530 nm and PI was detected at greater than 575 nm. The apoptotic rate was percentage of apoptotic cells in total cells [22].
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
RNA was extracted from tissues and cells by Trizol (Life Technologies, MD, USA). cDNA was generated by a reverse transcription kit (Thermo, Massachusetts, USA). miR-449a, KDM3A, and HIF-1α levels were measured by SYBR Green method on the ABI7500 fluorescent quantitative PCR instrument, and normalized to U6 and β-actin. Data analysis was conferred to 2−ΔΔCt method. Primers were found in Table 1 [23].
Western blot assay
The extracted total protein in cells and tissues by radio-immunoprecipitation assay (RIPA) buffer was quantified by bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Subsequently, total protein was separated with 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a 0.45-µM polyvinylidene fluoride membrane (MilliPore, MA, USA). The protein membrane incubated with the specific antibodies were developed by enhanced chemiluminescence and exposed by Image Quant LAS 4000C (GE Company, USA). Primary antibodies included KDM3A (1:200, 12,835–1-AP, Proteintech), HIF-1α (1:1000, 610,958, BD Biosciences), Cleaved-PARP (ab32064, Abcam, MA, USA), Cleaved-CASP3 (9661, Cell Signaling Technology) and β-actin (1:1000, sc-47778, Santa Cruz Biotechnology) while secondary antibody included anti-rabbit immunoglobulin G (IgG; 7074, 1:2000, Cell Signaling Technology). β-actin was the internal control [24].
Dual luciferase reporter gene assay
The binding sites of miR-449a and KDM3 were firstly analyzed by RNA22 and their targeting relation was validated by dual luciferase reporter gene experiment. KDM3A mRNA 3'untranslated region (3'UTR) and the mutant sequence were cloned into the pGL3 dual luciferase reporter gene vector (Promega, WI, USA) to construct wild-type and mutant KDM3A 3'UTR vectors. The constructed vectors with miR-449a mimic/NC-mimic were co-transfected into A549 cells through Lipofectamine 2000 (Invitrogen). The luciferase activity was measured by the dual luciferase reporter system (Glomax20/20, ATCC, Manassas, VA, USA) [25].
Co-immunoprecipitation (Co-IP) assay
Cells were lysed with IP lysis buffer (500 μL) on ice and centrifuged at 13,500 r/min. The supernatant was reacted with the antibody overnight, added with protein A + G agarose (P2055, Beyotime, China) and centrifuged at 13,500 r/min. After washing 3 times with cold lysis buffer, the immunoprecipitate was combined with sodium dodecyl sulfate, heated to 98 °C and treated with sodium dodecyl sulphate polyacrylamide gel electrophoresis. After that, the protein was transferred to a 0.22-μm polyvinylidene fluoride membrane, immersed in 10% skimmed milk and combined with the primary antibodies KDM3A (1:200, 12,835–1-AP, Proteintech) and HIF-1α (1:1000, 610,958, BD Biosciences). Then, the membrane was washed 3 times with Tris-buffered saline with Tween-20 and cultured with IgG antibody (7074, 1:2000, Cell Signaling Technology). The pellet was rinsed with RIPA buffer and then subjected to Western blot analysis. Normal IgG was used as a NC [26].
Tumor xenografts in nude mice
Twenty-five BALA/C nude mice (4–6 weeks old, 18–20 g) from Hunan SJA laboratory Animal Co., Ltd. (Changsha, China) were subcutaneously injected with A549 cells (1 × 103 cells) into the right armpit. Phosphate-buffered saline (PBS)-treated A549 cells were used as NC. The average diameter of tumors was measured weekly. After 8 w, all mice were euthanized and tumor weight was measured [27].
Statistical analysis
The independent t-test was applied to compare data between two groups. All data were analyzed by statistical software SPSS 17.0 (SPSS, Chicago, USA) and expressed as mean ± standard deviation. P < 0.05 was considered a significant difference.