Patients
120 paired tumor tissues and para-carcinoma non-tumor tissues were obtained from patients with lung adenocarcinoma who had undergone surgical resection without receiving any preoperative adjuvant therapy in the Second Affiliated Hospital, Zhejiang University School of Medicine from May 2013 to April 2015. Written informed consent was obtained from each patient, and the Ethics Committee approved the present study of the Second Affiliated Hospital, Zhejiang University School of Medicine. The pathological parameters of all patients were presented in Table 1.
Quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR)
The RT-qPCR analysis was used to analyze the abnormal expressions of SCARA5 in tissues and cells, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) functioned as the internal control. Briefly, total RNA was isolated and extracted from tissues or cells using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) under the manufacturer’s instructions. Then, RNA was reverse transcribed into cDNA with the ExScript RT-PCR kit (Takara, Dalian, China). The expression of SCARA5 was detected by SYBR Green PCR Master Mix kit (Applied Biosystems Life Technologies, Foster City, CA, USA) following the protocol. All the amplification and detections were performed in the CFX96 Real-Time PCR system (Bio-Rad, Hercules, CA, USA), and the thermo-cycling profiles were described as follows: 95℃ for 10 seconds, 40 cycles of 95℃ for 5 seconds, and 62℃ for 30 seconds. Statistical analysis was calculated using the 2-△△CT method.
Immunohistochemistry (IHC)
Tissues gathered from 120 patients with lung adenocarcinoma were fixed and embedded in paraffin. Immunohistochemistry was carried out using a rabbit anti-SCARA5 antibody (1:100; ab6118894; Abcam, Shanghai, China). Briefly, the 4 μm tissue sections were deparaffinized and rehydrated. Then, 3% H2O2 and 4% bovine serum albumin (BSA; Gibco; Thermo Fisher Scientific, Waltham, MA, USA) were applied to block endogenous peroxidase and nonspecific endogenous antigens, respectively. Afterward, the treated sections were incubated with an anti-SCARA5 antibody overnight at 4℃. After washing, the sections were then incubated with IgG H&L secondary antibody (1:1000; ab150077; Abcam, Shanghai, China) at room temperature for 40 min. Last, sections were visualized with DAB (ml016974; Mlbio, Shanghai, China) followed by counterstaining with hematoxylin (ml015306, Mlbio, Shanghai, China) and analyzed with light microscopy.
Cell culture and transfection
Human normal bronchial epithelial cell line BEAS-2B and human lung adenocarcinoma cell line A549 were purchased from American Type Culture Collection (ATCC, Manassas, VA, United States). Following the protocol, these two cell lines were cultured in PRMI-1640 medium supplemented with 10% fetal bovine serum and penicillin-streptomycin (Solarbio Life Science, Beijing, China) at a humidified atmosphere with 5% CO2 at 37℃.
A549 cells were plated in a 24-well plate and transfected with pcDAN3.1 or pcDNA3.1-SCARA5 plasmids using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, United States) as per the manufacturer’s protocol. pcDNA3.1 and pcDNA3.1-SCARA5 plasmids were designed and synthesized by GenePharma (Shanghai, China) under the manufacturer’s instructions.
Cell viability measured by CCK-8 assay
A549 cells were plated in a 96-well plate with a density of 1×104 cells/well. After the cells reached 70% confluency, cells were transfected with pcDNA3.1 or pcDNA3.1-SCARA5. Then, at 0 hour, 12 hours, 24 hours, and 48 hours, 10 μL CCK-8 reagent was added to each plate and incubated for another 30 minutes at room temperature. Finally, the cell viability was analyzed by measuring the absorbance at 450 nm using a Microplate Reader.
Cell proliferation rated evaluated by EdU staining
The transfected cells were inoculated in a 96-well plate with a density of 4×103 cells/well. Then, 100 μL 50 μM EdU reagent was added to each well and cultured for 2 hours. After washed by PBS twice, 50 μL PBS containing 4% paraformaldehyde solution was added to each well and cultured for 30 minutes at room temperature. Subsequently, 100 μL Apollo® 567 Imaging Kit (Riobio) and Hoechst 33342 were added and cultured at 37℃ for 30 minutes. Afterward, 100 μL PBS containing 0.5% Triton X-100 was added and cultured for another 5 minutes at room temperature. Finally, all images were captured with fluorescence microscopy.
Cell apoptosis detected by flow cytometry analysis
After transfection, cells were cultured in a 6-well plate at 37℃ in a humidified atmosphere. At 48 hours post-incubation, cells were stained with Annexin V/PI using an apoptosis kit. Finally, the cell apoptosis rate was analyzed using flow cytometry following the instructions.
Apoptosis-related proteins determined by western blot analysis
At 24 hours post-transfection, total proteins were isolated from cells by RIPA buffer and quantified using a Protein BCA Assay Kit following the manufacturer’s protocol. Then, proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane. Afterward, the membranes were blocked with 5% non-fat milk at 37℃ for 2 hours and incubated with primary antibodies (anti-AKT, ab38449, 1:1000, Abcam, Shanghai, China; anti-p-AKT, ab38449, 1:1000, Abcam, Shanghai, China; anti-Bcl-2, ab32124, 1:1000, Abcam, Shanghai, China; anti-Bax, ab32503, 1:1000, Abcam, Shanghai, China; and anti-GAPDH, AB9485, 1:2500, Abcam, Shanghai, China) at 4℃ overnight. After washing with TBST three times, the membranes were then probed with secondary antibodies (goat-anti-rabbit IgG H&L, ab7090, 1:1000, Abcam, Shanghai, China) at 37℃ for 1 hour. Finally, an ECL solution was added to detect protein bands, and ImageJ software was used to quantify the results.
Statistical analysis
All analyses were performed using SPSS 17.0 (Chicago, IL, USA) to analyze the data. The expression level of SCARA5 was presented as a mean ± standard deviation via the student’s t-test. The 2 test was used to determine the correlation of SCARA5 expression and pathological factors in lung adenocarcinoma. The ROC curve defined by the log-rank test and the overall survival by Kaplan-Meier analysis was applied to determine the diagnostic and prognostic value of SCARA5 in lung adenocarcinoma, respectively. A P value < 0.05 was considered to be statistically significant.