2.1 Bioinformatics analysis
GEPIA platform (http://gepia.cancer-pku.cn) and UALCAN datebase (http://ualcan.path.uab.edu) were used to analysis the expression of CHMP4C in NSCLC tissues and the corresponding paracancerous tissues, and comfirm the effect of CHMP4C on the prognosis of NSCLC patients. The tumor types in this analysis were LUAD (lung adenocarcinoma) and LUSC (lung squamous cell carcinoma).
2.2 Patient information and tissue specimens
We collected primary NSCLC specimens who underwent surgical treatment at the Affiliated Hospital of North Sichuan Medical College between January 2019 to December 2020. Clinical data of these patients were also obtained. The study included a total of 80 NSCLC tissues and corresponding normal paracancerous tissues (located at least 2 cm away from the tumor margin). None of these patients had received preoperative chemotherapy or radiotherapy. The research was approved by the Medical Ethics Committee of our hospital, and it was assigned the ethical number 2022ER225-1.
2.3 Immunohistochemical staining (IHC)
Samples from 80 patients with NSCLC were collected, including 40 cases with LUAD and 40 cases with LUSC. After routine dewaxing, rehydrated and antigen repair, endogenous peroxidase was blocked using peroxidase blocker at room temperature for 10 minutes and goat serum was added to close. Then, primary antibody was added dropwise and incubated at 4℃ overnight. On the next day, biotin-labeled secondary antibody was incubated at 37℃ for 30 minutes on a shaker and rinsed with PBS. DAB color development for 3 minutes, hematoxylin re-staining for 3 minutes, water washing until returning to blue, graded ethanol to dehydrate, xylene making transparent, neutral gum sealing.
Immunohistochemical staining results and staining cell scores: We invited two experienced associate chief pathologists to evaluate independently. The IRS scoring system was used to obtain a composite score by multiplying the staining intensity of each section by the positive cell rate. Staining intensity: 0: no staining; 1: weak staining (light yellow); 2: moderate staining (brownish yellow); 3: strong staining (brown). Percentage of positive cell area (positive rate): 0: no positive cell; 1: positive cells ≤ 10%; 2: positive cells 11%-50%; 3: positive cells 51–80%; 4: positive cells > 80%. Photos were taken by using the Leica Flexcam C1 (Wetzlar, Germany) camera.
2.4 Cell line and cell culture
Two human non-small cell lung cancer cell lines (H1299, SKMES1) and one normal lung epithelial cell line (BEAS-2B) used in these experiments were purchased from Wuhan Pricella Life Sciences Co (Pricella, Wuhan, China). BEAS-2B and H1299 cells were cultured in RPMI 1640 medium supplemented 10% fetal bovine serum (10%FBS) and 1% antibiotics (100 U/mL penicillin, 100 mg/L streptomycin). According to the instructions, SKMES1 cells were cultured in MEM medium. All cells were incubated in a cell incubator at 37°C with 5% CO2. The reagents used for cell culture were purchased from Wuhan Pricella Life Sciences Co (Pricella, Wuhan, China).
2.5 Establishment the NSCLC cell lines with stable knockdown of CHMP4C
Lentivirus LV-CHMP4C-RNAi (shCHMP4C) and Negative control lentivirus (NC), which carry green fluorescent protein (GFP) and puromycin resistance gene, along with their corresponding viruses, were obtained from GENE (GENE, Shanghai, China). The shRNA sequence targeting human CHMP4C was as follows: 5'-GCAGAATAAGCGAGCTGCATT-3'; Negative control (NC) sequence: 5'-TTCTCCGAACGTGTCACGT-3'. Firstly, H1299 and SKMES1 cells were separately seeded in six-well plates and then transfected using HiTransG P (GENE, Shanghai, China) when the cell density reached 40%-50%. After transfection, complete medium containing 4ug/ml puromycin was used to select transfected cells.
The remaining cells, after 7–10 days of selection, were considered as cells with stable knockdown of CHMP4C and were maintained in complete medium containing 2ug/ml puromycin for long-term culture. The experimental groups included: Negative control group: shNC; Stable knockdown CHMP4C group: shCHMP4C; Negative control combined with cisplatin group: shNC + DDP; Stable knockdown CHMP4C combined with cisplatin group: shCHMP4C + DDP.
2.6 Quantitative real-time PCR (QPCR)
Total RNA was extracted from cells according to the instructions of Trizol reagent (Invitrogen, Carlsbad, USA). Complementary cDNA was synthesized from total RNA using HiScript® III RT SuperMix reagent kit (Vazyme, Nanjing, China) and PCR was accomplish by using SYBR qPCR premix kit (Vazyme, Nanjing, China). Relative expression was calculated using the 2− ΔΔCT method.
The primers used to amplify the coding sequences were as follows:
GAPDH Forward: 5’-GGAGTCCACTGGCGTCTTCA-3’,
Reverse 5’-GTCATGAGTCCTTCCACGATACC-3’;
CHMP4C Forward: 5’-AGAAGCCCTGGAGAACTCAC-3’,
Reverse: 5’- CTTGGGCAGTATCCTGTTGC-3’.
2.7 Western blot analysis
The cells from each experimental group were collected and lysed with RIPA solution containing protease inhibitor (Beyotime, Shanghai, China) and protein phosphatase inhibitor mixture (Solarbio, Beijing, China). The protein concentration in the lysates was quantified using a BCA kit (Beyotime, Shanghai, China). To perform western blot analysis, the equal amount of proteins were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The separated proteins were then transferred onto PVDF membranes (Millipore, USA) and subsequently blocked with a protein-free rapid sealing solution (Epizyme, Shanghai, China) for 20 minutes. Next, the PVDF membranes were incubated with specific primary antibodies overnight at 4°C: CHMP4C (OmnimAbs, 1:1000), PI3K (FineTest, 1:1000), p-PI3K (Affinity, 1:1000), AKT (Cell Signaling Technology, 1:1000), p-AKT (Cell Signaling Technology, 1:1000), caspase-3 (FineTest, 1:1000), Bad (Beyotime, 1:1000), β-actin (FineTest, 1:3000). On the second day, the PVDF membranes were washed 3 times with Tris-buffered saline solution with Tween (TBST). After washing, the membranes were incubated with the corresponding secondary antibodies coupled with Horseradish Peroxidase (HRP) (Beyotime, 1:3000) for 1 hours at room temperature. They were observed by an enhanced chemiluminescence system. The expression of relevant proteins was analyzed using Image J software.
2.8 Cell counting Kit-8 assay
Stably transfected cells were seeded into 96-well plates at a density of 5×104 cells per milliliter and incubated in a 5% CO2 incubator at 37°C. Five replicate wells were set up for each sample. The cells were subsequently treated with or without cisplatin (Solarbio, Beijing, China). Following the treatment, the Cell counting Kit-8 (CCK-8) (APExBIO, USA) was used to assess cell viability according to the manufacturer’s instructions. Specifically, 10 uL of CCK-8 reagent was added to each well every 24 hours. Then, the plates were placed back in the 5% CO2 incubator at 37 ℃ for 2 hours. Cell viability was detected at 0 hour, 24 hours, 48 hours and 72 hours, respectively. The absorbance (OD value) at 450 nm was measured using a Microplate Reader. All experiments were repeated at least three times.
2.9 Cell clone formation test
In the experiment, 1000 stably transfected H1299 and SKMES1 cells were inoculated in six-well plates and incubated in a 5% CO2 incubator at 37℃ for 10–14 days. When the macroscopic clones were observed, the medium in the wells was discarded, and the cells were washed three times with PBS. Then, the cells were fixed with 4% paraformaldehyde for 20 minutes. After fixation, the cells stained with 0.1% crystal violet for 20 minutes at room temperature. The stained cells were observed and counted under the Leica Flexcam C1 (Wetzlar, Germany) camera.
2.10 Cell cycle assays
After the teatment, the cells were harvested and washed with pre-cooled phosphate-buffered saline (PBS).The cells were fixed by incubating them in 70% ethanol overnight at 4°C. After fixation, the dyeing solution containing Rnase and Propidium Iodide (PI) (KeyGEN BioTECH, Jiangsu, China) were added to the cells, and incubated for 1 hours at room temperature away from light. The cell cycle distribution was measured using a Flow Cytometry at 488 nm excitation wavelength.
2.11 Cell apoptosis assays
After collecting the cells, they were washed twice with cold PBS. The cells were resuspended in 1×Binding Buffer and incubated with Annexin V-APC/PI for 10 minutes at room temperature in the dark. The samples were analyzed by flow cytometry.
2.12 statistical analysis
SPSS 26.0 and Graphpad Prime 8.0 were used for data analysis and making statistical chart. All experiments were repeated at least three times independently, and the measurement data was expressed as mean±standard deviation (±SD). Comparisons between two groups were made by t-test, and comparisons between multiple groups were made by one-way ANOVA. To assess the correlation between CHMP4C expression and the clinical characteristics of patients, χ2 test was performed. p < 0.05 was deemed to be statistically significant.