Identification of KIN expression and Survival prognosis analysis based on bioinformatics databases
Tumor Immune Estimation Resource (TIMER2) was performed to compare the mRNA expression of KIN17 in normal and tumor tissues[20]. Following that, KIN17's protein expression was examined utilizing the Clinical Proteomic Tumor Analysis Consortium (CPTAC)[21]. Z-values for the LUAD denote SD from the median for all samples. After being first standardized within each sample profile, the log2 spectral count ratio values from CPTAC were then normalized across samples. Besides, in The Cancer Genome Atlas (TCGA)[22], the Kaplan–Meier plotter[23], and GEPIA2 database, KIN17’s prognostic values for LUAD patients, such as disease free survival (DFS) was carried out.
Enrichment analysis of KIN-related genes
The investigation of the network of molecular interactions was done employing the STRING website[24]. Additionally, the "ClusterProfiler" R package ran KEGG enrichment examinations on genes that were comparable to PDHA1.
Cell lines and culture
From the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), one human non-tumorigenic lung epithelial cell line (BEAS-2B) and two human NSCLC cell lines (H522, H1299) have been bought. In DMEM (Gibco, China) which has 1 percent antibiotics (penicillin–streptomycin) and 10 percent fetal bovine serum (FBS, Gibco, China), all the cells were maintained and grown. At 37°C, the cells were grown in a CO2 incubator.
shRNA-mediated gene silencing in H1299 cell
Guangzhou Boyao Biotechnology Co., Ltd. (Guangzhou, China) created the pLV.i lentiviral constructs encoding control sh-NC and three distinct shRNAs targeting human KIN17 (sh-KIN17-c, sh-KIN17-b, and sh-KIN17-a). HEK-293T cells were transfected with the lentivirus helper plasmids and the construct. The successfully produced lentiviral particles were concentrated and transfected into NSCLC cells in vitro (maintained under the polybrene containing a complete medium). Selected stable cells received puromycin addition for a further 48 hours. The following tests used stable cells.
Cell proliferation assay
Following the instructions of the manufacturer, the CCK-8 assay was employed to calculate the rate of cell proliferation. 96-well plates were used, and 5 × 103 cells in total were seeded into each well before being cultured in a cell incubator. CCK-8 solution (10 µL, Abcam, UK) was incorporated at the given times, incubated with the grown cells in the incubator for a further two hours, and then absorbance (450 nm) analysis with a microplate reader was performed (Thomas Scientific, NJ, USA).
Cell migration and invasion assays
Cells were grown in six-well plates in the migration assay (6 × 104 cells/well) with ~ 100% confluent. A 200‑µl pipette tip was utilized to make a linear scratch in the cell monolayer. The wound width was determined using Image J software (version 1.8.0; National Institutes of Health). A light microscope was employed to take pictures of the wound after 24 hours (Nikon Corporation; magnification x100). The migration distance was determined by subtracting the scratch's width at 0 h from its width at 24 h. By normalizing the control group, the relative migration rate was computed.
The cells (4 × 105) were plated in the top chamber in the invasion assay with a membrane coated with Matrigel. Conditioned medium was filled in the bottom chambers. The number of migrating cells on the membrane's lower side was measured as previously described after a 48-hour incubation period. After fixing the cells for 0.5 hours at room temperature with 4 percent paraformaldehyde, the cells were stained for 30 minutes at 37°C with 0.1 percent crystal violet. In three random fields, the number of cells moving through the membrane was observed. This was done using a 100x magnification light microscope.
Apoptosis assay and cell cycle analysis
The cells were transfected for 24 and 48 hours, respectively, in the apoptotic experiment before being washed. annexin V (Annexin V-FITC-PI kit; Beyotime, Shanghai, China) and propidium iodide (PI) were used to stain samples of 5 × 104 to 5 × 105 cells in accordance with the instructions of the manufacturer. A BD FACSVerse was utilized to evaluate stained cells right away (Becton Dickinson, Franklin Lakes, NJ, USA).
Cells (1 × 107) were gathered, PBS-washed, and fixed in ethanol (70 percent) for 2 hours at 4°C for the cell cycle analysis. Following a PBS wash, the cells were incubated in PI (10 g/mL) for 30 min at 4°C after being incubated with RNase A (10 mg/L) for 30 min at 37°C. Flow cytometry was employed for examining the distribution of cell cycles. NovoExpress software (BD Biosciences, San Jose, CA, USA)was used to assess the outcomes.
Quantitative real-time PCR (qRT-PCR)
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was employed to extract total cellular RNA in accordance with the recommendations of the manufacturer. Then, using oligo (dT) primers, 1 µg of RNA was reverse-transcribed into cDNA. According to the manufacturer's instructions, qRT-PCR was performed using SYBR Green qPCR Master Mix (Roche, Shanghai, China). The initial denaturation at 95°C for 10 minutes is followed by 40 cycles of 95°C for 15 seconds and 60°C for 45 seconds is the qRT-PCR amplification procedure. The reactions were carried out in triplicate, and the comparative CT (2−△△CT) method was used for relative quantification. The particular primers used for the amplification of β-actin, WNT, and β-catenin are listed in Supplementary Table S1.
Western blot (WB)
When the cells were removed, RIPA lysis buffer was used to extract the total protein (Beyotime, China). To the lysis buffer, protease inhibitors (Beyotime, China) were incorporated (1:100). The lysates were centrifuged for 15 minutes at 4˚C, 850 x g. A loading buffer (Beyotime Institute of Biotechnology) that contains 100 mM dithiothreitol was combined with the supernatant after being collected. The proteins (30 µg/lane) were separated using SDS-PAGE (15%), and the total protein was quantified utilizing a Protein Concentration Determination BCA kit from the Beyotime Institute of Biotechnology. The isolated proteins were then put onto PVDF membranes (EMD Millipore) and blocked for 2 hours at room temperature with 5 percent BSA (Beyotime, China). The following primary antibodies were applied to the membranes and incubated overnight at 4°C: anti‑β-catenin (cat. no. A302-010A-T; 1:1000 dilution), anti‑WNT1 (cat. no. MA5-15544; 1:1000 dilution), anti‑E‑cadherin (cat. no. PA5-32178; 1:1000 dilution), anti‑Vimentin (cat. no. PA5-27231; 1:5000 dilution), anti‑KIN (cat. no. PA5-40419; 1:2000 dilution), anti‑β-actin (cat. no. PA1-988; 1:5000 dilution), and anti‑N‑cadherin (cat. no. PA5-19486; 1:1000 dilution). ThermoFisher provided all of the antibodies. The membranes were then rinsed with TBS that contain 0.1 percent Tween-20 and incubated for 1.5 hours at room temperature with either a donkey anti-rabbit IgG secondary antibody (cat. no. BA1055, 1:5,000 dilution; Boster, China) or a horseradish peroxidase‑conjugated donkey anti‑rabbit IgG secondary antibody (cat. no. BA1055; 1:5,000 dilution; Boster, China) after primary incubation. Using Image-Pro Plus version 6, the protein bands were measured and visualized employing the Odyssey Western Blot Analysis system (Tanon, China).
Immunofluorescence
The cells were permeabilized with 0.1 percent Triton X-100 (BioFRoxx, cat. no. EZ1609D315) for 30 minutes at room temperature after being fixed with 4˚C 4 percent polyphosphate formaldehyde for 14 minutes. The cells were then blocked with 10 percent goat serum (Bioss, cat. no. C01-03001) for 30 minutes at room temperature after this procedure was carried out three times. Anti‑E‑cadherin (cat. no. PA5-32178; 1:200 dilution), anti‑Vimentin (cat. no. PA5-27231; 1:200 dilution), FITC‑labeled secondary fluorescent antibodies (goat anti‑rabbit IgG; cat. no. ab6717; Abcam; 1:200 dilution), and anti‑N‑cadherin (cat. no. PA5-19486; 1:400 dilution) were incorporated to the cells after incubating overnight at 4°C with primary antibodies. After being incubated for 0.5 hours at 37 degrees Celsius, the film was sealed, DAPI was incorporated in the dark, and the results were seen under a fluorescence microscope (magnification, x200). For each sample, three random fields were examined.
Immunohistochemistry (IHC) staining
As mentioned earlier[25], tissue samples were embedded in paraffin, cut into sections of 4 µm thickness, deparaffinized in xylene, rehydrated using graded ethanols, and blocked in 3 percent hydrogen peroxide for 10 min at 25 degrees Celsius. Specific primary antibodies N‑cadherin (cat. no. PA5-19486; 1:400 dilution), E-cadherin (cat. no. PA5-32178; 1:200 dilution), and Vimentin (cat. no. PA5-27231; 1:200 dilution) were incubated with tumor sections for 12 hours at 4 degrees Celsius. The secondary antibody (Alexa Fluor 488, 1:2000 dilution) was then incubated with the tumor tissues at 25 degrees Celsius for 2 hours. Antibodies were bought from Abcam in Cambridge, MA, USA. The Olympus IX73 microscope (Tokyo, Japan) was used to take the pictures, which were at a magnification of about × 40 magnifications.
Nude mice xenograft experiments
Under the direction of Guangzhou Forevergen Biosciences, the animal experiment was thoroughly evaluated, conducted, and approved. Guangzhou Forevergen Biosciences (Guangzhou, China) provided adult male nude mice (BALB/c, 4 weeks old). NSCLC cells stably transfected with the corresponding plasmids (control sh-NC group and sh-KIN group) were unilaterally subaxillarily subcutaneously injected into four-week-old nude mice (1 × 107 cells per mouse) for inducing tumors. During 30 days, the mice were observed daily to track the development of tumors. Measurements of the tumor's length (L) and width (W) were taken, and the tumor's volume (V) was computed using the formula V(mm3) = 0.5 × (W)2 × (L). The tumors from each mouse were weighed and employed for immunohistochemistry (IHC) examination at the end of the analysis.
Statistical analysis
All statistical analyses used the R (v.4.2.2) software. With the help of GraphPad Prism 8 and SPSS 22.0, all data were examined. The number of stars in TIMER2 indicates the statistical significance determined by the Wilcoxon test. In order to determine the relationship between patient prognosis and KIN17 expression levels, the Kaplan-Meier technique was employed. P < 0.05 was deemed statistically significant difference.