2.1 Expression of SIK2 in Thyroid Cancer Tissues
The Cancer Cell Line Encyclopedia (CCLE; https://portals.broadinstitute.org/CCLE) [30] from the Broad Institute provides gene data for numerous human cancer cell lines and has become one of the standard databases in cancer genomics. Therefore, SIK2 mRNA expression levels in thyroid cancer were downloaded from the CCLE database. For protein expression analysis, the Human Protein Atlas (HPA;https://www.proteinatlas.org)[31] and immunohistochemistry(IHC) experiments were used to detect the protein expression of SIK2 in male and female patients with papillary thyroid carcinoma.
2.2 Prognostic Analysis
To assess the clinical significance of SIK2 in papillary thyroid carcinoma, a prognostic analysis was conducted. The TIMER2.0 database (https://cistrome.shinyapps.io/timer/)[32] was utilized for this purpose. Various clinical parameters, including tumor burden, ethnicity, gender, stage, and overall survival (OS) of patients, were analyzed.Furthermore, the OS outcomes at different follow-up time points, including 1 year, 3 years, 5 years, 10 years, and 15 years, were presented to provide a comprehensive understanding of the prognostic implications of SIK2 expression in papillary thyroid carcinoma.
2.3 Reverse Transcription Quantitative Polymerase Chain Reaction(RT-qPCR):
Obtain approximately 0.02g of tissue and thoroughly homogenize it in 1 mL of Trizol (Thermo, 15596026, USA) using a homogenizer. Utilize the total tissue mRNA as a template for cDNA reverse transcription. Briefly centrifuge the reaction mixture, followed by incubation at 50°C for 50 minutes and subsequent incubation at 85°C for 5 minutes.Conduct a search on the National Center for Biotechnology Information (NCBI) to obtain the sequence of the target gene. Employ Primer5 software for primer design in order to perform real-time quantitative PCR. The thermal cycling conditions for the quantitative polymerase chain reaction are as follows: initial denaturation at 95°C for 10 seconds, followed by denaturation at 95°C for 10 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 15 seconds.The primers employed in this study are as follows:SIK2:Forward primer:AAGGAGCGAAGGAGCGAAG,Reverse primer: GATGTCGTAGAACCCCACCC,Product length:181bp. β-Actin:Forward primer: ACAGCCTCAAGATCATCAGC, Reverse primer:GGTCATGAGTCCTTCCACGAT, Product length: 104 bp.
2.4 Western blotting(WB)
Take 0.025 g of tissue and homogenize it in 300 uL of RIPA lysis buffer. Repeat the homogenization process and centrifuge at 4°C, 12,000 rpm for 15 minutes. After gel preparation, initiate electrophoresis at a constant voltage of 75V for 130 minutes. Terminate the electrophoresis when the bromophenol blue dye reaches the bottom of the gel. Perform a constant current transfer at 300 mA. Once the transfer is complete, remove the membrane and wash it once in 1x PBST (pH 7.5, 1x, from China Abiowell, AWI0130). Prepare a 5% skim milk solution in 1x PBST (from China Abiowell, AWB0004) and immerse the membrane in it for 90 minutes at room temperature.Dilute the primary antibodies (β-actin: USA, proteintech, 66009-1-Ig, Mouse, 1:5000; SIK2: UK, abcam, ab53423, Rabbit, 1 µg/ml) in 1x PBST according to the specified ratio. Incubate the membrane with the diluted primary antibodies overnight at 4°C, followed by 30 minutes at room temperature the next day. Dilute the HRP-conjugated secondary antibodies (Goat anti-Mouse IgG (H + L) Secondary Antibody, China Abiowell, AWS0001, 1:5000; Goat anti-Rabbit IgG (H + L) Secondary Antibody, China Abiowell, AWS0002, 1:5000) in 1x PBST. Incubate the membrane with the diluted secondary antibodies at room temperature for 90 minutes.For ECL detection, incubate the membrane with ECL chemiluminescent substrate for 1 minute, remove excess liquid with filter paper, and capture the image using a gel imaging system.
2.5 The enzyme-linked immunosorbent assay(ELISA)
Dilute the BSA (China Abiowell, AWB0104, 02A230220) standard solution to concentrations of 2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.12 mg/ml, 0.0625 mg/ml, and 0 mg/ml (0 represents the diluent). The total volume of BCA working solution is calculated as ((number of BSA standard samples + number of unknown samples) × number of replicates × volume of BCA working solution per sample (200 µl)). Prepare the BCA working solution by mixing BCA-A and BCA-B in a 50:1 volume ratio, ensuring thorough mixing. Add 25 µl of the diluted BSA standard solution and 25 µl of the protein sample to their respective wells in a pre-labeled 96-well microplate. Add 200 µl of BCA working solution to each well, mix thoroughly, cover the plate with a lid, and incubate at 37°C for 30 minutes. Use a microplate reader to measure the absorbance of each sample and the BSA standard solution within the range of 540–590 nm, and record the values accordingly. Plot a standard curve and calculate the protein concentration in the samples.
2.6 Cell culture and transfection:
TPC-1 cells were cultured in RPMI-1640 medium (Sigma, R8758) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% antibiotics. The cells were maintained in a humidified incubator at 37°C with 5% CO2. Logarithmically growing cells were divided into the following groups: control group, where cells were not subjected to any treatment; empty vector group, where TPC-1 cells were transfected with pHG-LVsh-NC; SIK2 knockdown group, where TPC-1 cells were transfected with pHG-SIK2-sh (Honorgene). Sterile centrifuge tubes were prepared, and each tube was filled with 95 µl of serum-free RPMI-1640 medium. Then, 3 µl of prepared cell suspension and 5 µl of lipofectamine 2000 (Invitrogen, 11668019) were added to the respective tubes. After 5 minutes of incubation at room temperature, the contents of the two tubes were mixed gently and incubated for an additional 20 minutes at room temperature. Subsequently, 800 µl of RPMI-1640 basal medium was added and mixed thoroughly. The resulting mixture was evenly distributed into the designated transfection wells. The cells were then incubated at 37°C for 6 hours to assess the success of transfection.
2.7 Cell Counting Kit-8 (CCK-8) assay:
TPC-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at 37°C with 5% CO2 in a humidified incubator. After transfection, the cells from each group were harvested, counted, and seeded into 48-well plates at a density of 1 × 104 cells per well in a volume of 300 µl. Each group had three replicate wells, and 30 µl of CCK-8 reagent was added to each well. The plates were then incubated at 37°C with 5% CO2 for an additional 4 hours. After incubation, the absorbance at 450 nm was measured using a microplate reader to analyze the optical density (OD) values.
2.8 Wound healing assay:
A 6-well plate was taken and marked on the backside with a ruler to create evenly spaced horizontal lines. A bottle of cells was obtained and digested using trypsin (Abiowell, AWC0232). After cell counting, approximately 5×105 cells were added to each well. The cells were treated according to the grouping method mentioned above. Once the cells covered the entire well, a pipette tip was used to create vertical scratches perpendicular to the previously drawn horizontal lines. The cells were washed three times with sterile PBS (Abiowell, AWC0409) to remove the scratched cells, and serum-free RPMI-1640 medium was added. Images of the scratches were captured at 0 hours, and at 24 hours and 48 hours after incubation at 37°C with 5% CO2, three fields of view were recorded for each time point. The data were analyzed using ImageJ (v. 1.8.0; National Institutes of Health).
2.9 Cell cycle and Apoptosis analysis:
The cells were resuspended in 1 ml of pre-chilled PBS, centrifuged at 800 rpm for 5 minutes, and the supernatant was aspirated. Then, 400 µl of PBS was gently added to the cells to dissociate them into single cells. Next, 1.2 ml of pre-chilled 100% ethanol was added dropwise to achieve a final concentration of 75% ethanol, and the cells were fixed overnight at 4°C. The fixed samples were then centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in 1 ml of pre-chilled PBS, centrifuged at 800 rpm for 5 minutes, and the cell pellet was collected. This process was repeated twice to remove the ethanol. Subsequently, 150 µl of propidium iodide (PI) working solution (propidium iodide, Dalian Meilun, MB2920) was added, and the cells were stained in the dark at 4°C for 30 minutes. The stained cells were transferred to a flow cytometry tube for analysis. PI was excited by a 488 nm argon laser and detected through a 630 nm bandpass filter. A total of 10,000 cells were collected based on the FSC/SSC scatter plot using gating techniques to exclude cell aggregates and debris. The percentage of cells in each phase of the cell cycle was analyzed based on the fluorescence histogram of PI.
Cells were collected by digestion with trypsin without EDTA. The cells were washed twice with PBS, centrifuged at 2000 rpm for 5 minutes each time, and approximately 3.2×105 cells were collected. Then, 500 µl of Binding buffer was added to suspend the cells, followed by the addition of 5 µl of Annexin V-APC and thorough mixing. Subsequently, 5 µl of Propidium Iodide was added and mixed. The reaction was carried out at room temperature, protected from light, for 10 minutes. Within 1 hour, the samples were analyzed using a flow cytometer.
2.10 Enrichment analyses
The potential mechanisms of SIK2 were revealed by gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses. First, SIK2-related genes were gathered based on STRING (https://cn.string-db.org/)[33] and GeneMania (https://genemania.org/)[34]. Second, GO and KEGG analyses were carried out by R 4.1.3( https://www.r-project.org/). Third, the top 20 KEGG pathways, biological processes (BP), molecular functions (MFs), and the top 10 cellular components (CC) were determined and visualized by GraphPad Prism 9.5.1.
2.11 Immune infiltration analysis
The relationship between immune infiltration and SIK2 and THCA was investigated by utilizing the TISIDB database(http://cis.hku.hk/TISIDB/). TISIDB was employed to elucidate the associations between immune inhibitory factors, immune stimulatory factors, MHC molecules, lymphocytes, and SIK2 expression. Additionally, it demonstrated the correlations with several commonly observed immune cell types, which were found to be statistically significant (P < 0.05).OS analysis of SIK2 was applied by the Kaplan–Meier plotter (https://kmplot.com/analysis/)[35, 36],which derived data from the cancer genome atlas (TCGA:https://cancergenome.nih.gov), gene expression omnibus (GEO: https://www.ncbi.nlm.nih.gov/geo/), and european genome-phenome archive (EGA: https://ega-archive.org/) databases. Furthermore, the impact of modulating immune cell factors on OS was analyzed. Forest plots were generated to display the 95% (Confidence Interval)CI and (Hazard Ratio)HR. The forest plots were created using GraphPad Prism 9.5.1 based on the results obtained from Kaplan-Meier survival analysis.
2.12 Statistical analysis.
All experiments were repeated at least three times. Data are expressed as the mean ± SD of triplicate experiments. All statistical analyses were carried out using GraphPad Prism9.5.1. The differences between two groups were compared using an unpaired Student's t‑test, while those among multiple groups with one‑way ANOV A followed by Tukey's post hoc test. P < 0.05 was considered to indicate a statistically significant difference.