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The high FKBP1A expression in WBCs as a potential screening biomarker for pancreatic cancer – Scientific Reports

Bioinformatics analysis

Three Gene Expression Omnibus (GEO) datasets were selected from the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/geo/), including GSE172103, GSE125158 and GSE151945. The GSE125158 gene expression microarray, which included blood samples from pancreatic cancer patients, was compared with GSE172103 which is the expression profile of activated macrophages in pancreatic cancer tissues and GSE151945 which is the expression profile of endothelial cells in pancreatic cancer tissues. GEO datasets were analyzed for the gene expression levels using the CU-DREAM (Connection Up- or Down-Regulation Expression Analysis of Microarrays Extra, website: http://pioneer.netserv.chula .ac) program and calculated p-values and odds ratios. All upregulated genes from GSE125158, GSE172103, and GSE151945 were analyzed. We manually observed the gene function of all upregulated genes (Fig. 1). Three candidate genes with significant p-values were then selected from the list of upregulated genes to observe gene expression in WBCs of pancreatic cancer patients.

Study population

This cross-sectional case–control study was performed at the Faculty of Medicine, Chulalongkorn University. All samples were recruited from King Chulalongkorn Memorial Hospital composed of the experimental set collected during March 2017 to March 2021 including 51 healthy normal and 27 pancreatic cancer patients, and the validation set collected during April 2021 to April 2022 including 11 healthy normal and 7 pancreatic cancer patients. All cancer cases were staged according to the revised Tumor, Node and Metastasis (TNM) classification criteria by a pathologist. Healthy normal samples were collected from the patients without a family history of cancer, immune disorders, jaundice and chronic diseases. The demographic data of all samples is presented in Table 1. All participants in this study were of Asian descent and provided their written informed consent to participate in this study.

Table 1 The demographic data of experimental set consisted of healthy normal controls (N = 51) and pancreatic cancer (N = 27) samples which divided by stage I to stage IV in males and females.

Sample size calculation

We used the preliminary results from GSE172103, GSE125158 and GSE151945 to find the appropriate sample size with the following formula26,27:

$${text{N}} = left[ {{{left( {{{text{Z}}_{{{upalpha /2}}}} + {{text{Z}}_{upbeta }}} right)}^2}left( {{{upsigma }^2}_{text{d}}} right)} right]/{left( {{{bar{text{x}}}_{text{d}}}} right)^2}$$

N = sample size, d = Different of value in each group, ({bar{text{x}}_{text{d}}}) = Different of mean in each group, σ2d = Different of variance in each group, Zα/2 = Standard normal variate for level of significance, Zβ = Standard normal variate for power.

We calculated and found that the sample size for our study was 12.51 samples, confirming that this study has recruited enough samples for the experimentation.

Blood sample collection

Two ml of EDTA blood was extracted from all patients. All samples were collected in 4 × 6 ml K3 EDTA tubes (VACUETTE, Greiner Bio-One GmbH, Kremsmünster, Austria) and provided by King Chulalongkorn Memorial Hospital. All samples were centrifuged for 15 min at 3000 rpm to separate the WBC layer (buffy coat). Then, 100 μl of the WBC layer was transferred to a 1.5 ml Eppendorf tube. The cells were washed with 1 ml PBS for 15 min at 1700 rpm 16 °C. All subjects participating in blood collection were given a self-administered questionnaire to collect their medical history, which was carefully recorded. All samples were obtained under a research protocol approved by the Ethics Committee, Faculty of Medicine, Chulalongkorn University, Thailand (approval number: IRB 034/59. The collection of blood samples from all participants was performed based on the World Health Organization (WHO) guidelines. This study was conducted in accordance with the Declaration of Helsinki. Signed informed consent was obtained.

RNA extraction

The WBC pellets were mixed with 500 μl of TRIzol reagent (ThermoFisher Scientific, MA, USA) and incubated at room temperature for 10 min, then 200 μl of chloroform was added and incubated at room temperature for 3 min. The sample was then separated into three phases by centrifugation at 8500 rpm at 4 °C for 15 min. The colorless upper aqueous phase was transferred to a new RNA 1.5 ml Eppendorf tube, supplemented with 4 μl of glycogen (20 mg/mL) and 500 μl of 100% isopropanol, incubated for 10 min at room temperature, then centrifuged at 8500 rpm at 4 °C for 10 min. Supernatants of the centrifuged tubes were discarded. The RNA pellets were washed with 1 ml of 75% ethanol and mixed by vortexing. The samples were then centrifuged at 7000 rpm at 4 °C for 5 min. The supernatants were discarded and the RNA pellets were dried by vacuum for 8 min. The RNA pellets were resuspended with 20 μl of DEPC water. Finally, RNA concentration and integrity were evaluated by Nanodrop and bioanalyzer.

Complementary DNA (cDNA) synthesis

The cDNA was synthesized using RevertAid First Strand cDNA Synthesis (Thermo Scientific) following the manufacturer’s protocol. Briefly, the amount of RNA templates was adjusted into 0.5–1 μg in each reaction. The samples were added to the 1 μl of oligo dT and incubated at 65 °C for 5 min. The following solutions including primer 1 μl, nuclease-free water up to 12 μl, 5X reaction buffer 4 μl, Ribolock RNAse inhibitor 1 μl, 10 mM dNTP mix 2 μl, RevertAid M-MuLV RT 1 μl were added to the samples. After mixing and brief centrifuging, the samples were incubated for 60 min at 42 °C followed by 5 min at 70 °C. The product of the first strand cDNA synthesis can be used directly in PCR or quantitative real-time PCR.

Primer design and preparation

Primers were designed using reference gene sequences from NCBI database including CR542168.1 for FKBP1A, NM_001130081.3 for PLD1 and NM_001330675.2 for PSMA4. The primers were synthesized by U2Bio. The details of the primer sequence, melting temperature, and product length are described in Table 2. Prior to quantitative PCR, conventional PCR and electrophoresis for finding optimal temperature for each primer was conducted.

Table 2 Details of forward and reverse primer sequences of three candidate genes which used qRT-PCR analysis including FKBP1A, PLD1, and PSMA4.

Quantitative real-time PCR (qRT-PCR)

The quantitative real-time PCR contained 10 µl SensiFast Lo-ROX reagent (Bioline), 0.8 µl of forward and reverse mixture primers, 1 µl of cDNA sample, 0.1 µl of Taq polymerase, and 8.1 µl distilled water in a total volume of 20 µl. The reactions were operated by QuantStudio 5 (Thermo Fisher Scientific) following with the manufacturer’s protocol. The amplification conditions were as follows: denaturation at 95 °C for 2 min with 40 cycles, annealing at 60 °C, respectively for 30 s. The positive signals from the amplified product were detected at the end of the annealing step. Duplications were in all samples. In this study, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was selected as the housekeeping gene or the reference gene. GAPDH gene was analyzed alongside all candidate genes.

The amplification results were calculated with the following formula:

$$Delta Delta {text{Ct}} = Delta {text{Ct}};left( {text{a target sample}} right) – Delta {text{Ct}};left( {text{a reference sample}} right)$$

The results were then represented in the folds of change (the equation is in the form of 2−ΔΔCt) of the candidate gene expression in the sample against the reference sample30.

ROC analysis

The Receiver Operating Characteristic (ROC) curves were calculated with MedCalc program version 22.009 (Belgium). Ct values of each candidate gene were entered into the dataset. The sensitivity, specificity, and area under curve (AUC) were calculated for all the datasets.

Statistical analysis

The box plot graph and summary of the dataset (including t-test results of Ct mean of candidate gene) were analyzed with GraphPad Prism version 8.4.3. (MA, USA). The p-value < 0.05 was the cut-off for each analysis calculated by unpaired t-tests.

Ethics approval

All samples were obtained under a protocol approved by the Ethics Committee, Faculty of Medicine, Chulalongkorn University, Thailand (approval number: IRB 034/59). The blood sample collection from all participants was performed in accordance with the WHO guidelines. This study was conducted in accordance with the Declaration of Helsinki. The participants provided their written informed consent to participate in this study.