Salivary CPLANE1 Levels as a Biomarker of Oral Squamous Cell Carcinoma

Materials and Methods

Patients. This study was approved by the Aichi Gakuin University ethics committee (approval number: 66, 74). According to the Declaration of Helsinki, all patients and HVs provided written consent for the use of their samples in the current study. This study included 42 patients with OSCC, 37 with OPMDs, and 42 HVs who visited the Department of Maxillofacial Surgery at Aichi Gakuin University Dental Hospital or Japanese Red Cross Nagoya Daiichi Hospital from December 2015 to March 2020. All patients with OSCC underwent primary surgical treatment, including tumor resection, neck dissection, and primary reconstruction with vascular microsurgery. Patients were staged according to the 7^th edition of the UICC classification as follows: stage I and II, 23 patients; stage III and IV, 19 patients. The OSCC patient population included 30 men and 12 women of ages ranging from 27 to 91 years (63.6±16.6, mean ± SD). OPMDs patients included 33 with oral leukoplakia (OL) and 4 with oral lichen planus without dysplasia (OLP). The OPMDs patient population included 20 men and 17 women of ages ranging from 29 to 91 years (65.8±14.7). HVs were selected among the individuals from hospital staff and their families without smoking habits, drinking, or any systemic history. The HVs population included 23 men and 19 women of ages ranging from 27 to 91 years (58.1±18.7). The detailed characteristics of the research subjects is shown in Table I.

Saliva collection. Two types of saliva were obtained to preserve the DNA and RNA. Saliva DNA samples were collected using the Orangene® DNA Self-Collection kit (DNA Genotek Inc., Ontario, Canada). Saliva collection was completed upon patient waking up before food and liquid consumption. Saliva samples were collected using the spitting method and the patient was instructed to collect 2 ml of saliva within 15 min. Saliva RNA samples were collected using the Orangene^® RNA Self-Collection kit (DNA Genotek Inc.) (15). Samples were preferentially collected in the morning to avoid biochemical changes in the saliva. Patients were prohibited from ingesting water or food 1 h prior sample collection to prevent food and water interference with salivary enzyme levels.

Bacterial DNA extraction from saliva. The extraction of total bacterial DNA from saliva samples was carried out according to the manufacturer’ s protocol (DNA Genotek Inc.). In brief, saliva samples were incubated at 50°C for 1 h. Orangene^® purification solution (PT-L2P-5, 20 μl) was added to 500 μl of the saliva sample and vortexed for a few sec. Then, the samples were placed on ice for 10 min and centrifuged at 14,000 × g for 5 min. Next, 600 μl of 100% ethanol was added to the supernatant, followed by incubation at room temperature for 10 min. Subsequently, the samples were centrifuged at 14,000 × g for 2 min. The resulting pellets were washed with ethanol (250 μl, 75%) for 1 min. After complete extraction using ethanol, the DNA pellets were dissolved in 100 μl of LoTE buffer (10 mmol/l Tris hydrochloride, 1 mmol/l ethylenediaminetetraacetic acid buffer, pH 8) and stored at –20°C.

Total RNA extraction. A total RNA recovery from saliva was performed according to the protocol of the Oragene^® RNA Self-Collection kit (DNA Genotek Inc.) (15). mRNA was then reverse transcribed into cDNA using a QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany).

Microarray analysis. For microarray analysis, cDNA was synthesized using a Low Input Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA, USA). Complementary RNA (cRNA) was purified using RNeasy mini spin columns (Qiagen, Hilden, Germany), and its quality was tested using the Agilent 2100 BioAnalyzer series II. The concentration was determined by measuring the absorbance using a NanoDrop 1000, and the samples were stored at –80°C. cRNA was hybridized to microarrays using a Gene Expression Hybridization Kit (Agilent Technologies). The processed arrays were scanned using a microarray scanner (Agilent Technologies). Data were digitized with Agilent Feature Extraction software and normalized using a 75^th percentile shift.

Real-time quantitative reverse transcription PCR (qRT-PCR). To confirm the results obtained using the expression array, quantitative PCR was performed using the saliva samples of OSCC patients (n=42), OPMDs patients (n=37), and HVs (n=42). qRT-PCR was performed to determine the relative expression levels of CPLANE1 using TB Green Premix Ex Taq II (Takara Bio, Nojihigashi, Kusatsu, Japan). It was performed using a Biosystems7500 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The primer sequences for CPLANE1 were 5’-ACCTTGGCCACATGGAACTG-3’ (forward primer) and 5’-GCAGACTATGACACGGAGCA-3’ (reverse primer). GAPDH was used for gene expression normalization. The primer sequences for GAPDH were 5’-TTAGCACCCCTGGCCAAGGT-3’ (forward primer) and 5’-GGCCATCCACAGTCTTCTGG-3’ (reverse primer). The reaction conditions were as follows: 95°C for 30 s for preincubation, followed by 40 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 30 s, and extension at 72°C for 20 s (16).

Bioinformatics analysis of gene expression. The gene expression of CPLANE1 in normal (n=43) and oral cancer samples (n=444) was analyzed using the Gene Expression database of Normal and Tumor tissues (GENT2). Gene expression data were downloaded from the GEO public repository using the U133Plus2 (GPL570) platform and U133A (GPL96) (17).

DNA isolation and bacterial 16S rDNA sequencing using Next Generation Sequencer. The DNA library consisted of the first primer set (515F/806R) and the second primer set (Forward: 5’-AATGATACGGCCGACCACCGAGATCTACAC-Index2-ACACTCTTTCCCTACACGACGC-3’, Reverse: 5’-CAAGCAGAAGACGGCATACGAGAT-Index1-GTGACTGGAGTTCAGACGTGTG-3’). These were constructed by two-step PCR using a unique barcode primer of the bacterial 16S rRNA V4 region. The first PCR reaction was performed at 94°C for 2 min, followed by denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and elongation at 72°C for 30 s, with a final elongation step at 72°C for 5 min. The second PCR reaction was performed at 94°C for 2 min, followed by denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, and 72°C for 5 min. The final extension step was performed for 10 cycles. In both PCR assays, 10 μl of the reaction system contained 2 μl of DNA template, 0.1 μl of 5 U Taq DNA polymerase (Takara Bio, Mountain View, CA, USA), 1.0 μl of 10× PCR buffer, 0.8 μl of dNTP mixture (2.5 mM each), 5.1 μl of double distilled water, and 0.5 μl of each primer.

Sequence analysis was performed using the Illumina MiSeq pyrosequencing platform (Illumina, San Diego, CA, USA). Quality filtering and error correction were performed using the FASTX toolkit. Sequences that passed quality filtering were merged using the paired-end merge script FLASH. The merged sequences were filtered by fragment length, and only fragments with 246-260 bases were used for further analysis. Sequences that passed all filtering were checked for chimeric sequence detection using the USEARCH Uchime algorithm. The non-chimeric sequences were clustered into operational taxonomic units (OTUs) using quantitative insights into microbial ecology (QIIME) with a 97% identity threshold. Taxonomic assignment was performed against the 16S rRNA reference sequence in the Human Oral Microbiome database (HOMD). Most of the sequences could be identified at the phylotype/species level. Sequences with less than 97% identity were classified to the genus level and not at the species level. The OSCC patient group, OPMDs patient group, and HVs group libraries were constructed from clonal analysis.

Statistical analysis. To compare salivary expression levels of CPLANE1 between the OSCC, OPMDs, and HV groups, the Mann–Whitney test and Student’s t-test were used. The differential expression of each marker was used to construct receiver operating characteristic (ROC) curves. The area under the ROC curve (AUC) was obtained by numerical integration. A p-value of <0.05 was considered statistically significant. All statistical analyses were performed using R software (The R Foundation for Statistical Computing) on EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan).