2.1. Ethics statement
The experimental protocols of the present study were approved by the ethics committee of Mashhad University of Medical Sciences (IR.MUMS.DENTISTRY.REC.1399.166), and all subjects visiting as outpatients were enrolled in the Department of the Research Council of Mashhad University of Medical Sciences, Faculty of Dentistry, Mashhad,Iran. They were well informed about the purpose of this study and signed written informed consent.
2.2. Participantsand sample collections
This cross-sectional study was conducted in 15 months (from January 4, 2021, to March 21, 2022). Eighty subjects were recruited from the Dental Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
In a sterile RNase/DNase free falcon tube, 1.5 CC of fresh human saliva was collected between 6 am and 11 am and kept on ice. Next, thesamples stored at -80°C. The total sample size for the study was 80participants inthree groups: OLP (n = 27), OSCC (n = 28), and healthy control (n = 25).
2.3. Inclusion Criteria
Participants were randomely selected with definitive clinical diagnosisin OSCC and OLP. The healthy controls had no medical history of any malignancies or thesystemic diseases. The candidate patients had not undertaken therapy before including surgery, chemotherapy, and radiotherapy. Three groups were similar in age and sex.
2.4. Exclusion Criteria
Participants with a history of other malignancies and a reluctance to cooperate to continue the project, samples that did not have the required quality were excluded from the study.
2.5. Salivary RNA extraction
In this study, microRNA was extracted from saliva without the use of specific kits with high quality and purity, which was one of the important achievements of this study. The procedure was as follows:
The 300-500 µl saliva samples were centrifuged for 20 minutes at 12000 RPM and 4°C, then the supernatant was discarded. We applied RNXplus (Sina Clon, IRAN) reagent and changed the procedure instructions. To begin with, each pellet was given 800 µl RNXplus and vortexed for 15 seconds to homogenize it. Then we incubated them for 3-4 minutes at room temperature. After that, each tube received 200 µl of chloroform (Merck Company), which was vortexed for 15 seconds before being incubated at room temperature for 3-5 minutes. The materials were centrifuged for 20 minutes at 12000 RPM and 4°C. Each sample had about 600µl of the upper aqueous layer transferred into a fresh 1.5 ml DNase and RNase-free microtube. We repeated this step: 200 μl chloroform (Merck Company) was added to each tube and vortexed for 15 seconds, and incubated at room temperature for 3-5 minutes. Then centrifuged at 12000 RPM and 4°C for 20 minutes, and approximately 500 μl of the upper aqueous layer were carefully transferred to the new 1.5 ml DNase/ RNase-free microtubes. After that, for RNA precipitation, 500 µl of cold absolute ethanol (Merck Company) was added to each tube and the microtubes were turned upside down for a few seconds. The microtubes were incubated overnight at -20°C. Then, the samples were centrifuged for 20 minutes at 12000 RPM and 4°C. The supernatant was dispersed. The pellet was washed in 1 ml of cold 80% molecular-grade ethanol and centrifuged for 20 minutes at 12000 RPM and 4°C. This step was repeated two times. The pellet was air dried for 3–4 minutes at room temperature, and then it was resuspended in 20 μl of DEPC water and incubated for 5 minutes at room temperature. Finally, we collected material from the bottom of microtubes using a rapid spin.
The purity of the extracted RNA was determined by assessment of 260/280 nm absorbance ratio of wave length using nanodrop 1000 spectrophotometer (NanoDrop, Thermo Scientific 2000, USA). The RNAs with suitable quantification (100 ng/µl) and purity (1.5-2 ratio) are utilized for complementary DNA (cDNA) synthesis.
2.6. Reverse transcription
CDNA was synthesized by the Adscriptc DNA synthesis Kit (REF: 22701, Bio-Tech, Addbio, Korea). The cDNA synthesis was performed in 20 µl total volume according manufacture instructure including 10µl of 2X Reaction Buffer, 2μl of 10 mM dNTP mixture, 6μl of RNA, and 1 μl of RT primer (1 pM) for U6 or miR-Let-7a-5p and 1 µl of 20X enzymeby ABI thermocycler (One Step, USA). The temperature cycling protocol were preincubation in25 °C for 10 minutes, reverse transcription (RT) at 50 °C for 60 minutes, RT inactivation at 80 °C for 5 minutes, and holding at 12 °C.
2.7. QuantitativeReal-time PCR (qPCR)
The qPCR was performed to quantitate the relative expression of miR-let7a-5p and U6 using SYBR Green master mix (REF: 22701, Bio-Tech, addbio, Korea) with target primers (Table 1). All reactions were performed in duplicate in 20 µl total volumes in separate wells. Each reaction contained 0.5 µL of each primer (10 pM), 10 µl of SYBR Green master mix (XX), 7μl distilled sterile water, and 2 μlof cDNA. The qPCR temperature process was used by Light Cycler 96 (Roche, Germany) following preincubation step at 95 °C for 30 seconds, and 50 cycles at 95 °C for 5 seconds for denaturation and 60 °C for 30 seconds for annealing. Differential expression was analyzed by the ΔΔCT method. The U6 housekeeping miR was used as the reference gene (Figure 1). Moreover, we performed the analysis of the melting curve to confirm specific target amplification.
2.8. Statistical analyses
All data analysis was performed using SPSS software (Version 25). The Data normalized by threshold cycle number ΔCt, and the relative miRNA expression level was calculated as 2^-(ΔCt) which is commonly used in miRNA expression. The p-value less than 0.05 was considered significant.