Patients and Samples
This study was fulfilled in the Pathology Ward of Namazi Hospital, Shiraz, Iran (2016-2018). To conduct this study, a total number of 44 formalin-fixed paraffin-embedded (FFPE) samples were collected from LT patients with HCC. Macro-dissection was also performed. In brief, there were attempts to adopt a standard procedure to dissect the middle region of the tumor to harvest a proper sample, surrounded by the cancerous cells. The harvesting procedure was then confirmed microscopically to ensure the nonexistence of non-cancerous cells in each sample.
The samples were divided into three groups, i.e., 14 samples used for LNA-array probe (the 1st study group), 20 samples taken from non-rejected LT patients (the 2nd study group), and the rest (the 3rd group), 10 samples belonged to post-transplant samples from patients with acute rejection (Figure 1). All the protocols applied in this study were in complete agreement with the Helsinki Declaration and its later amendment. The local Ethics Committee of Shiraz University of Medical Sciences also approved each stage of this study. After explaining the study objectives, written informed consent was further obtained from each patient.
De-Paraffinization of FFPE Samples
Using a microtome, 10 cuts of 5μm of each sample were prepared. The sample cuts were collected in a 2ml RNase-free microtube for de-paraffinization. De-paraffinization was then started by adding 1ml Xylene (Merck, Germany), which continued by vortexing for 5 seconds. The samples were subsequently put in a rotator for 30 minutes and then centrifuged at 12000 rpm/15 minutes. After discarding the supernatant, 1ml of ethanol was added. Next, the samples were incubated at room temperature for 5 minutes. Then, they were centrifuged at 12000 rpm/15 minutes. Removing the supernatant, 30μl of acetone was added to each sample and left to dry. Afterwards, the samples were digested by 10μl of Proteinase K (Vivantis, Malaysia) enzyme and 200μl of enzyme buffer at 37oC left overnight. Finally, 10μl of Proteinase K enzyme was added to the samples and incubated at 50oC for 3 hours and 10 minutes at 72oC.
RNA Isolation and Quality Control
All the samples were RNA isolated, using Trizol™ (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Sample RNA Quality Control was further performed by an Agilent 2100 Bioanalyzer, to provide an electropherogram for each sample. In addition to the measurement of the rRNA ratio in a traditional manner (28S/18S), the Bioanalyzer provided an RNA Integrity Number (RIN) value (ranging from 0 to 10) and a reliable impression of RNA quality. The recommended RIN values were higher than 7 for good array performance. The NanoDrop instrument was also used to measure the concentration (A260), protein contamination (ratio A260/A280), and contamination with buffer components, or organic compounds (ratio A260/A230) accurately.
Sample Preparation for LNA-Array Probe
Labeling, Hybridization, and Normalization
Using the miRCURY LNATM miRNA Hi-Power Labeling Kit (Exiqon, Denmark), 400ng total RNA were labeled by Hy3TM and Hy5TM fluorescent label for labeling the samples and the references, respectively to produce highly efficient and uniform labeling. The samples (labeled with Hy3TM) and the references (labeled with Hy5TM) were then mixed pairwise and recruited for hybridization. The hybridization procedure was performed using Tecan’s HS 4800™ hybridization station (Tecan, Austria) followed by miRCURY LNA™ miRNA array instruction manual. To control the labeling procedure, spike-in oligonucleotides created range-expected signals. A total number of 42 different spike-in controls were also spiked into the labeling reaction.
Later, the labeled specimens were hybridized by miRCURY LNATM miRNA array 7th Gen (Exiqon, Denmark) using the manufacturer’s protocol. After hybridization, the microarray slides were scanned and stored in an ozone-free environment (below 2.0 ppb). After that, the miRCURY LNA™ miRNA array slides were scanned via the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene® 9 (miRCURY LNA™ miRNA Array Analysis Software, Exiqon, Denmark).
The quantified signals of the background were also corrected (Normexp with offset value 10) and normalized using the global Locally Weighted Scatterplot Smoothing (LOWESS) regression algorithm. Data normalization was further performed to minimize the differences between colors in an intensity-dependent manner. It should be noted that the LOWESS uses a locally established regression to smooth the log ratio/log mean-intensity (M/A) scatterplot towards a linear distribution.
Probe Preparation and Threshold Filtering
To prepare the probes, the data registered in the miRBase database (www.mirbase.org) was used. The background threshold was also calculated for each individual micro-array slide as 1.2 times the 25th percentile of the overall signal intensity of the slide. MiRNAs with intensity above the threshold less than 20% of the samples were additionally removed from the final database. In the present study, a total number of 1648 of probes were discarded through this filtering procedure.
qRT-PCR
Poly A Polymerization, cDNA Synthesis, and qRT-PCR
To this end, 5µg of each isolated total RNAs was used for poly A polymerization by means of MiRNA cDNA Synthesis Kit (Parsgenome, Iran). Then, 2 µg of the product was applied for the next step, i.e., cDNA synthesis using specific reverse primers for each miRNA. Finally, these miR specific cDNAs were employed as templates for quantitative SYBR Green Real-time analysis (Parsgenome, Iran). The expression level of the selected miRNA molecules was further determined employing SYBR Green Real-time PCR (ABI, USA). In addition, U6 miRNA primers were included in the mentioned kit as an internal control.
The mix used for qRT-PCR for each reaction contained SYBR Green Premix (10µl of Ex taq, Takara, Japan), Rox reference dye (0.4µl), forward and reverse primers (1µl of 10pM mix of each primer pairs), and template (2µl of synthesized cDNA, between 0.1 to 100ng). Nuclease free water also added to this reaction up to 20µl total volume. The program employed for qRT-PCR was one cycle 95oC -5 minutes, followed by 40 cycles of 95oC- 5 seconds, 61oC- 20 seconds, and 72oC- 30 seconds, followed by melting curve analysis for specificity of each reaction.
Statistical Analysis
The data were coded and imported into the IBM SPSS Statistics software (version 24). The quantitative variables were then compared using the non-parametric Kruskal-Wallis test for a comparison between more than two groups and the Mann-Whitney U test was employed to compare two groups. The calculations for expression comparison in LNA-array tested samples were also executed by the R/Bioconductor software, using the LIMMA package. The P-value cut-off score for a significant difference was considered less than 0.05.
Ethical Considerations
Informed consent was obtained from all human adult participants as well as parents or legal guardians of the minor. All the protocols applied in this study were in complete agreement with the Helsinki Declaration and its later amendment. Besides, the local Ethics Committee of Shiraz University of Medical Sciences approved each stage of this study.