Study population
Study population consisted of 15 individuals referred to surgery centers of Al-Zahra and Sina hospitals in Isfahan and Tehran, respectively. This work was conducted from April 2019 to April 2020. The HCC stage was determined by a specialist according to the eighth edition of the Cancer Staging Manual by the American Joint Committee on Cancer (AJCC)[14]. The sampling was carried out at least one month after the last radiotherapy, chemotherapy, and other therapeutic approaches. None of the patients was on preoperative radiotherapy, chemotherapy and other medical interventions, which could affect the immune system and cytokine productions at the time of the sampling. Experimental protocols were approved by the Ethics Committee of Tehran University of Medical Sciences (ethic code: IR.TUMS.VCR.REC1396.4790) and preformed according to the declaration of Helsinki. All participants were informed before entering the study and informed consent was obtained from the subjects.
Sample collection
LR was performed by a surgical team and approximately 70% of the total liver was removed. Liver tissues were collected from patients in several times following surgical resection. Briefly, in the first place, the sample was obtained after starting surgery opened the abdominal cavity and before LR. Malignant tissues were removed and remained liver tissue was subjected to sampling (30, 60, and 90 minutes after LR). The samples were stored at -196 °C for the next experiments.
RNA extraction and complementary deoxyribonucleic acid (cDNA) synthesis and Real-time PCR
The total RNAs were isolated from the frozen liver tissues using a RNA isolation kit based on the manufacturer’s instructions (Yekta Tajhiz, Iran). cDNA was synthetized and to assess the mRNA levels of IL-1α, IL-1β, IL-6, IL-10, TNF-α, and TGF-β, real time-PCR was carried out using an ABI7700 machine (Applied Biosystems, Foster City, USA). Each reaction was initiated at 95 °C for 10 minutes followed by 40 cycles of 95 °C for 30 seconds and 60˚C for 40 seconds. All analyses were done in triplicate. Real-time PCR was carried out in a reaction mixture (10 μL) consisted of 4 μL of 2X Real-Time PCR master mix (SYBR® Premix Ex Taq™ II; Bio Fact, Korea), 2 μL of forward and reverse primers (10 pM), 1 μL of cDNA template, 2.7 μL of DNase-RNase free water, and 0.3 μL of ROX. The temperature and cycling parameters mentioned above were used to determine the expression levels of IL-1α, IL-1β, IL-6, IL-10, TNF-α, and TGF-β1. The melting curves were automatically generated in 60˚C to 95˚C temperature range. The primer sequences and other information of real-time PCR are shown in supplementary Table 1. The expression levels of the genes were normalized to the expression levels of β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes as the endogenous controls.
Cytokine assay
To evaluate the effect of LR on the serum levels of cytokines in HCC patients, peripheral blood samples were obtained from patients before and after 30, 60, 90 and 180 minutes of LR. The serum levels of TNF-α and IL-6 were measured using an enzyme-linked immunosorbent assay (ELISA) kit.
Statistical analysis
Data analysis was performed by GraphPad Prism 6 (GraphPad Software, USA). The results are shown as standard error of the mean (SEM). One-way ANOVA and paired t-tests were used to compare the groups with normal distribution. Data with non-parametric distribution were analyzed using Mann–Whitney and Kruskal–Wallis tests. The significance level was considered as P <0.05.