Cell Journal (Yakhteh)

261-272

Authors: Parisa Sahranavardfard, Zahra Madjd, Amirnader Emami Razavi, Alireza Ghanadan, Javad Firouzi, Pardis Khosravani, Saeid Ghavami, Esmaeil Ebrahimie, Marzieh Ebrahimi

Objective Epithelial-mesenchymal transition (EMT) and the stemness potency in association with BRAF mutation are in dispensable to the progression of melanoma. Recently, microRNAs (miRNAs) have been introduced as the regulator of a multitude of oncogenic functions in most of tumors. Therefore identifying and interpreting the expression patterns of these miRNAs is essential. The present study sought to find common miRNAs regulating all three important pathways in melanoma development. Materials And Methods In this experimental study, 18 miRNAs that importantly contribute to EMT and have a role in regulating self-renewal and the BRAF pathway were selected based on current literature and cross-analysis with available databases. Subsequently, their expression patterns were evaluated in 20 melanoma patients, normal tissues, serum from patients and control subjects, and melanospheres. Pattern discovery and integrative regulatory network analysis were used to find the most important miRNAs in melanoma progression. Results Among 18 selected miRNAs, miR-205, -141, -203, -15b, and -9 were differentially expressed in tumor samples than normal tissues. Among them, miR-205, -15b, and -9 significantly expressed in serum samples and healthy donors. Attribute Weighting and decision trees (DT) analysis presented evidence that the combination of miR-205, -203, -9, and -15b can regulate self-renewal and EMT process, by affecting CDH1, CCND1, and VEGF expression. Conclusion We suggested here that miR-205, -15b, -203, -9 pattern as the key miRNAs linked to melanoma status, the pluripotency, proliferation, and motility of malignant cells. However, further investigations are required to find the mechanisms underlying the combinatory effects of the above mentioned miRNAs.

288-293

Authors: Saber Khazaei, Abbasali Khademi, Mohammad Hossein Nasr Esfahani, Mozafar Khazaei, Mohammad Hossein Nekoofar, Paul M.h. Dummer

Objective The aim of present study was to isolate and differentiate human adipose-derived stem cells (ASCs) into odontoblast-like cells. Materials And Methods In this experimental study, human adipose tissues were taken from the buccal fat pad of three individuals (mean age: 24.6 ± 2.1 years). The tissues were transferred to a laboratory in a sterile culture medium, divided into small pieces and digested by collagenase I (2 mg/mL, 60-90 minutes). ASCs were isolated by passing the cell suspension through cell strainers (70 and 40 µm), followed by incubation at 37ºC and 5% CO2in Dulbecco’s modified eagle medium (DMEM) supplemented with fetal bovine serum (FBS 5%) and penicillin/streptomycin (P/S). After three passages, the ASCs were harvested. Subsequently, flow cytometry and reverse transcriptase polymerase chain reaction (RT-PCR) were used to detect expression levels of NANOG and OCT4 to evaluate stemness. Then, a differentiation medium that included high-glucose DMEM supplemented with 10% FBS, dexamethasone (10 nM), sodium β-glycerophosphate (5 mM) and ascorbic acid (100 µM) was added. The cells were cultivated for four weeks, and the odontogenic medium was changed every two days. Cell differentiation was evaluated with Alizarin red staining and expressions of collagen I (COL1A1), dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP1). Results The ASCs were effectively and easily isolated. They were negative for CD45 and positive for the CD105 and CD73 markers. The ASCs expressed OCT4 and NANOG. Differentiated cells highly expressed DSPP, COL1A1 and DMP1. Alizarin red staining revealed a positive reaction for calcium deposition. Conclusion ASCs were isolated successfully in high numbers from the buccal fat pad of human volunteers and were differentiated into odontoblast-like cells. These ASCs could be considered a new source of cells for use in regenerative endodontic treatments.

303-312

Authors: Maesumeh Eslami, Shahrbanoo Oryan, Mehdi Rahnema, Mohammad Reza Bigdeli

Objective Choroid plexus epithelial cells (CPECs) have the epithelial characteristic, produce cerebrospinal fluid, contribute to the detoxification process in the central nervous system (CNS), and are responsible for the synthesis and release of many nerve growth factors. On the other hand, studies suggest that normobaric hyperoxia (HO) by induction of ischemic tolerance (IT) can protect against brain damage and neurological diseases. We examined the effect of combination therapy of encapsulated CPECs and HO to protect against ischemic brain injury. Materials And Methods In this experimental study, six groups of adult male Wistar rats were randomly organized: sham, room air (RA)+middle cerebral artery occlusion (MCAO), HO+MCAO, RA+MCAO+encapsulated CPECs, HO+MCAO+encapsulated CPECs, RA+MCAO+empty capsules. RA/HO were pretreatment. The CPECs were isolated from the brain of neonatal Wistar rats, cultured, and encapsulated. Then microencapsulated CPECs were transplanted in the neck of the animal immediately after the onset of reperfusion in adult rats that had been exposed to 60 minutes MCAO. After 23 hours of reperfusion, the neurologic deficit score (NDS) was assessed. Next, rats were killed, and brains were isolated for measuring brain infarction volume, blood-brain barrier (BBB) permeability, edema, the activity of superoxide dismutase (SOD), and catalase (CAT) and also, the level of malondialdehyde (MDA). Results Our results showed that NDS decreased equally in HO+MCAO, RA+MCAO+encapsulated CPECs, and HO+MCAO+encapsulated CPECs groups. Brain infarction volume decreased up 79%, BBB stability increased, edema decreased, SOD and CAT activities increased, and MDA decreased in the combination group of HO and transplantation of encapsulated CPECs in the ischemic brain as compared with when HO or transplantation of encapsulated CPECs was applied alone. Conclusion The combination of HO and transplantation of encapsulated CPECs for stroke in rats was more effective than the other treatments, and it can be taken into account as a promising treatment for ischemic stroke.

319-328

Authors: Mojtaba Eslami, Sahar Esfandyari, Marzieh Aghahossini, Zahra Rashidi, Shirzad Hosseinishental, Samane Brenjian, Aligholi Sobhani, Fardin Amidi

Objective Astaxanthin (AST) has been introduced as a radical scavenger and an anti-apoptotic factor that acts via regulating the nuclear factor-E2-related factor 2 (NRF2) and related factors. Here, we intended to examine the effect of AST on granulosa cells (GCs) against oxidative stress by examining NRF2 and downstream phase II antioxidant enzymes. Materials And Methods In this experimental study, we used cultured human primary GCs for the study. First, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test to evaluate cells viability after treatment with hydrogen peroxide (H2O2) and AST. The apoptosis rate and ROS levels were measured by flow cytometry. To determine NRF2 and phase II enzymes expression, we performed real-time polymerase chain reaction (PCR). Finally, we used western blot to measure the protein levels of NRF2 and Kelch-like ECsH-associated protein 1 (KEAP1). Enzyme activity analysis was also performed to detect NRF2 activity. Results This study showed that AST suppressed ROS generation (P<0.01) and cell death (P<0.05) in GCs induced by oxidative stress. AST also elevated gene and protein expression and nuclear localization of NRF2 and had an inhibitory effect on the protein levels of KEAP1 (P<0.05). Furthermore, when we used trigonelline (Trig) as a known inhibitor of NRF2, it attenuated the protective effects of AST by decreasing NRF2 activity and gene expression of phase II enzymes (P<0.05). Conclusion Our results presented the protective role of AST against oxidative stress in GCs which was mediated through up-regulating the phase II enzymes as a result of NRF2 activation. Our study may help in improving in vitro fertilization (IVF) outcomes and treatment of infertility.

335-340

Authors: Amir Hossein Jalali, Hossein Mozdarani, Hossein Ghanaati

Objective To evaluate the effect of contrast enhanced abdominopelvic magnetic resonance imaging (MRI), using a 3 Tesla scanner, on expression and methylation level of ATM and AKT genes in human peripheral blood lymphocytes. Materials And Methods In this prospective in vivo study, blood samples were obtained from 20 volunteer patients with mean age of 43 ± 8 years (range 32-68 years) before contrast enhanced MRI, 2 hours and 24 hours after contrast enhanced abdominopelvic 3 Tesla MRI. After separation of mononuclear cells from peripheral blood, using Ficoll-Hypaque, we analyzed gene expression changes of ATM and AKT genes 2 hours and 24 hours after MRI using quantitative reverse transcription polymerase chain reaction (qRT-PCR). We also evaluated methylation percentage of the above mentioned genes in before, 2 hours and 24 hours after MRI, using MethySYBR method. Results Fold change analysis, in comparison with the baseline, respectively showed 1.1 ± 0.7 and 0.8 ± 0.5 mean of gene expressions in 2 and 24 hours after MRI for ATM, while the results were 1.4 ± 0.6 and 1.4 ± 1 for AKT (P>0.05). Methylation of the ATM gene promoter were 8.8 ± 1.5%, 9 ± 0.6% and 9 ± 0.8% in before contrast enhanced MRI, 2 and 24 hours after contrast enhanced MRI, respectively (P>0.05). Methylation of AKT gene promoter in before contrast enhanced MRI, 2 hours and 24 hours after contrast enhanced MRI was 5.4 ± 2.5, 5 ± 3.2, 4.9 ± 2.9 respectively (P>0.05). Conclusion Contrast enhanced abdominopelvic MRI using 3 Tesla scanner apparently has no negative effect on the expression and promoter methylation level of ATM and AKT genes involved in the repair pathways of genome.

349-354

Authors: Nasrin Sereshki, Alireza Andalib, Mohadeseh Toghyani, Hossein Motedayyen, Mohammad Sadegh Hesamian, Abbas Rezaei, David Wilkinson

Objective The maternal immune response to paternal antigens is induced at insemination. We believe that pregnancy protective alloantibodies, such as anti-paternal cytotoxic antibody (APCA), may be produced against the paternal antigens in the context of stimulated immunity at insemination and that they increase during pregnancy. APCA is necessary for pregnancy. It is directed towards paternal human leucocyte antigens (HLAs) and has cytotoxic activity against paternal leucocytes. The present study aims to determine whether APCA is produced by the maternal peripheral blood mononuclear cells (PBMCs) in contact with the husband’s spermatozoa and to evaluate the relation of APCA production with HLA class I and II expressions by spermatozoa in fertile couples. Materials And Methods This cross-sectional study included 30 fertile couples with at least one child. The maternal PBMCs were co-cultured with the husband’s spermatozoa and the supernatant was assessed for the presence of IgG by ELISA. Cytotoxic activity of the supernatant on the husband’s PBMCs was assessed by the complement-dependent cytotoxicity (CDC) assay. Results IgG was produced in all co-cultures, and the mean level of supernatant IgG was 669 ng/ml. The cytotoxic activity of the supernatant was observed in all the supernatant obtained from the co-cultures. The mean percentage of APCA in supernatant was 73.93%. Conclusion Based on the results of this study it can be concluded that APCA may be a natural anti-sperm antibody (ASA), which can be produced during exposure to spermatozoa and may have some influence before pregnancy. Further research is required to determine the role of APCA before pregnancy.

366-366

Authors: Yan Gu, Zhanzhan Xiao, Jianlie Wu, Mingjin Guo, Ping Lv, Ning Dou

This article published in Cell J (Yakhteh), Vol 23, No 2, 2021, on pages 191-198, corresponding author and corresponding address were changed based on authors’ request. The authors would like to apologies for any inconvenience caused

382-388

Authors: Maryam Hezavehei, Bahare Shokoohian, Mohammad Hossein Nasr-Esfahani, Anastasia Shpichka, Peter Timashev, Abdolhossein Shahverdi, Massoud Vosough,

Coronavirus disease 2019 (COVID-19), as a severe respiratory disease, affects various tissues and organs. The specific SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), is highly expressed in male gonads. Thus, male reproductive tissues could be a potential target for virus colonization. We performed a comprehensive search in PubMed and Google Scholar to retrieve relevant articles published till 15 April 2021. The keywords used were: male fertility, male reproductive health, semen parameters, sex hormones, SARS-CoV-2, and COVID-19. Validated evidence about the adverse effects of the SARS-CoV-2 infection on the male reproductive system is limited and few studies have reported semen analysis results or presence of viral RNA in semen samples of infected men. Nevertheless, alterations in reproductive hormones such as decreased level of testosterone (T) with raised luteinizing hormone (LH) have been reported in some patients. Although the impact of SARS-CoV-2 infection on the male reproduction health remains unclear, evidence suggests that male gonads may be potentially vulnerable to SARS-CoV-2 infection. In this article, we discussed the possible impacts of COVID-19 on male gonads, sex hormones, and semen quality and suggested preventive solutions.

397-405

Authors: Esmat Ghalkhani, Mohammad Taghi Akbari, Pantea Izadi, Habibollah Mahmoodzadeh, Fatemeh Kamali

Objective: Metastasis might be latent or occur several years after primary tumor removal. Currently used methods for detection of distant metastasis have still some limitations. Blood tests may improve sensitivity and specificity of currently used screening procedures. The present study was designed to investigate promoter methylation status of DAPK1 and CAVIN3 genes in plasma circulating free DNA (cfDNA) samples in Iranian invasive ductal carcinoma (IDC) patients. We also investigated association of two gene promoter methylations with breast cancer (BC) and metastatic BC was also assessed. Materials and Methods: In this case-control study, MethySYBR assay was performed to determine DAPK1 and CAVIN3 promoter methylation status in breast IDC from 90 patients and 30 controls. Based on clinicopathological information, patient samples subdivided into stage I, II/III and IV groups (each group contained 30 individuals). Results: According to the results an increased promoter methylation level of the DAPK1 gene in BC patients was observed. It was found that as disease progressed, the percentage of methylation was changed while it was not significant. Methylation changes in metastatic and non-metastatic BC revealed that methylation levels were significantly increased in metastatic than non-metastatic group. Analysis revealed that promoter methylation of CAVIN3 gene in BC patients was significantly increased. The observed methylation changes from less to more invasive stages were not significant in the CAVIN3 gene. Moreover, promoter methylation was changed in metastatic rather than non-metastatic condition, although it was not significant. Conclusion: Promoter hypermethylation of DAPK1 and CAVIN3 genes in plasma are associated with the risk of BC and they can be potential diagnostic biomarkers along with current methods. Additionally, association of aberrant DAPK1 promoter methylation with metastasis suggests its potential usage as a non-invasive strategy for metastatic BC diagnosis.

414-420

Authors: Sayed Rasoul Zaker, Kamran Ghaedi

Objective: Breast cancer is one of the most frequent types of cancer with a gradually increasing incidence in developing countries. The aim of this study was to assess modulation of LINC02615 levels in breast cancer progress, using pairwise breast cancer and healthy control tissue samples with regard to the obesity and other conditions, as estrogen receptor (ER) expression. Materials and Methods: In this cohort study, the genes, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) in several important pathways of chromosomal instability, apoptosis and proliferation were analyzed through in silico studies pinpointing the important genes which were responsible for the breast cancer incidence. Then, the respective miRNAs and lncRNAs were selected by relevant databases. At the next step, Lncbase was used for interaction analysis of selected miRNAs and LncRNAs, which resulted in final selection of LINC02615. Total RNA was isolated from 24 pairwise breast cancer and healthy control tissue samples. Expression profile of LINC02615 was assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Correlation between LINC02615 expression and clinicopathological characteristics were analyzed using Pearson’s Chi-square test in breast cancer patients. Results: Data demonstrated that expression of LINC02615 was significantly downregulated in breast cancer tissues compared to the healthy controls (P=0.046). In particular, the relative LINC02615 expression was significantly different in breast cancer tissues especially in obese patients compared to those persons without obesity (P=0.047). Furthermore, a significant difference in LINC02615 level was found between the high and low ER expressions (P=0.014). However, the aberrant expression of LINC02615 was significantly related to physical activity and diabetes disease as well as the stress and age at menopause (P=0.028, P=0.046, P=0.047 and P=0.025, respectively). Conclusion: Taken together, we suggest that LINC02615 downregulation may be related to the risk of breast cancer in Iranian patients. Thus, it may serve as a novel biomarker for identification of breast cancer tissues.

429-434

Authors: Şahabettin Can Özyarım, Funda Karabağ Çoban

Objective: Colorectal cancer is one of the most prevalent consequences of cancer-bound decease worldwide and it remains one of the leading outcomes of cancer-bound decease. Boron is an important mineral that acts significant function in various biological courses. Some important chemical properties of boric acid support its utility in the treatment of cancer. The aim of this study is to evaluate the antiproliferative effects of boric acid in colon cancer. Materials and Methods: This experimental study effect of different concentrations of boric acid on the CCl-233 human colon adenocarcinoma cell lines was investigated, by analyzing proliferation assay (proliferation was applied to the cells for 24, 48 and 72 hours). Proliferation assay was performed using CCK8 Assay Kit. Vascular endothelial growth factor (VEGF) and poly (ADP-) ribose polymerase (PARP) analyses were performed using Sun-Red Human (VEGF) ELISA Kit and Sun-Red Human (PARP) ELISA Kit, respectively. Results: As a result of the studies, analysis of the cell viability showed that 50 mM boric acid decreased cell proliferation after 24, 48 and 72 hours. The maximal decrease in cell proliferation was found to occur at 48 hours. Therefore, PARP and VGEF analyses were performed at 48 hours. PARP values were significantly higher in cisplatin (P<0.05). In contrast, PARP levels were significantly lower (P<0.05) at two concentrations of boron (50-100 mM). In VEGF, analysis showed that boron levels were significantly different from cisplatin, but there was no significant difference between control groups. Conclusion: It is proposed that the molecular mechanisms leading to this type of cancer as well as the effect of boric acid on colon cancer should be clarified in more detailed ways for the early diagnosis and treatment of colon cancer.

445-450

Authors: Sajedeh Yadegari-Dehkordi, Seyed Mohammad Firoozabadi, Mehdi Forouzandeh Moghadam, Zeinab Shankay

Objective: The cell membrane is a major barrier for delivery of hydrophilic drugs and molecules into the cells. Although low voltage and high frequency electric fields (LVHF) are proposed to overcome the cell membrane barrier, the mechanism of membrane permeabilization is unclear. The aim of study is to investigate endocytosis pathways as a possible mechanism for enhancing uptake of bleomycin by LVHF. Materials and Methods: In this experimental study, MCF-7 cells were exposed to bleomycin or to electric fields with various strengths (10-80 V/cm), frequency of 5 kHz, 4000 electric pulse and 100 μs duration in the presence and absence of three endocytosis inhibitors-chlorpromazine (Cpz), amiloride (Amilo) and genistein (Geni). We determined the efficiency of these chemotherapeutic agents in each group. Results: LVHF, depending on the intensity, induced different endocytosis pathways. Electric field strengths of 10 and 20 V/cm stimulated the macropinocytosis route. Clathrin-mediated endocytosis was observed at electric field intensities of 10, 30, 60 and 70 V/cm, whereas induction of caveolae-mediated endocytosis was observed only at the lowest electric field intensity (10 V/cm). Conclusion: The results of this study imply that LVHF can induce different endocytosis pathways in MCF-7 cells, which leads to an increase in bleomycin uptake.

457-464

Authors: Mingyu Zhai, M.D., Mingming Han, Xiang Huang, Fang Kang, Chengwei Yang, Juan Li

Objective: The study was aimed to investigate the effects and potential mechanisms of Dexmedetomidine (Dex) on hypoxia/reoxygenation (H/R) injury in human renal tubular epithelial HK-2 cells. Materials and Methods: In this experimental study, HK-2 cells were divided into four groups: control group, Dex group, H/R group, and Dex+H/R group. The cells in control group received no treatment, and cells in Dex group were only treated with 0.1 nmol/L Dex. The cells in H/R group and Dex+H/R group were all treated with H/R (hypoxia for 24 hours and normoxia for 4 hours), and only the cells in Dex+H/R group were pre-administrated with 0.1 nmol/L Dex. Following treatments at 37˚C for 28 hours, cell viability and apoptosis were measured by MTT assay and flow cytometry, respectively. Also, the expressions of hypoxia-inducible factor 1 (HIF-1α), glucose-regulated protein 78(GRP78), C/EBP homologous protein (CHOP), caspase-12 and cleaved caspase-3 were determined by western blot. Results: The cell viability was significant decreased in H/R group compared with control group (P<0.05), while was significantly increased in Dex+H/R group compared with that in H/R group (P<0.05). However, the change tendency of the cell apoptosis was opposite to that of cell viability. Compared with H/R group, the expression of HIF-1α was evidently up-regulated, while GRP78, CHOP, capase-12 and cleaved caspase-3 expressions were all obviously downregulated in Dex+H/R group (P<0.05). In addition, the concentrations of malondialdehyde (MDA) in H/R group and Dex+H/R group were 1.68 ± 0.22 nmol/mgprot and 0.85 ± 0.16 nmol/mgprot, respectively. The superoxide dismutase(SOD) activity was higher in Dex+H/R group (121 ± 11 U/L), which which was more than twice larger than that in H/R group (57 ± 10 U/L). Conclusion: Dex could promote cell viability and inhibit apoptosis through up-regulating HIF-1α, reducing endoplasmic reticulum (ER) stress and mediating oxidative stress, thus ameliorating the H/R injury.

474-484

Authors: Hongchang Yang, Jing Zhou, Junlei Wang, Luoning Zhang, Quzhi Liu, Jing Luo, Hongyan Jia, Li Liu, Qiang Zhou

Objective: This study aimed to characterize the circulating exosomal microRNA (miRNA) profiles associated with acute soft tissue injury. Materials and Methods: In this experimental study, a total of 12 rats were randomly divided into control group and model group (n=6 for each group). The rats in the model group were used to establish an acute soft tissue injury following the mechanical injury of the leg. The exosomes from the peripheral blood of all the rats were isolated and then characterized by Nanosight NS300 particle size analyser (NTA), transmission electron microscopy (TEM) and western blot. Next, the exosomal miRNAs in the control and model groups were sequenced, and the differentially expressed miRNAs (DE-miRNAs) were identified using the DESeq algorithm. Functional analyses were performed using Gene Ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases. Finally, quantitative reverse-transcription polymersa chain reaction (qRT-PCR) was used to verify the expression of the DE-miRNAs. Results: TEM, NTA and western blot results showed that the exosomes were approximately 100 nm in size and exhibited cup-shaped morphology. A total of 628 miRNAs were obtained by sequencing. After that, 28 DE miRNAs (DEmiRNAs) were identified, including seven down-regulated miRNAs and 21 up-regulated miRNAs. These DE-miRNAs were linked to 7539 target genes with GO. Also, KEGG analyses demonstrated that these genes were enriched for phosphorylation, VEGF signaling pathway, and MAPK signaling pathway. Additionally, the consistency rate between the qRT-PCR and sequencing results was 83.33%, which showed a high relative reliability of the sequencing results. Conclusion: These findings suggest that these 28 exosomal miRNAs may be involved in the regulation of acute soft tissue injury, by one of critical biological processes (BP), phosphorylation. The findings provide valuable clues by utilizing exosomes as therapeutic targets for the effective treatment of acute soft tissue injury.