Cell Journal (Yakhteh)

11-16

Authors: Hossein Pirani; Ali Bakhtiari; Bahareh Amiri; Omid Reza Salehi

Objective: Exercise can attenuate mitochondrial dysfunction caused by aging. Our study aimed to compare 12 weeks of high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) on the expression of mitochondria proteins [e.g., AMP-activated protein kinase (AMPK), Estrogen-related receptor alpha (ERRα), p38 mitogen-activated protein kinase (P38MAPK), and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α)] in gastrocnemius muscle of old female rats. Materials and Methods: In this experimental study, thirty six old female Wistar rats (18-month-old and 270-310 g) were divided into three groups: i. HIIT, ii. MICT, and iii. Control group (C). The HIIT protocol was performed for 12 weeks with 16-28 minutes (2 minutes training with 85-90% VO2max in high intensity and 2 minutes training with 45-75% VO2max low intensity). The MICT was performed for 30-60 minutes with the intensity of 65-70% VO2max. The gastrocnemius muscle expression of AMPK, ERRα, P38MAPK, and PGC1α proteins were determined by Western blotting. Results: The expression of AMPK (P=0.004), P38MAPK (P=0.003), PGC-1α (P=0.028), and ERRα (P=0.006) in HIIT was higher than C group. AMPK (P=0.03), P38MAPK (P=0.032), PGC-1α (P=0.015), and ERRα (P=0.028) in MICT was higher than the C group. Also expression of AMPK (P=0.008), P38MAPK (P=0.009), PGC-1α (P=0.020) and ERRα (P=0.014) in MICT was higher than MICT group. Conclusion: It seems that exercise training has beneficial effects on mitochondrial biogenesis, but the HIIT training method is more effective than MICT in improving mitochondrial function in aging.

25-34

Authors: Masoomeh Masoomikarimi; Majid Salehi; Farshid Noorbakhsh; Samira Rajaei

Objective: Decellularized uterine scaffold, as a new achievement in tissue engineering, enables recellularization and regeneration of uterine tissues and supports pregnancy in a fashion comparable to the intact uterus. The acellular methods are methods preferred in many respects due to their similarity to normal tissue, so it is necessary to try to introduce an acellularization protocol with minimum disadvantages and maximum advantages. Therefore, this study aimed to compare different protocols to achieve the optimal uterus decellularization method for future in vitro and in vivo bioengineering experiments. Materials and Methods: In this experimental study, rat uteri were decellularized by four different protocols (P) using sodium dodecyl sulfate (SDS), with different doses and time incubations (P1 and P2), SDS/Triton-X100 sequentially (P3), and a combination of physical (freeze/thaw) and chemical reagents (SDS/Triton X-100). The scaffolds were examined by histopathological staining, DNA quantification, MTT assay, blood compatibility assay, FESEM, and mechanical studies. Results: Histology assessment showed that only in P4, cell residues were completely removed. Masson’s trichrome staining demonstrated that in P3, collagen fibers were decreased; however, no damage was observed in the collagen bundles using other protocols. In indirect MTT assays, cell viabilities achieved by all used protocols were significantly higher than the native samples. The percentage of red blood cell (RBC) hemolysis in the presence of prepared scaffolds from all 4 protocols was less than 2%. The mechanical properties of none of the obtained scaffolds were significantly different from the native sample except for P3. Conclusion: Uteri decellularized with a combination of physical and chemical treatments (P4) was the most favorable treatment in our study with the complete removal of cell residue, preservation of the three-dimensional structure, complete removal of detergents, and preservation of the mechanical property of the scaffolds.

45-50

Authors: Sara Seydabadi; Habib Nikukar; Fahimeh Ghotbizadeh Vahdani; Fariba Ramezanali; Maryam Shahhoseini; Raha Favaedi; Azadeh Ghaheri; Elham Etesami; Mohammadreza Zamanian

Objective: Preeclampsia (PE) is a pregnancy related disorder with prevalence of 6-7%. Insufficient trophoblastic invasion leads to incomplete remodeling of spiral arteries and consequent decrease in feto-placental perfusion. Altered placental expression of tissue inhibitors of matrix metalloproteinase (TIMPs) is considered to be involved in this process while the balance between matrix metalloproteinases (MMPs) and TIMPs contributes to remodeling of the placenta and uterine arteries by degradation and refurbishing of extracellular matrix (ECM). Therefore, TIMPs, fetal expression pattern was evaluated with the aim of its potential to be used as a determinant for the (early) detection of PE. Materials and Methods: In this case-control study, cell free fetal RNA (cffRNA) released by placenta into the maternal blood was used to determine expression patterns of TIMP1, 2, 3 and 4 in the severe preeclamptic women in comparison with the normal pregnant women. Whole blood from 20 preeclamptic and 20 normal pregnant women in their 28-32 weeks of gestational age was collected. The second control group consisted of 20 normal pregnant women in either 14 or 28 weeks of gestation (each 10). cffRNA was extracted from plasma and real-time polymerase chain reaction (PCR) was done to determine the expression levels of TIMP1, 2, 3 and 4 genes. Results: Statistical analysis of the results showed significant higher expression of TIMP1-4 in the preeclamptic women in comparison with the control group (P=0.029, 0.037, 0.037 and 0.049, respectively). Also, an increased level of TIMPs expression was observed by comparing 14 to 28 weeks of gestational age in the normal pregnant women in the second control group. Conclusion: An increased cffRNA expression level of TIMPs may be correlated with the intensity of placental vascular defect and may be used as a determinant of complicated pregnancies with severe preeclampsia.

62-72

Authors: Raziyeh Zareh-Khoshchehreh; Taravat Bamdad; Seyed Shahriar Arab; Mahdi Behdani; Mahmoud Biglar

Objective: Despite of antiviral drugs and successful treatment, an effective vaccine against hepatitis C virus (HCV) infection is still required. Recently, bioinformatic methods same as prediction algorithms, have greatly contributed to the use of peptides in the design of immunogenic vaccines. Therefore, finding more conserved sites on the surface glycoproteins (E1 and E2) of HCV, as major targets to design an effective vaccine against genetically different viruses in each genotype was the goal of the study. Materials and Methods: In this experimental study, 100 entire sequences of E1 and E2 were retrieved from the NCBI website and analyzed in terms of mutations and critical sites by Bioedit 7.7.9, MEGA X software. Furthermore, HCV-1a samples were obtained from some infected people in Iran, and reverse transcriptase-polymerase chain reaction (RTPCR) assay was optimized to amplify their E1 and E2 genes. Moreover, all three-dimensional structures of E1 and E2 downloaded from the PDB database were analyzed by YASARA. In the next step, three interest areas of humoral immunity in the E2 glycoprotein were evaluated. OSPREY3.0 protein design software was performed to increase the affinity to neutralizing antibodies in these areas. Results: We found the effective in silico binding affinity of residues in three broadly neutralizing epitopes of E2 glycoprotein. First, positions that have substitution capacity were detected in these epitopes. Furthermore, residues that have high stability for substitution in these situations were indicated. Then, the mutants with the strongest affinity to neutralize antibodies were predicted. I414M, T416S, I422V, I414M-T416S, and Q412N-I414M-T416S substitutions theoretically were exhibited as mutants with the best affinity binding. Conclusion: Using an innovative filtration strategy, the residues of E2 epitopes which have the best in silico binding affinity to neutralizing antibodies were exhibited and a distinct peptide library platform was designed.

500-505

Authors: Min Zheng; Wei-Hua Cai; Mou-Feng Wang; Yu-Jie Deng; Ling-ling Huang; Yong-Jin Cao

Objective: Breast cancer (BC) is the most common cancer, which is currently the leading cause of cancer death. Circular RNAs (circRNAs) play important roles in cancer, however, circRNAs serving as vital index in BC for guiding treatment have not yet been identified. The aim of our study is to explore a novel kind of potential biomarker for BC. Materials and Methods: In this retrospective study, the samples used for assays were two groups of breast tumor tissue obtained from four BC patients, including four pairs of tumor tissues and adjacent nontumor samples. The circRNA expression profiles were detected via microarray and validated by real-time quantitative polymerase chain reaction (PCR). Results: The differentially expressed circRNAs in tested samples were screened and analyzed by using human circRNA microarray. After analysis, considering a fold gene expression change of ≥2.0 and P<0.05, results suggested that 256 circRNAs were significantly up-regulated and 277 circRNAs were significantly down-regulated. Besides, the results of the real-time quantitative PCR assay showed that the expression of hsa_circ_0001583 was significantly up-regulated in BC groups (P<0.05) by real-time quantitative PCR. Therefore, we thought hsa_circ_0001583 might serve as a novel kind of biomarker for BC. Conclusion: Hsa_circ_0001583 showed significant up-regulation in BC patients with paired adjacent tissues. Many cancer immune pathways were related to has_circ_0001583, including autoimmune thyroid disease, chemokine and T-cell receptor signaling pathways.

515-521

Authors: Milad Mohammadi; Sara Khademi; Yazdan Choghazrdi; Rasoul Irajirad; Mohammad Keshtkar; Alireza Montazerabadi

Objective: Recently, development of multifunctional contrast agent for effective targeted molecular computed tomography (CT) imaging of cancer cells stays a major problem. In this study, we explain the ability of Triptorelin peptide-targeted multifunctional bismuth nanoparticles (Bi2S3@ BSA-Triptorelin NPs) for molecular CT imaging. Materials and Methods: In this experimental study, the formed nanocomplex of Bi2S3@ BSA-Triptorelin NPs was characterized using different methods. The MTT cytotoxicity test was performed to determine the appropriate concentration of nanoparticles in the MCF-7 cells. The X-ray attenuation intensity and Contrast to Noise Ratio (CNR) of targeted and non-targeted nanoparticles were measured at the concentrations of 25, 50, and 75 μg/ml and X-ray tube voltages of 90, 120 and 140 kVp. Results: We showed that the formed Bi2S3@ BSA-Triptorelin NPs with a Bi core size of approximately ~8.6 nm are nontoxic in a given concentration (0-200 μg/ml). At 90, 120, and 140 tube potentials (kVp), the X-ray attenuation of targeted cells were 1.35, 1.36, and 1.33-times, respectively, more than non-targeted MCF-7cells at the concentration of 75 μg/ml. The CNR values at 90, 120, and 140 kVp tube potentials were 171.5, 153.8 and 146.3 c/ϭ, respectively. Conclusion: These findings propose that the diagnostic nanocomplex of Bi2S3@ BSA-Triptorelin NPs can be applied as a good contrast medium for molecular CT techniques.

531-539

Authors: Kailing Pan; Xiaoya Zhao; Wenxia Xu

Objective: Drug resistance is the main hindrance to improve the prognosis of patients with gastric cancer. Amino acid metabolic reprograming is essential to satisfy the different requirements of cancer cells during drug resistance, of which serine deprivation could promote resistance to cisplatin in gastric cancer. As the key enzyme in the de novo biosynthesis of serine, phosphoglycerate dehydrogenase (PHGDH) inhibition could also induce cisplatin resistance in gastric cancer. This study aims to reveal the potential mechanisms of drug resistance induced by PHGDH inhibition via exploring the global mRNA expression profiles. Materials and Methods: In this experimental study, the viability and the apoptotic rate of gastric cancer cells were evaluated by using Cell Counting Kit-8 (CCK-8) analysis and flow cytometric determination, respectively. The identification of differentially expressed genes (DEGs) was tested by mRNA-sequencing (mRNA-Seq) analysis. The confirmation of sequencing results was verified using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Results: The inhibition of PHGDH significantly increased the viability and decreased the apoptotic rate induced by cisplatin in gastric cancer cells. mRNA-Seq analysis revealed that the combined treatment of NCT503 reduced the number of DEGs induced by cisplatin. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) showed that unfolded protein response, ECM receptor interaction and cell cycle signaling pathways were modulated by NCT503 treatment. Hub genes were identified by using protein-protein interaction network modeling, of which E1A binding protein p300 (EP300) and heat shock protein family A (Hsp70) member 8 (HSPA8) act as the vital genes in cisplatin resistance induced by the inhibition of PHGDH. Conclusion: These findings suggested that the inhibition of PHGDH promoted cisplatin resistance in gastric cancer through various intercellular mechanisms. And appropriate serine supplementation or the modulation of EP300 and HSPA8 may be of great help in overcoming cisplatin resistance in gastric cancer.

546-551

Authors: Ebrahim Shirzadeh; Fahimeh Piryaei; Hanieh Naddaf; Zahra Barabadi

The purpose of this experimental study was to investigate the genetic etiology of congenital cataract (CC) manifesting an autosomal recessive pattern of inheritance in four Iranian families. Affected individuals and their normal first-degree relatives in each family were included in the present study. The genomic DNA of the blood samples was extracted from all participants, and one affected member belonging to each family was subjected to Whole Exome Sequencing (WES). Using bidirectional Sanger sequencing, the identified variants were validated by co-segregation analysis. Two different mutations were detected in the FYCO1 gene encoding FYVE and coiled-coil domain-containing protein. A previously reported missense mutation, c.265C>T (p.Arg89Cys), was found in one Iranian family for the first time, and a combination of two variants in a single codon, c.[265C>T;267C>A] (p.Arg89X), was identified in the three other families. On the other hand, accompanying the c.265C>T mutation, the presence of the c.267C>A polymorphism leads to a premature stop codon. In-Silico Analysis of FYCO1 protein demonstrated that RUN domain will be interrupted so that the large part of functional protein will be eliminated due to this novel variant. FYCO1 has been proved to be involved in human lens development and transparency. Its mutations, therefore, result in CC. Herein, we reported the first autosomal recessive CC patients with c.265C>T (p.Arg89Cys) or c.[265C>T;267C>A] variant in Iranian population for the FYCO1 gene. FYCO1 mutations could be tracked for preventive objectives or even be targeted as therapeutic candidates via treatment approaches in the future.

434-441

Authors: Shabnam Fayezi; Parisa Fayyazpour; Zahra Norouzi; Amir Mehdizadeh

Primordial germ cells develop into oocytes and sperm cells. These cells are useful resources in reproductive biology and regenerative medicine. The mesenchymal stem cells (MSCs) have been examined for in vitro production of primordial germ cell-like cells (PGLCs). This study aimed to summarize the existing protocols for MSCs differentiation into PGLCs. In limited identified studies, various models of MSCs, including those derived from adipose tissue, bone marrow, and Wharton's jelly, have been successfully differentiated in to PGLCs. Although the protocols of specification induction are basically very similar, they have been adjusted to the mesenchymal cell type and the species of origin. The availability of MSCs has made it possible to customize conditions for their differentiation into PGLCs in several models, including humans. Refining germ cell-related signaling pathways during induced differentiation of MSCs will help to define extension to the protocols for PGLCs production.

449-457

Authors: Zahra Poursafavi; Saeid Abroun; Saeid Kaviani; Nasim Hayati Roodbari

Objective: Insulin insufficiency due to the reduced pancreatic beta cell number is the hallmark of diabetes, resulting in an intense focus on the development of beta-cell replacement options. One approach to overcome the problem is to search for alternative sources to induce insulin-producing cells (IPCs), the advent of mesenchymal stem cells (MSCs) holds great promise for producing ample IPCs. Encapsulate the MSCs with alginate improved anti-inflammatory effects of MSC treatment. This study aimed to evaluate the differentiation of wharton jelly-derived MScs into insulin producing cells using alginate encapsulation. Materials and Methods: In this experimental study, we established an efficient IPCs differentiation strategy of human MSCs derived from the umbilical cord’s Wharton jelly with lentiviral transduction of Pancreas/duodenum homeobox protein 1 (PDX1) in a 21-day period using alginate encapsulation by poly-L-lysine (PLL) and poly-L-ornithine (PLO) outer layer. During differentiation, the expression level of PDX1 and secretion of insulin proteins were increased. Results: Results showed that during time, the cell viability remained high at 87% at day 7. After 21 days, the differentiated beta-like cells in microcapsules were morphologically similar to primary beta cells. Evaluation of the expression of PDX1 and INS by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) on days 7, 14 and 21 of differentiation exhibited the highest expression on day 14. At the protein level, the expression of these two pancreatic markers was observed after PDX1 transduction. Results showed that the intracellular and extracellular insulin levels in the cells receiving PDX1 is higher than the control group. The current data showed that encapsulation with alginate by PLL and PLO outer layer permitted to increase the microcapsules’ beta cell differentiation. Conclusion: Encapsulate the transduced-MSCs with alginate can be applied in an in vivo model in order to do the further analysis.

465-472

Authors: hossein babaahmadi rezaei; alireza kheirolah; Faezeh Seif

Objective: In addition to the carboxy region, Smad2 transcription factor can be phosphorylated in the linker region as well. Phosphorylation of Smad2 linker region (Smad2L) promotes the expression of plasminogen activator inhibitor type 1 (PAI-1) which leads to cardiovascular disorders such as atherosclerosis. The purpose of this study was to evaluate the role of dual transactivation of EGF and TGF-β receptors in phosphorylation of Smad2L and protein expression of PAI-1 induced by endothelin-1 (ET-1) in bovine aortic endothelial cells (BAECs). In addition, as an intermediary of G protein-coupled receptor (GPCR) signaling, the functions of ROCK and PLC were investigated in dual transactivation pathways. Materials and Methods: The experimental study is an in vitro study performed on BAECs. Proteins were investigated by western blotting using protein-specific antibodies against phospho-Smad2 linker region residues (Ser245/250/255), phospho-Smad2 carboxy residues (465/467), ERK1/(Thr202/Thr204), and PAI-1. Results: TGF (2 ng/ml), EGF (100 ng/ml) and ET-1 (100 nM) induced the phosphorylation of Smad2L. This response was blocked in the presence of AG1478 (EGFR antagonists), SB431542 (TGFR inhibitor), and Y27632 (Rho-associated protein kinase (ROCK antagonist). Moreover, ET-1-increased protein expression of PAI-1 was decreased in the presence of bosentan (ET receptor inhibitor), AG1478, SB431542, and Y27632. Conclusion: The results indicated that ET-1 increases the phosphorylation of Smad2L and protein expression of PAI-1 via induced the transactivation pathways of EGFR and TGFR. This study is the first attempt to scrutinize the significant role of ROCK in the protein expression of PAI-1.

481-490

Authors: Maria Zahiri; Mansoureh Movahedin; Seyed Javad Mowla; Mehrdad Noruzinia; Morteza Koruji; Mohammad Reza Nowroozi; Fatemeh Asgari

Objective: Epigenetic and genetic changes have important roles in stem cell achievements. Accordingly, the aim of this study is the evaluation of the epigenetic and genetic alterations of different culture systems, considering their efficacy in propagating human spermatogonial stem cells isolated by magnetic-activated cell sorting (MACS). Materials and Methods: IIn this experimental study, obstructive azoospermia (OA) patient-derived spermatogonial cells were divided into two groups. The MACS enriched and non-enriched spermatogonial stem cells (SSCs) were cultured in the control and treated groups; co-culture of SSCs with Sertoli cells of men with OA, co-culture of SSCs with healthy Sertoli cells of fertile men, the culture of SSCs on PLA nanofiber and culture of testicular cell suspension. Gene-specific methylation by MSP, expression of pluripotency (NANOG, C-MYC and OCT-4), and germ cells specific genes (Integrin α6, Integrin β1, PLZF) evaluated. Cultured SSCs from the optimized group were transplanted into the recipient azoospermic mouse. Results: The use of MACS for the purification of human stem cells was effective at about 69% with the culture of the testicular suspension, being the best culture system. Upon purification, the germ-specific gene expression was significantly higher in testicular cell suspension and treated groups (P≤0.05). During the culture time, gene-specific methylation patterns of the examined genes did not show any changes. Our data from transplantation indicated the homing of the donor-derived cells and the presence of human functional sperm. Conclusion: Our in vivo and in vitro results confirmed that culture of testicular cell suspension and selection of spermatogonial cells could be effective ways for purification and enrichment of the functional human spermatogonial cells. The epigenetic patterns showed that the specific methylation of the evaluated genes at this stage remained constant with no alteration throughout the entire culture systems over time.

364-369

Authors: Fereshteh Mansoury; Nahid Babaei; Soheila Abdi; Maliheh Entezari; Abbas Doosti

Objective: Extremely low-frequency magnetic field (ELF-MF) exposure, as a targeted tumor therapy, presents several potential advantages. In this research, we investigated effects of different ELF-MF intensities on cell viability and expression levels of the mammalian target of rapamycin (mTOR) and hsa_circ_100338 in the normal fibroblast (Hu02) and human gastric adenocarcinoma (AGS) cell lines. Materials and Methods: In this experimental study, cell lines of AGS and Hu02, were cultured under the exposure of ELFMF with magnetic flux densities (MFDs) of 0.25, 0.5, 1 and 2 millitesla (mT) for 18 hours. The 3-(4, 5-dimethylthiazoyl-2- yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cell viability. Relative expression of mTOR and hsa_circ_100338 RNAs was estimated by quantitative real-time polymerase chain reaction (qRT-PCR) technique. Results: Viability of the normal cells was significantly increased at MFDs of 0.5, 1 and 2 mT, while viability of the tumor cells was significantly decreased at MFD of 0.25 and increased at MFD of 2 mT. Expression level of mTOR was significantly increased at the all applied MFDs in the normal cells, while it was significantly decreased at MFDs of 0.25 and 0.5mT in the tumor cells. MFDs of 1 and 2 mT in tumor cells inversely led to the increase in mTOR expression. hsa_circ_100338 was downregulated in MFD of 0.25 mT and then it was increased parallel to the increase of MFD in the normal and tumor cells. Conclusion: Results of the present study indicated that ELF-MF at MFDs of 0.25 and 0.5 mT can lead to decrease in the both mTOR and hsa_circ_100338 expression levels. Given the role of mTOR in cell growth, proliferation and differentiation, in addition to the potential role of hsa_circ_100338 in metastasis, expression inhibition of these two genes could be a therapeutic target in cancer treatment.

380-390

Authors: Abbas Heidari Moghadam; Vahid Bayati; Mahmoud Orazizadeh; Mohammad Rashno

Objective: The main objective of this study is to determine the myogenic effects of skeletal muscle extracellular matrix, vascular endothelial growth factor and human umbilical vein endothelial cells on adipose-derived stem cells to achieve a 3-dimensional engineered vascular-muscle structure. Materials and Methods: The present experimental research was designed based on two main groups, i.e. monoculture of adipose tissue-derived stem cells (ADSCs) and co-culture of ADSCs and human umbilical vein endothelial cells (HUVECs) in a ratio of 1:1. Skeletal muscle tissue was isolated, decellularized and its surface was electrospun using polycaprolactone/gelatin parallel nanofibers and then matrix topography was evaluated through H&E, trichrome staining and SEM. The expression of MyHC2 gene and tropomyosin protein were examined through real-time reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence, respectively. Finally, the morphology of mesenchymal and endothelial cells and their relationship with each other and with the engineered scaffold were examined by scanning electron microscopy (SEM). Results: According to H&E and Masson’s Trichrome staining, muscle tissue was completely decellularized. SEM showed parallel Polycaprolactone (PCL)/gelatin nanofibers with an average diameter of about 300 nm. The immunofluorescence proved that tropomyosin was positive in the ADSCs monoculture and the ADSCs/HUVECs coculture in horse serum (HS) and HS/VEGF groups. There was a significant difference in the expression of the MyHC2 gene between the ADSCs and ADSCs/HUVECs culture groups (P<0.05) and between the 2D and 3D models in HS/ VEGF differentiation groups (P<001). Moreover, a significant increase existed between the HS/VEGF group and other groups in terms of endothelial cells growth and proliferation as well as their relationship with differentiated myoblasts (P<0.05). Conclusion: Co-culture of ADSCs/HUVECs on the engineered cell-free muscle scaffold and the dual effects of VEGF can lead to formation of a favorable engineered vascular-muscular tissue. These engineered structures can be used as an acceptable tool for tissue implantation in muscle injuries and regeneration, especially in challenging injuries such as volumetric muscle loss, which also require vascular repair.

403-409

Authors: Nasrin Hadi; Seyed Morteza Seifati; Behnaz Nateghi; Parisa Ravaghi; Farinaz Khosravian; Faezeh Namazi; Maryam Fotouhi Firouzabad; Vahid Shaygannejad; Mansoor Salehi

Objective: Multiple sclerosis (MS) is a complex multifactorial neuro-inflammatory disorder. This complexity arises from the evidence suggesting that MS is developed by interacting with environmental and genetic factors. This study aimed to evaluate the miR-106a, miR-125b, and miR330- expression levels in relapsing-remitting multiple sclerosis (RRMS) patients. The miRNAs' impact on TNFSF4 and Sp1 genes through the NF-кB/TNF-α signaling pathway was analyzed by measuring the expression levels in case and controls. Materials and Methods: In this in silico-experimental study, we evaluated the association of miR-106a, miR- 125b, and miR330- with TNFSF4 and SP1 gene expression levels in 60 RRMS patients and 30 healthy controls by real-time polymerase chain reaction (PCR). Results: The expression levels of miR-330, miR-106a, and miR125-b in blood samples of RRMS patients were predominantly reduced. The expression of TNFSF4 in patients demonstrated a significant enhancement, in contrast to the diminishing Sp1 gene expression level in controls. Conclusion: Our findings indicated an association between miR-106a and miR-330 and miR125-b expression and RRMS in our study population. Our data suggested that the miR106-a, miR125-b, and mir330- expression are correlated with TNFSF4 and Sp1 gene expression levels.

417-423

Authors: Somayeh Feyzmanesh; Iman Halvaei; Nafiseh Baheiraei

Objective: The main goal was to evaluate the effects of alginate on human sperm parameters during cryopreservation. Materials and Methods: In this prospective study, twenty-five normozoospermic samples were divided into two groups, encapsulated with 1% alginate and the control group. The samples were then frozen by rapid freezing. Different sperm parameters including motility, normal morphology, viability, acrosome reaction, and DNA integrity, were examined before freezing and after thawing. Results: All sperm parameters had a significant decrease after thawing compared to before freezing. Our data showed a significant decrease in sperm motility of the alginate group but sperm viability, normal morphology, and DNA fragmentation were similar between the two groups. However, the rates of intact acrosome and native DNA were significantly lower in the control group compared to the alginate group (45.12 ± 11.1 vs. 55.25 ± 10.69 and 52.2 ± 11.92 vs. 68.12 ± 10.15, respectively, P<0.05). Conclusion: It seems that alginate

294-301

Authors: Beihai Ge; Xian Zhang; Wei Zhou; Yun Mo; Zhou Su; Guolong Xu; Qiang Chen

Objectives: This study aimed to explore biological function of long intergenic non-protein coding RNA 265 (LINC00265) in hepatocellular carcinoma (HCC) cells, and evaluate its potential function as a biomarker. Materials and Methods: In this experimental study, GEPIA database and Kaplan-Meier Plotter database were employed to analyze LINC00265 expression in HCC tissue samples and its predicting value for prognosis. LINC00265 expression in HCC tissues and cells was detected by quantitative real-time polymerase chain reaction (qRT-PCR). After overexpressing and knocking-down of LINC00265 in HCC cells, cell counting kit-8 (CCK-8) and 5-Ethynyl-2’- deoxyuridine (EdU) assays were adopted to detect proliferation of HCC cells. Transwell assay was used to detect migration and invasion of HCC cells. Interaction of LINC00265 with E2F transcription factor 1 (E2F1) was verified by the catRAPID online analysis tool, RNA pull-down experiment and RNA binding protein immunoprecipitation (RIP) assay. Binding of E2F1 to the promoter region of cyclin-dependent kinases 2 (CDK2) was detected by dual-luciferase reporter assay and chromatin immunoprecipitation. Regulatory effects of LINC00265 and E2F1 on CDK2 expression were probed by Western blot. Results: LINC00265 expression was increased in HCC tissues and cells. LINC00265 overexpression promoted proliferation, migration and invasion of HCC cells, and knocking-down LINC00265 worked oppositely. LINC00265 could bind to E2F1 and it could enhance combination of E2F1 and CDK2 promoter regions, thus promoting CDK2 transcription. LINC00265 overexpression promoted expression of CDK2 in HCC cells. Conclusion: Our data suggested that LINC00265 can promote malignant behaviors of HCC cells by recruiting E2F1 to the promoter region of CDK2.

309-315

Authors: Ashish Ranjan Sharma; Garima Sharma; Yeon-Hee Lee; Chiranjib Chakraborty; Sang-Soo Lee; Eun-Min Seo

Objectives: Osteoporosis is regarded as a silent disorder affecting bone slowly, leading to an increased risk of fractures. Lately, selenium has been found to be associated with the acquisition and maintenance of bone health by affecting the bone remodeling process. However, the mechanism of action of selenium on bone is poorly understood. Here, the objective of this study is to examine the protective effects and mechanism of sodium selenite on the differentiation process of osteoblasts as well as under oxidative stress-induced conditions by evaluating the expression of osteoblast differentiation markers in the sodium selenite and/or hydrogen peroxide (H2O2)-treated MC3T3-E1 cell line. Materials and Methods: In this experimental study, we confirmed the inducible osteogenic effect of sodium selenite on MC3T3-E1 cells. Moreover, we investigated the recovery of expression levels of osteogenic markers of sodium selenite in H2O2-treated MC3T3-E1 cells. Results: It was observed that sodium selenite could promote alkaline phosphatase (ALP) activity and collagen synthesis in pre-osteoblasts. Also, sodium selenite enhanced the mRNA expression levels of osteogenic transcriptional factors, like osterix (OSX) and runt-related transcription factor 2 (Runx2). In addition, the terminal differentiation markers, such as osteocalcin (OCN) and collagen 1� (Col1�) were also increased after the treatment of sodium selenite. Also treatment of sodium selenite recused the H2O2-induced inhibition of osteoblastic differentiation of pre-osteoblasts cells via the WNT signaling pathway, implicating its antioxidant activity. Furthermore, sodium selenite restored the H2O2 repressed �-catenin stability and axin-2 reporter activity in MC3T3-E1 cells. Conclusion: It may be concluded that sodium selenite can stimulate bone formation and rescue the oxidative repression of osteogenesis by activating WNT signaling pathways. Further detailed studies on the role of selenium and its ability to stimulate bone formation via the WNT signaling pathway may project it as a potential therapeutic intervention for osteoporosis.

323-329

Authors: Sahar Salahshouri; Fahimeh Akbarian; Marziyeh Tavalaee; Seyed Morteza Seifati; Mohammad Hossein Nasr Esfahani

Objectives: Transient receptor potential vanilloid 1 (TRPV1) is a heat-activated nonselective cation channel that plays important role in the spermatogenesis, capacitation, acrosome reaction and sperm/oocyte fusion. Considering the high testicular temperature and oxidative stress in varicocele condition, we aimed to assess expression of TRPV1 in sperm of infertile men. Materials and Methods: In this case-control study, twenty-five men with varicocele (grade II and III) as well as twentyfive fertile were recruited. Sperm parameters, protamine deficiency (Chromomycin A3), DNA damage (TUNEL), lipid peroxidation (BODIPY), TRPV1 gene expression (real time polymerase chain reaction), TRPV1 protein (flowcytometry and immunocytochemical techniques), and acrosome reaction were assessed between fertile and varicocele groups. Results: We observed a significant decrease in the sperm parameters, and also, an increased DNA damage, lipid peroxidation, and protamine deficiency in varicocele group. Although, the mRNA expression of TRPV1 was similar between varicocele and fertile groups, its expression at the protein level was significantly decreased in the varicocele group in comparison with fertile group. Additionally, the TRPV1 localization was changed from the equatorial region to the acrosomal region of the head, especially in the acrosomal region, which was more significant in the fertile group than the varicocele group after inducing acrosome reaction. Conclusion: In addition to the quality of sperm parameters, and chromatin integrity that were lower significantly in varicocele group, the expression of TRPV1 protein was also lower in varicocele condition that could be associated with reduced capacitation, acrosome reaction and sperm/oocyte fusion and thereby infertility.

337-345

Authors: Shahein Momenabadi; Abbas Ali Vafaei; Mahdi Zahedi Khorasani; Abedin Vakili

Objectives: This study was designed to determine the effects of pre-ischemic administration of oxytocin (OXT) on neuronal injury and possible molecular mechanisms in a mice model of stroke. Materials and Methods: In this experimental study, stroke was induced in the mice by middle cerebral artery occlusion (MCAO) for 60 minutes and 24 hours of reperfusion. OXT was given as intranasal daily for 7 consecutive days before ischemic stroke. Neuronal damage, spatial memory, and the expression levels of nuclear factor-kappa B (NF-�B), interleukin-1� (IL-1�), tumor necrosis factor-� (TNF-�), matrix metalloproteinase-9 (MMP-9), brain-derived neurotrophic factor (BDNF) and apoptosis were assessed 24 hours after stroke. Results: Pre-ischemic treatment with OXT significantly reduced the infarct size (P<0.01); but did not recover the neurological and spatial memory dysfunction (P>0.05). Moreover, OXT treatment considerably decreased the expressions of NF-�B, TNF-�, IL-1�, and MMP-9 (P<0.001) and enhanced the level of BDNF protein. OXT treatment also significantly downregulated Bax expression and overexpressed Bcl-2 proteins. Conclusion: The finding of this study indicated that administration of OXT before ischemia could limit brain injury by inhibiting MMP-9 expression, apoptosis, inflammatory signaling pathways, and an increase in the BDNF protein level. We suggested that OXT may be potentially useful in the prevention and/or reducing the risk of the cerebral stroke attack, and could be offered as a new prevention option in the clinics.Y�� AZ�E��4i