Cell Journal (Yakhteh)

503-509

Authors: Azam Kazemi, Saeid Abroun, Masoud Soleimani

Objective: Multiple myeloma (MM) is the clonal proliferation of neoplastic plasma cells in the bone marrow. Although bortezomib (BTZ) is a crucial drug for the treatment of MM, drug resistance is a major problem. OncomiR-19a plays an oncogenic role in many cancers, including MM; however, the function of miR-19a in the pathogenesis of MM and drug resistance has not been completely identified. The present research aims to investigate the inhibition of miR-19a by an antagomir to determine BTZ responsiveness, and determine if miR-19a can be a prognostic biomarker for MM. Materials and Methods: In this experimental study, viability and apoptosis of myeloma cells were analysed by the colorimetric 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide (MTT) and Annexin V/propidium iodide (PI) flow cytometry assays. Quantitative real-time polymerase chain reaction (qRT-PCR) was implemented to evaluate the expression levels of miR-19a, its targets SOCS3, STAT3, B-cell lymphoma 2 (BCL-2), PTEN and CDKN1A (antiapoptotic and cell cycle related genes) at the mRNA level. Results: miR-19a was downregulated and exacerbated in transfected cells treated with BTZ. The rate of apoptosis in the myeloma cells after BTZ treatment considerably increased, which indicated an increase in the mRNA of SOCS3, PTEN, BCL-2, and CDKN1. A decrease in STAT3 was also observed. Conclusion: OncomiR-19a, as a biomarker, may induce better responsiveness to BTZ in myeloma cell lines through its targets SOCS3, STAT3 and PTEN. In the future, this biomarker may provide new therapeutic targets for MM.

516-522

Authors: Mohammad Saeed Hakhamaneshi, Alina Abdolahi, Zakaria Vahabzadeh, Mohammad Abdi, Pedram Andalibi

Objective: Trimethylamine-N-Oxide (TMAO) is considered as a risk factor for atherosclerosis which further leads to inflammation during atherosclerosis. The exact mechanism(s) by which TMAO induces the inflammatory reactions remains to be determined. TMAO can cause the endoplasmic reticulum (ER) stress that triggers activation of Toll-Like Receptors (TLRs). In macrophages, this process stimulates the production of proinflammatory cytokines. This study designed to evaluate the expression level of TLR4 in TMAO-treated macrophages. Materials and Methods: In this experimental study, different concentrations of TMAO (37.5, 75, 150, and 300 μM) were exposed to murine macrophage (J774A.1 cell line) for 8, 18, 24, and 48 hours. The cells were also treated with 2.5 mM of 4-phenyl butyric acid as well as 2μg/ml of tunicamycin respectively as negative and positive controls for inducing ER-stress. We measured the viability of treated cells by the MTT test. Besides, the expression levels of TLR4 gene and protein were evaluated using western blotting and reverse transcription- quantitative polymerase chain reaction(RT-qPCR) analysis. One-Way ANOVA was used for statistical analysis. Results: No cell death was observed in treated cells. The cells treated with 150 and 300 μM doses of TMAO for 24 hours showed a significant elevation in the protein and/or mRNA levels of TLR4 when compared to normal control or tunicamycin-treated cells. Conclusion: Our results may in part elucidate the mechanism by which TMAO induces the macrophage inflammatory reactions in response to the induction of ER stress, similar to what happens during atherosclerosis. It also provides documentation to support the direct contribution of TLR4 in TMAO-induced inflammation.

532-537

Authors: Afsaneh Zare, Reza Fardid, Gholam Hossein Tamadon, Mohammad Amin Mosleh-Shirazi

Objective: Ionizing radiation is a tremendous risk factor for cancer development. MicroRNAs (miRNAs) are regulators that utilize cell pathways, which are implicated in human cancer prognosis. In addition, miRNAs respond to anti-cancer therapy and proliferation after irradiation. However, the changes in miRNA expression profiles in response to irradiation have not been comprehensively analysed. The present study was designed to assess potential changes that occur in miRNA expression following irradiation. Materials and Methods: In this experimental study, we used quantitative real-time polymerase chain reaction (qRTPCR) to measure the expressions of miR-155, miR-21, and let-7a in MCF-10A (normal breast cells) and MCF-7 (breast cancer cells) six hours after the cells were exposed to five different irradiation doses (50, 100, 400, 2000, and 4000 mGY). Results: After irradiation from the low to high doses, we observed an upsurge in miR-155 (more than 100%) expression and reduction in let-7a (more than 87%) expression. However, there was an increase and a reduction in miR-21 expression (more than 100%). Conclusion: Irradiation can play an important role in cancer development in normal breast cells (MCF-10A) at low dose irradiation. However, the results showed little difference at high doses of radiation.

544-551

Authors: Azar Shams, Ronak Shabani, Mohammad Najafi, Mahdi Karimi, Vahid Pirhajati, Mohammad Asghari Jafarabadi, Hamid Reza Asgari, Chad B. Maki, Seyed Mohsen Razavi, Morteza Koruji

Objective: In cancer treatments, smart gene delivery via nanoparticles (NPs) can be targeted for cancer cells, while concurrently minimizing damage to healthy cells. This study assessed the efficiency of poly lactic-co-glycolic acid (PLGA)-miR 143/206 transfection on apoptosis in mouse leukemia cancer cells (El4) and spermatogonial stem cells (SSCs). Materials and Methods: In this experimental study, neonatal mouse spermatogonia cells and EL4 cancer cell lines were used. MicroRNA-PLGA NPs were prepared, characterized, and targeted with folate. Several doses were evaluated to obtain a suitable miR dose that can induce appropriate apoptosis in EL4 cells, while not harming SSCs. Cells were treated separately at 3 doses of each miR (for miR 143, doses of 25, 50 and 75 nmol and for miR 206, doses of 50, 100 and 150 nmol). The experiments were performed at 24, 48 and 72 hours. Viability and apoptosis were investigated by MTT and Annexin Kits. Results: Based on MTT assay results, the optimal dose of miR 143 was 75 nmol (59.87 ± 2.85 % SSC and 35.3 ± 0.78 % EL4) (P≤0.05), and for miR 206, the optimal dose was 150 nmol (54.82 ± 6.7 % SSC and 33.92 ± 3.01% EL4) (P≤0.05). The optimal time was 48 hours. At these doses, the survival rate of the EL4 cells was below the half maximal inhibitory concentration (IC50) and SSC survival was above 50%. Annexin V staining also confirmed the selected doses(for miR 143 total apoptosis was 6.62% ± 1.8 SSC and 37.4% ± 4.2 EL4 (P≤0.05), and miR 206 was (10.98% ± 1.5 SSC and 36.4% ± 3.7 EL4, P≤0.05). Conclusion: Using intelligent transfection by NPs, we were able to induce apoptosis on EL4 cells and maintain acceptable SSC survival rates.

562-567

Authors: Elham Samei, Hossein Mozdarani, Farhad Samiei, Gholamreza Javadi

Objective: The aim of this study was to evaluate the effects of individual or combined use of two antioxidants, melatonin and famotidine on radiation induced apoptosis in leukocytes from breast cancer (BC) patients. Materials and Methods: In this experimental study, the DPPH assay was used to determine the appropriate doses of melatonin and famotidine for treatment of BC and control leukocytes. The leukocytes were cultured in complete RPMI- 1640 medium and treated with either agent for two hours. Cells were exposed to 4 Gy gamma rays generated from a Co-60 source at a dose rate of 0.85 Gy for 48 hours before harvesting. The cells were placed on slides and the neutral comet assay was performed. A total of 500 cells were stained with ethidium bromide and assessed for the amount of apoptosis under a fluorescent microscope x400 magnification. Results: We observed significantly more apoptosis following radiation alone in the leukocytes from BC patients compared with normal individuals (P<0.01). Individual use of famotidine and melatonin induced very low frequencies of apoptosis that was not significantly different from the control (P>0.05). However, when combined with radiation, there was a decreased frequency of apoptosis in leukocytes of both normal and BC patients (P<0.05). The effect of famotidine was more pronounced than melatonin. Conclusion: Melatonin, despite its potent antioxidant property, does not significantly affect radiation induced apoptosis in leukocytes derived from normal individuals; however, it has a moderately significant protective effect on in leukocytes derived from BC patients. Therefore, when used with radiation it might not intervene with the radiotherapy (RT) regimen of BC cancer patients. Famotidine is a good radioprotector for normal tissue. However, the efficacy of RT might be reduced with an accumulation of famotidine in tumour tissues.

576-583

Authors: Mohammad Ali Nilforoushzadeh, Nasser Aghdami, Ehsan Taghiabadi

Objective: Hair loss is a prevalent medical problem in both men and women. Maintaining the hair inductivity potential of human dermal papilla cells (hDPCs) during cell culture is the main issue in hair follicle morphogenesis and regeneration. The present study was conducted to compare the effects of different concentrations of exosomes derived from human adipose stem cells (hASCs) and platelet-rich plasma (PRP) on the proliferation, migration and expression of alkaline pholphatase (ALP), versican, and smooth muscle alpha-actin (α-SMA) in human DPCs. Materials and Methods: In this experimental study, hDPCs, human hair DPCs and outer root sheet cells (ORSCs) were separated from healthy hair samples. The protocol of exosome isolation from PRP and hASCs comprises serial low speed centrifugation and ultracentrifugation. The effects of different concentrations of exosomes (25, 50, 100 μg/ml) derived from hASCs and PRP on proliferation (MTS assay), migration (scratch test) and expression of ALP, versican and α-SMA (real time-polymerase chain reaction) in human DPCs were evaluated. Results: The flow cytometry analysis of specific cytoplasmic markers showed expression of versican (77%) and α-SMA (60.8%) in DPCs and K15 (73.2%) in ORSCs. According to NanoSight Dynamic Light Scattering, we found the majority of ASCs and PRP-exosomes (ASC-Exo and PRP-Exo) to be 30-150 nm in size. For 100 μg/ml of ASCs-Exo, the expressions of ALP, versican and α-SMA proteins increased by a factor of 1.2, 2 and 3, respectively, compared to the control group. The findings of our experiments illustrated that 100 μg/ml of ASCs-Exo compared to the same concentration of PRP-Exo significantly promote DPC proliferation and migration in culture. Conclusion: This study introduced the potential positive effect of ASC-Exo in increasing the proliferation and survival of DPCs, while maintaining their hair inductivity. Thus, ASCs-Exo possibly provide a new effective procedure for treatment of hair loss.

593-597

Authors: Zahra Tabatabaei, Khadijeh Karbalaie, Parham Habibzadeh, Mohammad Ali Farazi Fard, Mohammad Ali Faghihi, Mohammad-Hossein Nasr Esfahani

Congenital disorders of glycosylation (CDG) are a heterogeneous group of systemic disorders characterized by defects in glycosylation of lipids and proteins. One of the rare subtypes of CDG is CDG-Ij (MIM # 608093), which is caused by pathogenic mutations in DPAGT1, a gene encoding UDP-N-acetylglucosaminedolichyl-phosphate N-acetylglucosaminephosphotransferase enzyme. This enzyme catalyzes the first step of oligosaccharide synthesis in glycoprotein biosynthesis pathway. Preimplantation genetic testing for monogenic disorders (PGT-M) is a diagnostic technique that can reveal the genetic profile of embryos before implantation phase of in vitro fertilization (IVF). Currently, this approach is performed using next generation sequencing (NGS) technology. Herein, with the help of whole-exome and Sanger sequencing, we detected a novel missense mutation (NM_001382, c.1217 A>G) in DPAGT1 gene in two families with consanguineous marriage. Using different online bioinformatics tools including MutationTaster, I-Mutant v2.0, T- Coffee, and CADD v1.0, this mutation was predicted pathogen. Finally, after performing PGT-M followed by successful pregnancy, a normal child was born in one of these families. In conclusion, we identified a novel pathogenic mutation in DPAGT1 in a family with multiple members affected by CDG, which extends the range of pathogenic variants associated with CDG and therefore facilitates early detection of the disease.