Cell Journal (Yakhteh)

170-175

Authors: Zohreh Hashemain, Amir Amiri-Yekta, Mona Khosravifar, Faezeh Alvandian, Maryam Shahhosseini, Saman Hosseinkhani, Parvaneh Afsharian

Objective: Estrogen, a female hormone maintaining several critical functions in women's physiology, e.g., folliculogenesis and fertility, is predominantly produced by ovarian granulosa cells where aromatase enzyme converts androgen to estrogen. The principal enzyme responsible for this catalytic reaction is encoded by the CYP19A1 gene, with a long regulatory region. Abnormalities in this process cause metabolic disorders in women, one of the most common of which is polycystic ovary syndrome (PCOS). The main purpose of this research was to determine the effect of the promoters on aromatase expression in cells with normal and PCOS characteristics. Materials and Methods: In this experimental study, four promoters of the CYP19A1 gene, including PII, I.3, I.4, and PII/ I .3 promoter fragments, were cloned upstream of the luciferase gene and transfected into normal and PCOS granulosa cells. Subsequently, the effect of follicle-stimulating hormone (FSH) on the activity of these regulatory regions was examined in the presence and absence of FSH. Western blotting was used to confirm aromatase expression in all groups. Data analysis was performed using ANOVA and paired sample t test, compared by post-hoc least significant difference (LSD) test. Results: Luciferase results confirmed the intense activity of PII promoter in the presence of FSH. Moreover, the study demonstrated reduced activity of PII promoter in normal granulosa cells, possibly due to the regulatory region of I.3 next to PII. Conclusion: FSH stimulates transcription of aromatase enzyme by affecting PII promoter, a process regulated by the inhibitory role of the I.3 region in PII activity in granulosa cells. Given the distinct role of these promoters in normal and PCOS granulosa cells, the importance of nuclear factors residing in these regions can be discerned.

182-187

Authors: Negin Hadisi, Hadi Abedi, Majid Shokoohi, Seval Tasdemir, Shahriyar Mamikhani, Shahla Meshgi, Arian Zolfagharzadeh, Leila Roshangar

Objective: COVID-19 is an infectious disease that has become pandemic with a high mortality rate. This study aims to provide new insight into the relations between SARS-CoV-2 and the Endocrine system. Materials and Methods: In this cross-sectional study, we have hospitalized 60 patients with a positive SARA-CoV-2 PCR test. The information of complete blood count and endocrine hormones was obtained when the patients were admitted to the hospital or for a maximum of 4 days onset the hospitalization. Results: Of 60 patients with COVID-19, forty-four (73.33%) had at least one abnormality mean item >×3. In total, 26 (43.33%), 21 (35%), 18 (30%), 13 (21.67%), 31 (51.67%), 12 (20%), 30 (50%), 25 (41.67%) patients having estradiol, follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin, progesterone, testosterone, cortisol and thyroid stimulating hormone (TSH) abnormal test results, respectively. There was no change in creatinine levels. FSH has shown drastic changes in both sexes’ intensity (F: 769, P<0.0001). Although TSH had many abnormalities in women, analysis has shown no significant P value (P=0.4558). Furthermore, prolactin and testosterone mean level in men and the estradiol mean level in women have shown no significant P value (P=0.2077, P=0.1446, P=0.1351, respectively). Conclusion: Results suggest that COVID-19 affects directly or non-directly glands and related hormones.

196-203

Authors: Mohammad Reza Haghshenas, Nasrollah Erfani, Soolmaz Khansalar, Bijan Khademi, Mohammad Javad Ashraf, Mahboobeh Razmkhah, Abbas Ghaderi

Objective: Salivary gland tumors (SGTs) show some aggressive and peculiar clinicopathological behaviors that might be related to the components of the tumor microenvironment, especially mesenchymal stem cells (MSCs)-associated proteins. However, the role of MSCs-related proteins in SGTs tumorigenesis is poorly understood. This study aimed to isolate and characterize MSCs from malignant and benign tumor tissues and to identify differentially expressed proteins between these two types of MSCs. Materials and Methods: In this experimental study, MSC-like cells derived from benign (pleomorphic adenoma, n=5) and malignant (mucoepidermoid carcinoma, n=5) tumor tissues were verified by fluorochrome antibodies and flow cytometric analysis. Differentially expressed proteins were identified using two-dimensional polyacrylamide gel electrophoresis (2DE) and Mass spectrometry. Results: Results showed that isolated cells strongly expressed characteristic MSCs markers such as CD44, CD73, CD90, CD105, and CD166, but they did not express or weakly expressed CD14, CD34, CD45 markers. Furthermore, the expression of CD24 and CD133 was absent or near absent in both isolated cells. Results also discovered overexpression of Annexin A4 (Anxa4), elongation factor 1-delta (EF1-D), FK506 binding protein 9 (FKBP9), cytosolic platelet-activating factor acetylhydrolase type IB subunit beta (PAFAH1B), type II transglutaminase (TG2), and s-formylglutathione hydrolase (FGH) in MSCs isolated from the malignant tissues. Additionally, heat shock protein 70 (Hsp70), as well as keratin, type II cytoskeletal 7 (CK-7), were found to be overexpressed in MSCs derived from the benign ones. Conclusion: Malignant and benign SGTs probably exhibit a distinct pattern of tissue proteins that are most likely related to the metabolic pathway. However, further studies in a large number of patients are required to determine the applicability of identified proteins as new targets for cancer therapy.

212-214

Authors: Hiromitsu Tanaka, Hisayo Nishida-Fukuda, Morimasa Wada, Keizo Tokuhiro, Hiroaki Matsushita, Yukio Ando

HASPIN acts in chromosome segregation via histone phosphorylation. Recently, HASPIN inhibitors have been shown to suppress growth of various cancer cells. Pancreatic cancer has no symptom in the early stages and may progress before detection. So, the 5-year survival rate is low. Here, we reported that administration of the HASPIN inhibitor, CHR-6494, to mice bearing pancreatic BxPC-3-Luc cancer cells significantly suppressed growth of BxPC-3-Luc cells. CHR-6494 might be a useful agent for treating pancreatic cancer.

112-119

Authors: Mei Li, Zhubin Li, Juanrong Song, Xu Li, Pengtao Zhai, Xudong Mu, Fakai Qiu, Le Yao

Objective: The aim of the recent study was to investigate the effects of miR-205 on reversing Doxorubicin (DOX) resistance, as chemotherapeutic agents through up-regulation of PTEN in human liver cancer HepG2 cells. Materials and Methods: In this experimental study, the drug resistance in liver cancer cells via drug efflux inhibition and enhancing apoptosis by the regulation of PTEN and multi-drug resistance/ P-glycoprotein (MDR/P-gp) expression was revealed. Using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, effect of DOX on cell proliferation was evaluated after miR-205 transfection in HepG2 and HepG2/DOX cells. Activity of P-gp on drug efflux was measured by the Rhodamine 123 (Rho-123) assay. PTEN mRNA expression levels were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry was used to measure the apoptotic ratio of HepG2/DOX cells. Results: miR-205 overexpression considerably inhibited the HepG2/DOX cells viability (P<0.05). qRT-PCR results revealed that PTEN is a pivotal regulator in PI3K/Akt/P-gp axis. Overexpression miR-205 resulted in up-regulation PTEN and ultimately down-regulation of P-gp. This inhibits drug resistance, proliferation and induces apoptosis in HepG2/DOX cells (P<0.05). Whilst, treatment with 10 μM of special inhibitors, including LY294002 (PI3K) or PD098059 (MAPK), increased Rho 123-associated MFI, treatment with 10 μM of SF1670 (PTEN) almost abolished the effect of miR-205 overexpression (P<0.05). Finally, we found that miR-205 was down-regulated in HepG2/DOX cells, and its overexpression led to enhancing apoptosis with re-sensitization of HepG2/DOX cell lines to DOX through PTEN/PI3K/ Akt/MDR1 pathway. Conclusion: These findings may introduce miR-205 as a predictive biomarker and a potential treatment target for liver cancer therapy during MDR.

127-132

Authors: Sara Momenzadeh, Fereshteh Karamali, Atefeh Atefi, Mohammad Hossein Nasr-Esfahani

Objective: Degeneration of the photoreceptors due to retinal disorders can affect vision, and even lead to blindness. Recently therapeutic progress in retinal degeneration, using human embryonic stem cells (hESCs), has been facing technical challenges, demanding the development of simple and standardized protocols. In addition to the designing of the protocols, characterization of the obtained cells is highly required for confirming the reliability of the applied methods for future medical applications. Previously, we showed that human stem cells from apical papilla (SCAP) have stromal cell-derived inducing activity (SDIA). Materials and Methods: In this experimental study, we developed an efficient retinal differentiation protocol, based on the co-culture of confluent hESCs and SCAP in the absence of exogenous molecules, such as activators or inhibitors of molecular signaling pathways. This experimental procedure resulted in the generation of self-forming neural retina (NR)-like structures containing retinal progenitor cells (RPCs) within 4 weeks. Results: We have focused on the characterization of the derived RPCs, as a crucial step towards further verification of the efficiency of our previously suggested protocol. The differentiated cells expressed eye-field markers, PAX6, RAX, LHX2, and SIX3, and also generated neurospheres by a floating culture system for one week. Conclusion: We have reported that the treatment of hESC-derived RPCs by the Notch pathway-inhibitor induced the generation of photoreceptor precursor cells (PPCs). The presented method demonstrates the fact that a co-culture of hESCs and SCAP without exogenous molecules provides an efficient approach to produce RPCs for the treatment of retinal disease, and act as an in vitro model for the development of human retina.

140-147

Authors: Xueling Cheng, Mali Sha, Wenjin Jiang, Linjing Chen, Meihua Song

Objective: Dysregulation of long non-coding RNAs (lncRNAs) is associated with the progression of non-small cell lung cancer (NSCLC). This study aimed to investigate the role of long intergenic non-protein coding RNA 174 (LINC00174) in NSCLC. Materials and Methods: In this experimental study, LINC00174 expression in NSCLC tissues and cell lines was investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Besides, cell counting kit-8 (CCK-8), 5-bromo-2'-deoxyuridine (BrdU). Transwell and Flow Cytometry assays were applied to detect the regulatory function of LINC00174 on the growth, migration and apoptosis of NSCLC cells. Bioinformatics analysis, dual luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay predicted and verified the targeting relationship between LINC00174 and miR-31-5p, and between miR-31-5p and the 3´-untranslated region (3´UTR) of large tumor suppressor kinase 2 (LATS2), respectively. Western blotting was performed to detect the regulatory function of LINC00174 and miR-31-5p on LATS2 protein expression. Results: Compared with that in normal lung tissues, LINC00174 expression in NSCLC tissues and cell lines was reduced. LINC00174 expression was negatively associated with the TNM stage of the patients. Functional experiments showed that LINC00174 overexpression inhibited NSCLC cell multiplication and migration, and induced apoptosis. Furthermore, LINC00174 targeted miR-31-5p and repressed its expression. Additionally, LINC00174 upregulated LATS2 expression through competitively binding to miR-31-5p. Conclusion: LINC00174, as a competitive endogenous RNA, elevates LATS2 expression by adsorbing miR-31-5p, thereby inhibiting the viability and migration of NSCLC cells, and promoting apoptosis.

155-162

Authors: Arsalan Jalili, Abbas Hajifathali, Ahmad Bereimipour, Elham Roshandel, Nasser Aghdam

Objective: Different Cell Culture medias can affect the expansion of T cells. The aim of this study is to assess signaling pathways, protein interactions and genes in T cells cultured in different common T cell expansion medias to select the best candidate. Materials and Methods: In this in silico observational study, with the use of bioinformatics analysis and the use of enrichment databases, gene expression profiles were investigated using microarray analysis. Results: The results of this study were the joint selection of 26 upregulated genes and 59 downregulated genes that were involved in SREBP control of lipid synthesis, co-stimulatory signal during T-cell activation mitosis and chromosome dynamics, telomeres, telomerase, and cellular aging signal pathways. Conclusion: Using bioinformatics analyzes, integrated and regular genes were selected as common genes CD80, LST1, ATM and ITM2B 4-1BBL, Akt inhibitor, interleukin 7 and 15 expansion media.

62-68

Authors: Massoud Vosough, Sepideh Nikfam, Shukoofeh Torabi, Bahareh Sadri, Hadi Ahmadi Amoli, Ali Basi, Maryam Niknejadi, Nikoo Hossein-Khannazer, Seyedeh-Esmat Hosseini, Soura Mardpour, Vajiheh Azimian, Neda Jaroughi, Nasser Aghdami, Hamid Reza Amirzehni, Amir Anushirvani, Reza Malekzadeh, Hossein Baharvand, Mehdi Mohamadnejad

Objective: Perianal fistulas in Crohn’s disease (CD) are the main challenges in inflammatory bowel diseases (IBDs). Some of the fistulas are refractory to any therapeutic strategy. The aim of this study was to evaluate the therapeutic effects of mesenchymal stromal cells (MSCs) as a novel promising modality for the treatment of fistulizing CD. Materials and Methods: This case series clinical interventional study was conducted from 2014 to 2017 at Shariati Hospital, an IBD referral center in Tehran, Iran. Refractory adult patients with CD who had draining perianal fistulas were enrolled in this study. All patients were examined by a colorectal surgeon and the fistula imaging studies were performed by pelvic magnetic resonance imaging (MRI). After autologous bone marrow (BM) aspiration and MSCs isolation, the cells were cultured and passaged under current good manufacturing practice (cGMP) conditions. Four intra-fistula injections of cells, each containing 40×106 MSCs suspended in fibrin glue, were administered by an expert surgeon every 4 weeks. Procedure safety, feasibility and closure of the perianal fistulas at week 24 were assessed. Clinical examination and MRI findings were considered as the primary end points. Results: In total, 5 patients (2 males and 3 females) were enrolled in this study. No adverse events were observed during the six-month follow-up in these patients. Both the Crohn’s Disease Activity Index (CDAI) and Perianal Disease Activity Index (PDAI) scores decreased in all patients after cell injections and one patient achieved complete remission with closure of fistulas, discontinuation of fistula discharge, and closure of the external opening. Conclusion: Local injection of MSCs combined with fibrin glue is potentially a safe and effective therapeutic approach for complex perianal fistulas in patients with CD.

76-84

Authors: Sahar Samieyan Dehkordi, Seyed Hadi Mousavi, Marzieh Ebrahimi, Shaban Alizadeh, Amir Abbas Hedayati Asl, Monireh Mohammad, Bahareh Aliabedi

Objective: Acute myeloid leukemia (AML) is characterized by abnormalities of differentiation and growth of primary hematopoietic stem cells (HSCs) in the blood and bone marrow. In many studies, miR-625-5p has been shown to inhibit downstream pathways from affecting the metastasis and invasion of the integrin-linked kinase (ILK) signaling pathway. It has been proved that the expression of miR-625-5p decreases in AML cell lines. This study aimed to investigate the effect of miR-625-5p upregulation on the invasion of KG1 ell line in vitro. Materials and Methods: In this experimental study, we investigated the impact of upregulation of miR-625-5p on invasion via the ILK/AKT pathway in the KG1 cell line. After transfection using the viral method, the cellular invasion was assessed by invasion assay and the levels of miR-625-5p genes and protein were evaluated by quantitative polymerase chain reaction (qPCR) and western blotting. Moreover, CXCR4 level was assessed by flow cytometry. Results: The invasion significantly reduced in MiR-625-5p-transfected KG1 cells (P<0.01) that was concomitant with remarkably decreasing in the expression levels of ILK, NF-κB, and COX2 genes compare with the control group (P<0.01). Incontrast, MMP9, AP1, and AKT significantly increased (P<0.01, P<0.001 and P<0.01, respectively) and GSK3β did not change significantly in MiR-625-5p-transfected KG1 cells. The protein level of NF-κB decreased (P<0.01) and MMP9 increased, however it was not significant. Moreoever, the expression of CXCR4 was significantly lower (P<0.01) in comparison with the control group. Conclusion: miR-625-5p leads to a reduction in cell invasion in the AML cell line through ILK pathway. Therefore, it could be a breakthrough in future AML-related research. However, further studies are needed to support this argument.

91-98

Authors: Yongqian Xu, Xiaona Dong, Baoli Ma, Pingping Mu, Xiang Kong, Dongmei Li

Objective: This study aims to investigate the biological function of circular RNA (circRNA) circ_0000228 in the cervical cancer (CC). Materials and Methods: In this experimental study, the GSE113696 dataset was downloaded from the Gene Expression Omnibus (GEO). GEO2R was employed to obtain differentially expressed circRNA between CC tissues and matched paracancerous tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were employed to detect circ_0000228, microRNA-337-3p (miR-337-3p) and transforming growth factor, beta receptor I (TGFBR1) expression levels in the CC tissues and cells. Following gain-of-function and loss-of-function models establishment, CCK-8 and BrdU tests were conducted to examine cell proliferation. Transwell experiment was executed to examine CC cells migration and invasion. A lung metastasis model was utilized to determine the ability of circ_0000228 on the lung metastasis. Bioinformatics analysis, dual-luciferase reporter experiment and RNA immunoprecipitation (RIP) assay were applied to verify the targeting relationship among miR-337-3p, circ_0000228, and TGFBR1. Results: Circ_0000228 expression in the CC tissues and cells was up-modulated. Circ_0000228 overexpression markedly enhanced cell proliferation, migration, and invasion, while knocking down circ_0000228 remarkably repressed cell proliferation, migration, and invasion. MiR-337-3p could be adsorbed by circ_0000228. TGFBR1 was identified as a target gene of miR-337-3p that indirectly and positively modulated by circ_0000228 in the CC cells. Conclusion: Circ_0000228 up-modulates TGFBR1 by targeting miR-337-3p to enhance CC cell proliferation, migration and invasion. Also, Circ_0000228 is a promising therapeutic target for the CC.

103-104

Authors: Milad Zandi, Emad Behboudi, Parisa Zeinali, Saber Soltani, Mohammad Reza Shojaei

Following SARS-CoV-2 China epidemic in the December 2019, researches have attended to the genome of novel coronavirus. Hidden corners of SARS-CoV-2, maybe a shiny way to discover its pathogenicity and virulence. To design therapeutic agents, it is critical to map the complete repertoire of viral-translated proteins. Ribosome profiling is considered as a snapshot of all active ribosomes in a cell at a specific time point.

715-722

Authors: Marziyeh Pooladi; Mohamadreza Sharifi; Gholamreza Dashti

Objective The role of adiponectin in sperm function is inconclusive and there is a paucity of evidence. Obesity shows an ambiguous influence on sperm motility, and male subfertility. The aim of this study was to compare the role of adiponectin and sperm functional parameters among obese and non-obese men. Materials and Methods In this comprehensive study, 64 male patients were included, and were classified as non-obese [body mass index (BMI)< 24.9 kg/m2, n=32] and obese (BMI >25 kg/m2, n=32) groups. Sperm analysis, was conducted using World Health Organization (WHO) 2010 standards. Real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were used for the analysis of adiponectin gene expression and protein levels, respectively. Sperm viability was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl tetrazolium bromide (MTT test), Acridine orange (AO) test was utilized to detect DNA denaturation, and sperm chromatin dispersion (SCD) technique was used to investigate the fragmentation of DNA. Results In obese men, adiponectin gene expression (P<0.0001) and protein levels (P<0.001) were significantly lower compared to the non-obese group. Additionally, sperm motility, was significantly lower in the obese group. The rapid progressive (RP) motility was less in obese men in comparison to the non-obese group (P<0.001). Sperm count and morphology were not significantly different in the two groups. DNA denaturation and DNA fragmentation were significantly more frequent in the obese group than in non-obese men (P<0.05) and (P<0.01), respectively. The obese men showed significantly lower sperm viability compared to the non-obese group (P<0.05). Conclusion This study showed no significant correlation between the evaluated variables (sperm parameter, sperm viability, DNA fragmentation and integrity), and obesity in men. Based on these results, adiponectin may potentially play positive role in sperm function for acquiring fertility.

732-740

Authors: Chenyu Li; Shaozhen Ying; Xiaolin Wu; Tongjian Zhu; Qing Zhou; Yue Zhang; Yongsheng Liu; Rui Zhu; He Hu

Objective C1q/TNF-related proteins 1 (CTRP1) is a recently identified adiponectin associated with obesity-linked disorders and adverse cardiovascular events. The effect of CTRP1 on cardiac fibrosis has not yet been fully elucidated; thus, we aimed to explore this association. Materials and Methods In this experimental study, a mouse model of cardiac fibrosis was established by administering isoproterenol (ISO) (subcutaneously injecting 10 mg/kg/day for 3 days and then 5 mg/kg/day for 11 days). Mice were also injected with recombinant CTRP1 protein (200 μg/kg) 14 days after the final ISO administration. Adult mouse fibroblasts were isolated and stimulated with transforming growth factor (TGF) β1, followed by treatment with recombinant CTRP1. Primary bone marrow-derived macrophages were isolated from C57BL/6J mice and treated with recombinant CTRP1 as well. Results CTRP1 level was increased in mouse plasma and heart tissue 2 weeks after ISO injection. Our findings indicated that recombinant CTRP1 injection aggravated ISO-induced cardiac fibrosis and dysfunction. However, recombinant CTRP1 did not alter TGFβ1-induced fibroblast proliferation and activation or collagen transcription. Recombinant CTRP1 exacerbated ISO-induced macrophage infiltration and inflammatory response. We determined that macrophages treated with recombinant CTRP1 showed increased pro-inflammatory cytokine release. Fibroblasts co-cultured with macrophages treated with recombinant CTRP1 showed increased proliferation and collagen transcription. We also found that CTRP1 upregulated the NADPH oxidase 2 (NOX2)/p38 pathway in macrophages. When we inhibited p38 signaling, the pro-inflammatory effect of CTRP1 on macrophages was counteracted. Fibroblasts co-cultured with macrophages treated with a p38 inhibitor also showed limited proliferation and collagen transcription. Conclusion: Cardiac fibrosis was aggravated with the activation of the NOX2/p38 pathway in macrophages after CTRP1 treatment.

748-756

Authors: Mohammad Mardani; Rasoul Ganji; Nazem Ghasemi; Mohammad Kazemi; Shahnaz Razavi

Objective Multiple sclerosis (MS) is known as a nerve tissue disorder, which causes demyelination of central nervous system (CNS) fibers. Cell-based treatment is a novel strategy for the treatment of demyelinating diseases such as MS. Adipose-derived stem cells (ADSCs) have neuroprotective and neuroregenerative effects and pregnenolone as a neurosteroid has remarkable roles in neurogenesis. We intend to examine the impact of intraventricular transplantation of human ADSCs and systemic injection of pregnenolone on the remyelination of a rat model cuprizone-induced demyelination. Materials and Methods This experimental study was performed on 36 male Wistar rats that received a regular diet and a cuprizone diet for 3 weeks for M.S. induction. Through lipoaspirate surgery, human-ADSCs (hADSCs) were obtained from a patient. Six groups of rats (n=6): healthy, MS, sham, pregnenolone injection, ADSCs transplantation, and pregnenolone injection/ADSCs transplantation were included in this study. For assessment of remyelination, transmission electron microscopy (TEM), immunohistochemistry staining, real-time reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay (ELISA) were performed. Results TEM outcomes revealed an increase in the thickness of the fibers myelin in the treatment groups (P<0.05). We also observed a significant upregulation of MBP, PDGFR-α, and MOG after treatment with hADSCs and pregnenolone compared to other study groups (P<0.001). These results were confirmed by immunostaining analysis. Moreover, there was no significant difference between the ADSCs/pregnenolone group and the control group regarding the level of MBP, A2B5, and MOG proteins in ELISA. Conclusion Our data implied that the remyelination and cell recovery were more improved by intraventricular ADSCs transplantation and pregnenolone injection after inducing a rat model of MS.

764-773

Authors: Tongmiao Wang; Jingwen Liu; Jianhua Chen; Bo Qin

Objective We aimed to generate induced pluripotent stem cells (iPSCs)-derived retinal pigmented epithelium (RPE) cells from peripheral blood mononuclear cells (PBMCs) and age-related macular degeneration (AMD) patient to provide potential cell sources for both basic scientific research and clinical application. Materials and Methods In this experimental study, PBMCs were isolated from the whole blood of a 70-year-old female patient with AMD and reprogrammed into iPSCs by transfection of Sendai virus that contained Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC). Flow cytometry, real-time quantitative polymerase chain reaction (qPCR), karyotype analysis, embryoid body (EB) formation, and teratoma detection were performed to confirm that AMD-iPSCs exhibited full pluripotency and maintained a normal karyotype after reprogramming. AMD-iPSCs were induced into RPE cells by stepwise induced differentiation and specific markers of RPE cells examined by immunofluorescence and flow cytometry. Results The iPSC colonies started to form on three weeks post-infection. AMD-iPSCs exhibited typical morphology including roundness, a large nucleus, sparse cytoplasm, and conspicuous nucleoli. QPCR data showed that AMDiPSCs expressed pluripotency markers (endo-OCT4, endo-SOX2, NANOG and REX1). Flow cytometry indicated 99.7% of generated iPSCs was TRA-1-60 positive. Methylation sequencing showed that the regions of OCT4 and NANOG promoter were demethylated in iPSCs. EBs and teratomas formation assay showed that iPSCs had strong differentiation potential and pluripotency. After a series of inductions with differentiation mediums, a monolayer of AMDiPSC-RPE cells was observed on day 50. The AMD-iPSC-RPEs highly expressed specific RPE markers (MITF, ZO-1, Bestrophin, and PMEL17). Conclusion A high quality iPSCs could be established from the PBMCs obtained from elderly AMD patient. The AMDiPSC displayed complete pluripotency, enabling for scientific study, disease modeling, pharmacological testing, and therapeutic applications in personalized medicine. Collectively, we successfully differentiated the iPSCs into RPE with native RPE characteristics, which might provide potential regenerative treatments for AMD patients.

779-781

Authors: Tie-Ying Qiu; Jin Huang; Li-Ping Wang; Bi-Song Zhu

In this article which was published in Cell J, Vol 23, No 1, Spring 2021, on pages 51-60, the authors discovered that Figures 1B, 2D, 2F, 5B, and 5D some errors that occurred accidentally during figure organization in this article. The figures below have been corrected. The authors would like to apologies for any inconvenience caused.

647-656

Authors: Elham Adham Foumani; Shiva Irani; Yalda Shokoohinia; Ali Mostafaie

Objective Breast cancer is one of the major causes of mortality among women. Due to many side effects of the existing chemotherapeutic agents, the research of anti-cancer drugs, including natural products, is still a big challenge. Here, we investigated the effects of colchicine on apoptosis of two breast cancer cell lines ( human MCF-7 and mouse 4T1). Materials and Methods In this experimental study, we evaluated the apoptotic effects of colchicine on (MCF-7) and (4T1), as well as a human cancer-associated fibroblast cell line as a control group. Extraction and chromatographic techniques were applied to isolate colchicine from Colchicum autumnale L. To compare the isolated colchicine with pure standard colchicine, we used the H-NMR technique. The methyl thiazolyl tetrazolium (MTT) assay, quantitative reverse transcriptase-polymerase chain reaction, Western blotting and annexin V/PI staining were used to evaluate the apoptotic effects of the isolated and standard colchicine. Results Similar to standard colchicine, the isolated colchicine inhibited cell proliferation significantly in cancer cell lines. Colchine inhibited proliferation and induced apoptosis on a dose-dependent manner. The medicine modified the expression of genes-related to apoptosis by up-regulation of P53 ,BAX, CASPASE-3, -9 and down-regulation of BCL-2 gene, which led to an increase in the BAX/BCL-2 ratio. Conclusion We showed that isolated colchicine from Colchicum autumnale and pure standard colchicines modulate the expression levels of several genes and therefore exerting their anticancer effects on both human (MCF-7) and mouse (4T1) breast cancer cells. Based on these results, we suggest that colchicine can be a potential candidate for prevention and treatment of breast cancer.

665-672

Authors: Liumin Kang; Jie Sun; Jie Liu; Feng Xu; Qilin Zhu; Xiaohua Shi

Objective: Reportedly, long non-coding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) is involved in regulating colorectal cancer (CRC) progression. However, the function and detailed downstream mechanism of CASC2 in CRC progression are not fully elucidated. The aim of the study was to investigate the potential function and molecular mechanism of CASC2 in CRC progression. Materials and Methods: In this experimental study, quantitative real-time polymerase chain reaction (qRT-PCR) was adopted to probe CASC2, microRNA-18a-5p (miR-18a-5p) and B cell translocation gene 3 (BTG3) mRNA expression in CRC tissues and cell lines. After CASC2 was overexpressed in Colo-678 and HCT116 cell lines, methylthiazol tetrazolium (MTT) and 5-bromo-2’-deoxyuridine (BrdU) assays were employed to examine the proliferation of CRC cells. Transwell migration and invasion assays were executed to evaluate the metastatic potential of CRC cells. The targeting relationships among CASC2, miR-18a-5p and BTG3 were validated by dual luciferase reporter gene assay. Western blot assay was applied to examine the regulatory effects of CASC2 and miR-18a-5p on BTG3 protein expression. Results: CASC2 was decreased in CRC tissues and cell lines, and its low expression in CRC tissues was associated with larger tumor size and lymph node metastasis. CASC2 overexpression restrained proliferative, migrative and invasive capabilities of CRC cells. CASC2 could function as a molecular sponge for miR-18a-5p and repress the expression of miR-18a-5p. Furthermore, the inhibitory effects of CASC2 on the malignant phenotypes of CRC cells was counteracted by miR-18a-5p mimics. Additionally, CASC2 could positively regulate BTG3 expression via suppressing miR-18a-5p. Conclusion: CASC2 inhibits CRC development by suppressing miR-18a-5p and raising BTG3 expression.

681-688

Authors: Mojtaba Ghobadi; Babak Arji; Maryam Yadegari; Mansour Esmailidehaj; Farshad Homayouni Moghadam; Mohammad Ebrahim Rezvani

Objective: Ferulic acid (FA) is a phenolic compound that exhibits neuroprotective effects in the central nervous system (CNS). This study was conducted to evaluate the potential effects of FA on the cognitive and motor impairments in the cuprizone-induced demyelination model of multiple sclerosis (MS). Materials and Methods: In this experimental study, demyelination was induced in mice by feeding them with chow containing cuprizone (CPZ) 0.2% for 6 weeks. Mice in the control group received normal chow. Mice in the CPZ+Veh, CPZ+FA10, and CPZ+FA100 groups received saline, and FA at a dose of 0, 10, or 100 mg/kg (intraperitoneal, I.P., daily) respectively. After cognitive and motor assessments, under anaesthesia, animal brains were removed for evaluating the histological, apoptosis, and molecular changes. Results: The results showed that FA increased freezing behaviour in contextual (P<0.05) and cued freezing tests (P<0.05). FA also reduced the random arm entrance (P<0.01) and increased spontaneous alternations into the arms of Y-maze compared to the CPZ+Veh group (P<0.05). Time on the rotarod was improved in rats that received both doses of FA (P<0.01). Demyelination, apoptosis, and relative mRNA expression of p53 were lower in the FA-treated groups relative to the CPZ+Veh group (P<0.01). In addition, FA increased mRNA expression of brain-derived neurotrophic factor (Bdnf), Olig2, and Mbp (P<0.05) but decreased GFAP mRNA expression compared to the CPZ+Veh group (P<0.01). Conclusion: The results of this study showed that FA plays a significant neuroprotective role in CPZ models of demyelination by reducing neuronal apoptosis and improving oligodendrocytes (OLs) growth and differentiation.

697-704

Authors: Shahla Chaichian; Abolfazl Mehdizadeh Kashi; kobra Tehermanesh; Vahid Pirhajati Mahabadi; Sara Minaeian; Neda Eslahi

Objective: One of the challenges in gene therapy is the transfer of the gene to the target cell. MicroRNAs (miRNAs) regulate gene expression after transcription by binding directly to the messenger and play a vital role in cell behaviors and the pathogenesis of some diseases. This study was aimed at developing poly (lactic-co-glycolic acid) (PLGA)- based nanoparticles (NPs) for gene delivery to endometriotic cyst stromal cells (ECSCs). Materials and Methods: In this experimental study, endometriosis cells were isolated from women with severe endometriosis (DIE) and digested by the enzymatic method (40 μg/ml DNAase I and 300 μg/ml collagenase type 3). PLGA-based NPs were synthesized and characterized. The size of sole PLGA NPs and PLGA/miRNA were 60 ± 4 nm and 70 ± 5.1 nm respectively. Poly lactic-co-glycolic-based NPs were used as vector carriers for miRNA 503 transfection in endometriosis cells. The cells were divided into the five groups of control and four doses (25, 50, 75, and 100 μm) of miRNA 503/PLGA at 12, 24, 48, and 72 hours. Viability and apoptosis were evaluated by the MTT assay and Annexin Kits. Data were analyzed by one-way analysis of variance. Results: The results show that the size of PLGA/miRNA complex with dynamic light scattering (DLS) was 70 ± 5.1 nm and zeta potential values of the PLGA/PEI/miRNA complexes were 27.9 mV. Based on the MTT assay results, the optimal dose of miRNA 503/PLGA was 75 μm, at which the viability of ECSCs was 52.6% ± 1.2 (P≤0.001), and the optimal time was 48 hours. The apoptotic rates of ECSCs treated with PLGA/miRNA503 (34.75 ± 4.9%) were significantly higher than those of ECSCs treated with PLGA alone (3.35 ± 2.58%, P≤0.01). Conclusion: Cell death increased with increasing the concentration of miRNA; thus, it can be suggested as a treatment for endometriosis.