CRISPR/Cas9 Ablated BCL11A Unveils the Genes with Possible Role of Globin Switching.

Purpose: Fetal hemoglobin (HbF) upregulation is a mitigating factor in ß-hemoglobinopathies therapy like ß-thalassemia and sickle cell diseases. Finding molecular mechanisms and the key regulators responsible for globin switching could be helpful to develop effective ways to HbF upregulation. In our prior in silico report, we identified a few factors that are likely to be responsible for globin switching. The goal of this study is to experimentally validate the factors. Methods: We established K562 cell line with BCL11A knock down leading to increase in HBG1/2 using CRISPR/Cas9 system. Then, using quantitative polymerase chain reaction (qPCR), we determined the expression level of the factors which were previously identified in our prior in silico study. Results: our analysis showed that BCL11A was substantially knocked down, resulting in the upregulation of HBG1/2 in the BCL11A-ablated K562 cells using CRISPR/Cas9 system. Additionally, the experimental data acquired in this study validated our prior bioinformatics findings about three potentially responsible genes for globin switching, namely HIST1H2Bl, TRIM58, and Al133243.2. Conclusion: BCL11A is a promising candidate for the treatment of ß-hemoglobinopathies, with high HbF reactivation. In addition, HIST1H2BL, TRIM58 and Al133243.2 are likely to be involved in the mechanism of hemoglobin switching. To further validate the selected genes, more experimental in vivo and in vitro studies are required.

The SW480 cell line, overexpressing PIWIL2 gene, maintains the expression of stemness and proliferation genes in the mice xenografts.

Aim: This study aims to confirm previous fundamental in vitro findings about the PIWIL2 gene by investigating the effects of its overexpression on cell cycle, proliferation, apoptosis, and stem cell expression markers in colorectal cancer cells (CRC cells) at in vivo level. Background: PIWIL2 has a critical role in maintaining cellular stemness and proliferation. PIWIL2 is an oncogene whose expression in CRC is associated with the occurrence, metastasis, and poor prognosis. Methods: SW480 cells harboring expression vectors with/without PIWIL2 were cultured and inoculated in BALB/c nude mice. Tumor formation and growth were monitored every 3 days. On the 28th day after inoculation, the tumors were harvested for their total RNA extraction, and the expression profiling of the candidate genes was performed by Real-time PCR. Results: Our results for the expression profiling of the xenografted tumors showed a significant increase in the expression of cancer stem cell markers, including CD24, CD133, and pluripotency marker SOX2 in the PIWIL2 over-expressing xenografts, compared to the control cell line. Moreover, PIWIL2 dramatically promoted the anti-apoptotic pathway by inducing STAT3 and BCL2-L1 genes in the PIWIL2 over-expressing xenografts, along with the up-regulation of Cyclin D1 and Ki-67 genes. Conclusion: This research supports our prior in vitro findings, highlighting the critical role that PIWIL2 plays in the development of CRC and its substantial promise as a leading candidate for CRC-targeted therapy.

PIWIL2 Regulates the Proliferation, Apoptosis and Colony Formation of Colorectal Cancer Cell Line.

Background: Tumor cells proliferation and apoptosis inhibition are the mechanisms through which the Colorectal Cancer (CRC) progression, metastasis and chemoresistance are promoted pathologically, offering clinical advantages for characterizing their molecular regulators. Objectives: In this study, to unravel the role of PIWIL2 as a potential CRC oncogenic regulator, we examined the effect of its overexpression on proliferation, apoptosis and colony formation of SW480 colon cancer cell line. Material and Methods: Established SW480-P (overexpression of PIWIL2) and SW480-control (SW480-empty vector) cell lines were cultured in DMEM containing 10% FBS with 1% penicillin-streptomycin. The total DNA and RNA was extracted for further experiments. Real-Time PCR and western blotting assay were performed to measure the differential expression of proliferation associated genes including the expression of cell cycle and anti-apoptotic genes as well as Ki-67 and PIWIL2 in both cell lines. Cell proliferation was determined using MTT assay, doubling time assay and the colony formation rate of transfected cells was measured with the 2D colony formation assay. Results: At the molecular level, PIWIL2 overexpression was associated with significant up-regulation of cyclin D1, STAT3, BCL2-L1, BCL2-L2 and Ki-67 genes. MTT and doubling time assay showed that PIWIL2 expression induced time-related effects on proliferation rate of SW480 cells. Moreover, SW480-P cells had markedly greater capacity to form colonies. Conclusions: PIWIL2 plays important roles to promote cancer cell proliferation and colonization via the cell cycle acceleration and inhibition of apoptosis, the mechanisms through which this gene seems to contribute to CRC development, metastasis and chemoresistance, hence potentially highlighting PIWIL2 targeted therapy as a valuable tool for CRC treatment.

Stem Cell Protein PIWIL2 Promotes EMT Process and Stem Cell-Like Properties in MCF7 Breast Cancer Cell Line.

Background: Piwi-like RNA-mediated gene silencing 2 (PIWIL2) is a member of AGO/PIWI gene family, which is enriched in cancer stem cells (CSCs). The purpose of this research was to investigate the overexpression of PIWIL2 and its role in the induction of EMT and CSC properties in MCF7 breast cancer cell line. Materials and Methods: MCF7 cells were transfected with the human gene PIWIL2 (Hili) under the control of CMV promoter utilizing the neon electroporation method. Subsequently, the selection was conducted using G418, and doubling time was calculated in the transformed and control cells. RT and real-time PCR were also performed to analyze the expression of epithelial and mesenchymal genes and those related to CSCs. Results: According to the observations from this study, transfecting MCF7 cells with PIWIL2 triggered the conversion of epithelial cells to mesenchymal cells and induced the genes specific for breast CSCs, which was coincident with 9-h reduction in the doubling time of the transfected cells. Furthermore, the molecular analyses revealed a significant reduction in the expression of epithelial markers, while a significant increase was detected in the expression of mesenchymal genes and many CSC biomarkers. Conclusion: PIWIL2 protein acts as a master regulatory protein that is able to manipulate the transcription through specific signaling pathways, which allow the cells to gain stem cell-like properties.

Optimization of 3D islet-like cluster derived from human pluripotent stem cells: An efficient in vitro differentiation protocol.

Development of an optimized protocol to produce sufficient functional human insulin-producing islet-like cluster is important as a potential therapeutic strategy for diabetes as well as in vitro studies. Here, we described a stepwise protocol for differentiation of the human induced pluripotent stem cell line (R1-hiPSC1) into the islet-like cluster using several growth factors and small molecules. Therefore, various differentiation steps have been adopted to maximize mimicking of developmental processes in order to form functional islet like cluster. The differentiation protocol enables us to generate 3D islet-like clusters with highly viable cells, which are insulin producer and glucose responsive. Transcriptome analysis of transcription factors and functional genes revealed high coordination between gene expressions and resembling to those reported during natural development of islet cell. This coordination was further confirmed by hierarchical clustering of genes during differentiation. Furthermore, the islet-like clusters were enriched with insulin producing cells and formed glucose responsiveness behavior upon stimulation with glucose. Our protocol provides a robust platform and well-behaved model for additional developmental studies and shed light our clusters as a good candidate for in vitro model. Further studies are needed to assess the hormonal content of this cluster as well as transplantation into the animal model.

Y Chromosome Genes May Play Roles in the Development of Neural Rosettes from Human Embryonic Stem Cells.

BACKGROUND: The human Y chromosome harbors genes that are mainly involved in the growth, development, sexual dimorphism, and spermatogenesis process. Despite many studies, the function of the male-specific region of the Y chromosome (MSY) awaits further clarification, and a cell-based approach can help in this regard. RESULTS: In this study, we have developed four stable transgenic male embryonic stem cell (ESCs) lines that can overexpress male-specific genes HSFY1, RBMY1A1, RPS4Y1, and SRY. As a proof of principle, we differentiated one of these cell lines (RPS4Y1 over-expressing ESCs) to the neural stem cell (rosette structure) and characterized them based on the expression level of lineage markers. RPS4Y1 expression in the Doxycycline-treated group was significantly higher than control groups at transcript and protein levels. Furthermore, we found Doxycycline-treated group had a higher differentiation efficiency than the untreated control groups. CONCLUSIONS: Our results suggest that the RPS4Y1 gene may play a critical role in neurogenesis. Also, the generated transgenic ESC lines can be widely employed in basic and preclinical studies, such as sexual dimorphism of neural and cardiac functions, the development of cancerous and non-cancerous disease models, and drug screening.

Bcl11a and the Correlated Key Genes Ascribable to Globin Switching: An In-silico Study.

BACKGROUND: Reactivation of HbF is a potential strategy to ameliorate symptoms of hemoglobinopathies such as sickle cell disease and b-thalassemia. After birth, there is a switch from fetal to adult hemoglobin, for which the molecular mechanisms and key regulators await further understanding in order to develop effective methods for HbF reactivation. Bcl11a, one of the major HbF reactivation regulators, demonstrates no significant changes at transcriptional levels in F erythroblasts compared to the non-HbF expressing cells. Therefore, it is possible that posttranscriptional regulation and epigenetic effects, for which the miRNAs play an important role, are the primary causes of the decreased Bcl11a protein level in adult erythroblasts. OBJECTIVE: This paper aims to determine the differentially expressed mRNAs and miRNAs of erythroblasts in HSCs from the fetal liver and bone marrow. METHODS: Raw high-throughput sequencing data (GSE110936, GSE90878) was downloaded from Gene Expression Omnibus (GEO) database. After RNAseq analysis, several data sets and tools were used to select key genes and examine selection validation. RESULTS: We selected 42 DEmRNAs and nine DEmiRs, including hsa-let-7f-5p, hsa-miR-21-5p, hsamiR- 22-3p, hsa-miR-126-5p, hsa-miR-146b-5p, hsa-miR-181a-5p, hsa-miR-92a-3p, hsa-miR-25-3p and hsa-miR-191-5p. Furthermore, hub genes including hist1h2bl, al133243.2, trim58, abcc13, bpgm, and fam210b were identified in the coexpression network, as well as RPS27A in the PPI network. Functional analysis revealed that these DEmRNAs and DEmiRs might play a role in gene expression regulation at multiple levels. Gene set enrichment analysis, in particular, revealed a possible role for genes in the globin switching process. CONCLUSION: According to our findings, a number of the DEmRNAs and DEmiRs may play significant roles in globin switching regulation and thus have the potential to be applied for HbF reactivation.

The Contribution of Y Chromosome Genes to Spontaneous Differentiation of Human Embryonic Stem Cells into Embryoid Bodies In Vitro.

OBJECTIVE: Sexual dimorphism in mammals can be described as subsequent transcriptional differences from their distinct sex chromosome complements. Following X inactivation in females, the Y chromosome is the major genetic difference between sexes. In this study, we used a male embryonic stem cell line (Royan H6) to identify the potential role of the male-specific region of the Y chromosome (MSY) during spontaneous differentiation into embryoid bodies (EBs) as a model of early embryonic development. MATERIALS AND METHODS: In this experimental study, RH6 cells were cultured on inactivated feeder layers and Matrigel. In a dynamic suspension system, aggregates were generated in the same size and were spontaneously differentiated into EBs. During differentiation, expression patterns of specific markers for three germ layers were compared with MSY genes. RESULTS: Spontaneous differentiation was determined by downregulation of pluripotent markers and upregulation of fourteen differentiation markers. Upregulation of the ectoderm markers was observed on days 4 and 16, whereas mesoderm markers were upregulated on the 8th day and endodermic markers on days 12-16. Mesoderm markers correlated with 8 MSY genes namely DDX3Y, RPS4Y1, KDM5D, TBL1Y, BCORP1, PRY, DAZ, and AMELY, which were classified as a mesoderm cluster. Endoderm markers were co-expressed with 7 MSY genes, i.e. ZFY, TSPY, PRORY, VCY, EIF1AY, USP9Y, and RPKY, which were grouped as an endoderm cluster. Finally, the ectoderm markers correlated with TXLNGY, NLGN4Y, PCDH11Y, TMSB4Y, UTY, RBMY1, and HSFY genes of the MSY, which were categorized as an ectoderm cluster. In contrast, 2 MSY genes, SRY and TGIF2LY, were more highly expressed in RH6 cells compared to EBs. CONCLUSION: We found a significant correlation between spontaneous differentiation and upregulation of specific MSY genes. The expression alterations of MSY genes implied the potential responsibility of their gene co-expression clusters for EB differentiation. We suggest that these genes may play important roles in early embryonic development.

Optimization of activin-A: a breakthrough in differentiation of human induced pluripotent stem cell into definitive endoderm.

The first step in differentiation of pluripotent stem cell toward endoderm-derived cell/organ is differentiation to definitive endoderm (DE) which is the central issue in developmental biology. Based on several evidences, we hypothesized that activin-A optimization as well as replacement of fetal bovine serum (FBS) with knockout serum replacement (KSR) is important for differentiation of induced pluripotent stem cell (iPSC) line into DE. Therefore, a stepwise differentiation protocol was applied on R1-hiPSC1 cell line. At first, activin-A concentration (30, 50, 70 and 100 ng/ml) was optimized. Then, substitution of FBS with KSR was evaluated across four treatment groups. The amount of differentiation of iPSC toward DE was determined by quantitative gene expression analyses of pluripotency (NANOG and OCT4), definitive endoderm (SOX17 and FOXA2) and endoderm-derived organs (PDX1, NEUROG3, and PAX6). Based on gene expression analyses, the more decrease in concentrations of activin-A can increase the differentiation of iPSC into DE, therefore, 30 ng/ml activin-A was chosen as the best concentration for the differentiation of R1-hiPSC1 line toward endoderm-derived organ. Moreover, complete replacement of FBS with gradually increased KSR improved the differentiation of iPSC toward DE. For this reason, the addition of 0% KSR at day 1, 0.2% at day 2 and 2% for the next 3 days was the best optimal protocol of the differentiation of iPSC toward DE. Overall, our results demonstrate that optimization of activin-A is important for differentiation of iPSC line. Furthermore, the replacement of FBS with KSR can improve the efficiency of iPSC differentiation toward DE.

Potential role of RAB6C-AS1 long noncoding RNA in different cancers.

BACKGROUND: Long noncoding RNAs (lncRNAs) refer to a group of non-protein-coding RNAs that are usually more than 200 nucleotides. These long transcripts play significant roles in diverse cellular processes, mostly through epigenetic mechanisms. Thus, dysregulation of lncRNAs is associated with various diseases, especially cancer. This study aims to investigate the probable roles of RAB6C-AS1 lncRNA in different cancers. METHODS: Real-time quantitative reverse transcription-polymerase chain reaction was applied for the analysis of RAB6C-AS1 lncRNA amplification in gastric cancer (GC) samples compared with normal ones. Also, several online and offline data sets and tools were used to analyze the relation between RAB6C-AS1 lncRNA and different cancers. RESULTS: The end result of our analyses indicated that RAB6C-AS1 was overexpressed in 40% of the investigated GC specimens. Also, the results demonstrated a true relation between RAB6C-AS1 overexpression and higher GC tumor grades. However, bioinformatic analyses showed that while RAB6C-AS1 possibly functions as an oncogene in some cancer types, including prostate and breast cancers, it might have a tumor suppressive function in some others including brain tumors. CONCLUSIONS: We found that RAB6C-AS1 lncRNA is mostly overexpressed in GC. Also, based on bioinformatic and systems biology analyses, RAB6C-AS1 might function either as an oncogenic factor or tumor suppressor in a tissue-specific manner. Thus, RAB6C-AS1 could be considered as a candidate biomarker for various malignancies, especially prostate and brain cancers. According to our results, RAB6C-AS1 has a notable prognostic value for patients with brain lower grade glioma.

Improvement of hepatogenic differentiation of iPS cells on an aligned polyethersulfone compared to random nanofibers.

The application of stem cells holds great promises in cell and tissue transplants. This study was designed to compare the hepatogenic differentiation of iPSCs on aligned PES/COL versus random. Aligned and random PES/COL nanofibrus scaffolds were fabricated by electrospining and their surface modified through plasma treatment and collagen coating. The scaffolds were characterized using scanning electron microscopy (SEM) and ATR-FTIR. Morphology and biochemical activities of the differentiated hepatocyte-like cells (HLCs) were examined after 5 and 20 days of differentiation. Real-Time RT-PCR and ICC showed no significant difference in the mRNA and protein levels of two important definitive endoderm specific markers, including Sox17 and Foxa2 between two scaffolds. However, Real-Time RT-PCR analysis indicated an increase in the expression of Cyp7A1 gene over the period of the differentiation procedure on the aligned nanofibers but there was no difference in other genes such as Albumin and CK19. Moreover, comparison of hepatogenic differentiation evaluated by Albumin production in conditioned media of HLCs differentiated on aligned PES/COL, showed increase expression of these markers after 20 days compared to that of the random nanofibers. Taken together, the results of this study may indicate that aligned PES/COL nanofibrous scaffolds can improve terminal differentiation of HLCs from iPSCs.

Hepatogenic Differentiation of Human Induced Pluripotent Stem cells on Collagen-Coated Polyethersulfone Nanofibers.

Many scientists have been fascinated with induced pluripotent stem cells (iPSCs) for cell replacement therapies. Nanofibrous biocompatible scaffolds have been shown to foster better cell adhesion and improve stem cell differentiation. In the current study, after fabrication using electrospinning technique and surface modifications, the characteristics of polyethersulfone (PES) nanofibers were determined by scanning electron microscopy, attenuated total reflection Fourier transform infrared spectroscopy, and 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide (MTT) assay. Then, the hepatogenic potential of iPSCs was evaluated using real-time reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC) after culture on collagen-coated polyethersulfone (PES/COL) scaffolds. After scaffolds characterization, analysis of two important definitive endoderm specific markers (Sox17 and Foxa2) using real-time RT-PCR and ICC indicated increase in their mRNA and protein levels after 5 days of hepatogenic induction. In addition, to determine hepatic differentiation of iPSCs cultured on PES/COL, the expression of albumin and α-fetoprotein was evaluated by ICC after 20 days. Real-time RT-PCR analysis showed increased expression of albumin, TAT, cytokeratin 19, and Cyp7A1 genes during the course of differentiation program. Finally, enzyme-linked immunosorbent assay analysis demonstrated an increased expression of albumin in the protein level after 28 days of differentiation. In conclusion, our results demonstrated that PES/COL nanofibrous scaffolds could be a proper substrate to significantly increase the hepatogenic differentiation potential of iPSCs and could also be introduced as a promising candidate for liver tissue engineering applications.

A novel in vitro model for cancer stem cell culture using ectopically expressed piwil2 stable cell line.

OBJECTIVE: Piwil2, a member of Ago/Piwi gene family containing Piwi and PAZ domains, has been shown to be ectopically expressed in different cancer cells, especially its remarkable expression in cancer stem cells (CSCs), and is also known to be essential for germ line stem cell self-renewal in various organisms. The hypothesis that CSC may hold the key to the central problem of clinical oncology and tumor relapse leads to more anticancer treatment studies. Due to emerging controversies and extreme difficulties in studying of CSC, like the cells using in vivo models, more attempts have expended to establish different in vitro models. However, the progress was slow owing to the problems associated with establishing proper CSC cultures in vitro. To overcome these difficulties, we prompted to establish a novel stable cell line over-expressing Piwil2 to develop a potential proper in vitro CSC model. MATERIALS AND METHODS: In this experimental study, mouse embryonic fibroblasts (MEFs) were isolated and electroporated with a construct containing Piwil2 cDNA under the control of the cytomegalovirus promoter (CMV). Stable transfectants were selected, and the established MEF-Piwil2 cell line was characterized and designated as CSC-like cells using molecular markers. Functional assays, including proliferation, migration, and invasion assays were performed using characterized CSC like cells in serum-free medium. Additionally, MEF-Piwil2 cell density and viability were measured by direct and indirect methods in normoxic and hypoxic conditions. RESULTS: The results of reverse transcriptase-polymerase chain reaction (RT-PCR), western blot, and immunocytochemistry revealed an overexpression for Piwil2 in the transfected Piwil2 cells both in the RNA and protein levels. Furthermore, analysis of the kinetic and stoichiometric parameters demonstrated that the specific growth rate and the yield of lactate per glucose were significantly higher in the MEF-Piwil2 group compared to the MEF cells (ANOVA, p< 0.05). Also, analysis of functional assays including migration and invasion assays demonstrated a significantly higher number of migrated and invaded cells in the MEF-Piwil2 compared to that of the MEF cells (ANOVA, p< 0.05). The MEF-Piwil2 cells tolerated hypoxia mimetic conditions (CoCl2 ) with more than 95% viability. CONCLUSION: According to the molecular and functional studies, it has been realized that Piwil2 plays a key role(s) in tumor initiation, progression and metastasis. Therefore, Piwil2 can be used not only as a common biomarker for tumor, but also as a target for the development of new anticancer drug. Finally, the main outcome of our study was the establishment of a novel CSC-like in vitro model which is expected to be utilized in understanding the complex roles played by CSC in tumor maintenance, metastasis, therapy resistance or cancer relapse.

In vitro-derived gametes from stem cells.

Sperm and eggs are essential cells for reproduction and fertility in mammals. Lack of sperm production is one of the leading causes of infertility, a major and growing problem in the developed world affecting 13 to 18% of reproductive-age couples. The birth of the first test tube baby by in vitro fertilization marked an advance in infertility treatment. Later on, several important new techniques called assisted reproductive technologies were developed to help couples who experience infertility. One limiting factor is the requirement of reproductive cells (gametes) for use in in vitro fertilization. For azoospermic men lacking sperm cells, producing gametes in vitro could be a new window to overcome infertility. In the past few years, several reports have been published on generating germ cells from stem cells, one of the epitomes of which was the report on functional in vitro-derived (IVD) germ cells. These mature haploid sperm cells from mouse embryonic stem cells were capable of egg fertilization and producing live offspring. In tandem with previous advancements in germ cell research, development of new technologies based on IVD gametes will change the future of infertility and provide a new basis for the establishment of novel therapeutic approaches to cure more complicated conditions of infertility. In addition, IVD gametogenesis provides an accessible system for studying the specification and differentiation of sperm cells and related processes such as meiosis, morphogenesis, and motility.

Pathways of proliferation and antiapoptosis driven in breast cancer stem cells by stem cell protein piwil2.

Cancer stem cell studies may improve understanding of tumor pathophysiology and identify more effective strategies for cancer treatment. In a variety of organisms, Piwil2 has been implicated in multiple roles including stem cell self-renewal, RNA silencing, and translational control. In this study, we documented specific expression of the stem cell protein Piwil2 in breast cancer with predominant expression in breast cancer stem cells. In patients who were evaluated, we determined that 90% of invasive carcinomas and 81% of carcinomas in situ exhibited highest expression of Piwil2. In breast cancer cells, Piwil2 silencing suppressed the expression of signal transducer and activator of transcription 3, a pivotal regulator of Bcl-X(L) and cyclin D1, whose downregulation paralleled a reduction in cell proliferation and survival. Our findings define Piwil2 and its effector signaling pathways as key factors in the proliferation and survival of breast cancer stem cells.

mtRF1a is a human mitochondrial translation release factor decoding the major termination codons UAA and UAG.

Human mitochondria contain their own genome, encoding 13 polypeptides that are synthesized within the organelle. The molecular processes that govern and facilitate this mitochondrial translation remain unclear. Many key factors have yet to be characterized-for example, those required for translation termination. All other systems have two classes of release factors that either promote codon-specific hydrolysis of peptidyl-tRNA (class I) or lack specificity but stimulate the dissociation of class I factors from the ribosome (class II). One human mitochondrial protein has been previously identified in silico as a putative member of the class I release factors. Although we could not confirm the function of this factor, we report the identification of a different mitochondrial protein, mtRF1a, that is capable in vitro and in vivo of terminating translation at UAA/UAG codons. Further, mtRF1a depletion in HeLa cells led to compromised growth in galactose and increased production of reactive oxygen species.