Two novel splice variants of SOX2OT, SOX2OT-S1, and SOX2OT-S2 are coupregulated with SOX2 and OCT4 in esophageal squamous cell carcinoma.

Long noncoding RNAs (lncRNAs) have emerged as new regulators of stem cell pluripotency and tumorigenesis. The SOX2 gene, a master regulator of pluripotency, is embedded within the third intron of a lncRNA known as SOX2 overlapping transcript (SOX2OT). SOX2OT has been suspected to participate in regulation of SOX2 expression and/or other related processes; nevertheless, its potential involvement in tumor initiation and/or progression is unclear. Here, we have evaluated a possible correlation between expression patterns of SOX2OT and those of master regulators of pluripotency, SOX2 and OCT4, in esophageal squamous cell carcinoma (ESCC) tissue samples. We have also examined its potential function in the human embryonic carcinoma stem cell line, NTERA2 (NT2), which highly expresses SOX2OT, SOX2, and OCT4. Our data revealed a significant coupregulation of SOX2OT along with SOX2 and OCT4 in tumor samples, compared to the non-tumor tissues obtained from the margin of same tumors. We also identified two novel splice variants of SOX2OT (SOX2OT-S1 and SOX2OT-S2) which coupregulated with SOX2 and OCT4 in ESCCs. Suppressing SOX2OT variants caused a profound alteration in cell cycle distribution, including a 5.9 and 6.9 time increase in sub-G1 phase of cell cycle for SOX2OT-S1 and SOX2OT-S2, respectively. The expression of all variants was significantly diminished, upon the induction of neural differentiation in NT2 cells, suggesting their potential functional links to the undifferentiated state of the cells. Our data suggest a part for SOX2OT spliced variants in tumor initiation and/or progression as well as regulating pluripotent state of stem cells.

Distinct gene expression patterns of SOX2 and SOX2OT variants in different types of brain tumours.

Numerous investigations have been recently published on the dysregulated expression of long-noncoding RNAs (lncRNAs) in various cancer types, emphasizing that abnormal lncRNA expression is a major contributor to tumourigenesis. A broad spectrum of lncRNAs is expressed in the central nervous system, where these RNAs seem to play key roles in brain development and function. In addition to expressing SOX2, a master regulator of pluripotency that lies within its third intron, lncRNA SOX2OT has a proposed role in regulating neural development. Based on our previous studies, alternative splicing of SOX2OT generates two alternatively spliced variants (SOX2OT-S1 and SOX2OT-S2). The present study investigated the expression patterns of SOX2OT variants and SOX2 in three principal types of brain tumours (gliomas, meningiomas and pituitary adenomas) and in four brain tumour cell lines (U87-MG, 1321N1, A172 and DAOY). Total RNAwas extracted from 34 human brain tumour specimens, and the expression profile of target genes was measured using a real-time reverse transcription PCR approach. Our data revealed distinct expression patterns for SOX2OT variants and SOX2 in the brain tumour samples, indicating their potential involvement in brain tumourigenesis. Moreover, our results highlighted the potential usefulness of SOX2OT-S1, SOX2OT-S2, and SOX2 in molecular diagnosis and brain tumour classification.

NEUROD2 function is dispensable for human pancreatic ß cell specification.

Introduction: The molecular programs regulating human pancreatic endocrine cell induction and fate allocation are not well deciphered. Here, we investigated the spatiotemporal expression pattern and the function of the neurogenic differentiation factor 2 (NEUROD2) during human endocrinogenesis. Methods: Using Crispr-Cas9 gene editing, we generated a reporter knock-in transcription factor (TF) knock-out human inducible pluripotent stem cell (iPSC) line in which the open reading frame of both NEUROD2 alleles are replaced by a nuclear histone 2B-Venus reporter (NEUROD2nVenus/nVenus). Results: We identified a transient expression of NEUROD2 mRNA and its nuclear Venus reporter activity at the stage of human endocrine progenitor formation in an iPSC differentiation model. This expression profile is similar to what was previously reported in mice, uncovering an evolutionarily conserved gene expression pattern of NEUROD2 during endocrinogenesis. In vitro differentiation of the generated homozygous NEUROD2nVenus/nVenus iPSC line towards human endocrine lineages uncovered no significant impact upon the loss of NEUROD2 on endocrine cell induction. Moreover, analysis of endocrine cell specification revealed no striking changes in the generation of insulin-producing b cells and glucagon-secreting a cells upon lack of NEUROD2. Discussion: Overall, our results suggest that NEUROD2 is expendable for human b cell formation in vitro.

Recent advances in gene therapy for bone tissue engineering.

Autografting, a major treatment for bone fractures, has potential risks related to the required surgery and disease transmission. Bone morphogenetic proteins (BMPs) are the most common osteogenic factors used for bone-healing applications. However, BMP delivery can have shortcomings such as a short half-life and the high cost of manufacturing the recombinant proteins. Gene delivery methods have demonstrated promising alternative strategies for producing BMPs or other osteogenic factors using engineered cells. These approaches can also enable temporal overexpression and local production of the therapeutic genes in the target tissues. This review addresses recent progress on engineered viral, non-viral, and RNA-mediated gene delivery systems that are being used for bone repair and regeneration. Advances in clustered regularly interspaced short palindromic repeats/Cas9 genome engineering for bone tissue regeneration also is discussed.

Increasing Gene Editing Efficiency for CRISPR-Cas9 by Small RNAs in Pluripotent Stem Cells.

Gene manipulations of human induced pluripotent stem cells (iPSCs) by CRISPR-Cas9 genome engineering are widely used for disease modeling and regenerative medicine applications. There are two competing pathways, non-homologous end joining (NHEJ) and homology directed repair (HDR) that correct the double-strand break generated by CRISPR-Cas9. Here, we improved gene editing efficiency of gene knock-in (KI) in iPSCs with minimum components by manipulating the Cas9 expression vector. Either we inserted short hairpin RNA expression cassettes to downregulate DNAPK and XRCC4, two main players of the NHEJ pathway, or we increased cell survival by inserting an anti-apoptotic expression cassette of miRNA-21 into the Cas9 vector. For an easy readout, the pluripotency gene SOX2 was targeted with a T2A-tdTomato reporter construct. In vitro downregulating DNAPK and XRCC4 increased the targeting efficiency of SOX2 KI by around twofold. Furthermore, co-expression of miRNA-21 and Cas9 improved the efficiency of SOX2 KI by around threefold. Altogether, our strategies provide a simple and valuable approach for efficient CRISPR-Cas9 gene editing in iPSCs.

Generation of an INSULIN-H2B-Cherry reporter human iPSC line.

Differentiating human induced pluripotent stem cells (hiPSCs) into insulin (INS)-producing ß-like cells has potential for diabetes research and therapy. Here, we generated a heterozygous fluorescent hiPSC reporter, labeling INS-producing ß-like cells. We used CRISPR/Cas9 technology to knock-in a T2A-H2B-Cherry cassette to replace the translational INS stop codon, enabling co-transcription and T2A-peptide mediated co-translational cleavage of INS-T2A and H2B-Cherry. The hiPSC-INS-T2A-H2B-Cherry reporter cells were pluripotent and showed multi-lineage differentiation potential. Cells expressing the ß-cell specific hormone INS are identified by nuclear localized H2B-Cherry reporter upon pancreatic endocrine differentiation. Thus, the generated reporter hiPSCs enable live identification of INS hormone-producing ß-like cells.

Generation of a homozygous ARX nuclear CFP (ARXnCFP/nCFP) reporter human iPSC line (HMGUi001-A-4).

The aristaless related homeobox (ARX) transcription factor plays a crucial role in glucagon-producing α-cell differentiation. Here, we generate an ARX reporter iPSC line by 3' fusion of an intervening viral T2A sequence followed by a nuclear-localized histone 2B-cyan fluorescent protein (nCFP). The resulting cells have a normal karyotype and preserved pluripotency. In vitro differentiation of the ARXnCFP/nCFP reporter iPSCs towards the endocrine lineage confirmed the specific co-expression of the reporter protein in human glucagon+ α-like cells. Thus, ARXnCFP/nCFP iPSC line will provide a powerful tool to monitor human α-cell progenitor differentiation as well as ARX+ α-like cell function in vitro.

In Utero Exposure to Gestational Diabetes Alters DNA Methylation and Gene Expression of CDKN2A/B in Langerhans Islets of Rat Offspring.

OBJECTIVE: DNA methylation, a major epigenetic reprogramming mechanism, contributes to the increased prevalence of type 2 diabetes mellitus (T2DM). Based on genome-wide association studies, polymorphisms in CDKN2A/B are associated with T2DM. Our previous studies showed that gestational diabetes mellitus (GDM) causes apoptosis in ß-cells, leading to a reduction in their number in pancreatic tissue of GDM-exposed adult rat offspring. The aim of this study was to examine the impact of intrauterine exposure to GDM on DNA methylation, mRNA transcription, as well as protein expression of these factors in the pancreatic islets of Wistar rat offspring. Our hypothesis was that the morphological changes seen in our previous study might have been caused by aberrant methylation and expression of CDKN2A/B. MATERIALS AND METHODS: In this experimental study, we delineated DNA methylation patterns, mRNA transcription and protein expression level of CDKN2A/B in the pancreatic islets of 15-week-old rat offspring of streptozotocin-induced GDM dams. We performed bisulfite sequencing to determine the DNA methylation patterns of CpGs in candidate promoter regions of CDKN2A/B. Furthermore, we compared the levels of mRNA transcripts as well as the cell cycle inhibitory proteins P15 and P16 in two groups by qPCR and western blotting, respectively. RESULTS: Our results demonstrated that hypomethylation of CpG sites in the vicinity of CDKN2A and CDKN2B genes is positively related to increased levels of CDKN2A/B mRNA and protein in islets of Langerhans in the GDM offspring. The average percentage of CDKN2A promoter methylation was significantly lower in GDM group compared to the controls (P<0.01). CONCLUSION: We postulate that GDM is likely to exert its adverse effects on pancreatic ß-cells of offspring through hypomethylation of the CDKN2A/B promoter. Abnormal methylation of these genes may have a link with ß-cell dysfunction and diabetes. These data potentially lead to a novel approach to the treatment of T2DM.

Generation of a human iPSC line harboring a biallelic large deletion at the INK4 locus (HMGUi001-A-5).

The INK4 locus is considered as a hot-spot region for the complex genetic disorders, including cancer, type 2 diabetes (T2D) and coronary artery disease (CAD). By CRISPR/Cas9 gene editing, we generated a human induced pluripotent stem cell (hiPSC) line (HMGUi001-A-5) deleting an 8 kb genomic DNA encompassing six T2D-associated SNPs at the INK4 locus. The resulting hiPSC line revealed a normal karyotype, preserved pluripotency and was able to differentiate towards germ layers, endoderm, mesoderm and ectoderm. Thus, the HMGUi001-A-5 line could provide a valuable cellular model to explore the molecular mechanisms linking these SNPs to T2D and other genetic disorders.

Generation of a heterozygous C-peptide-mCherry reporter human iPSC line (HMGUi001-A-8).

The peptide hormone insulin produced by pancreatic ß-cells undergoes post-transcriptional processing before secretion. In particular, C-peptide is cleaved from pro-insulin to generate mature insulin. Here, we introduce a C-peptide-mCherry human iPSC line (HMGUi001-A-8). The line was generated by CRISPR/Cas9 mediated heterozygous insertion of the mCherry sequence into exon 3 of the insulin locus. We demonstrate that the line is pluripotent and efficiently differentiates towards pancreatic ß-like cells, which localize a red fluorescent C-peptide-mCherry fusion protein in insulin containing granules. Hence, the HMGUi001-A-8 line is a valuable resource to purify derived ß-like cells and follow insulin-containing granules in real time.

Development and Clinical Translation of Approved Gene Therapy Products for Genetic Disorders.

The field of gene therapy is striving more than ever to define a path to the clinic and the market. Twenty gene therapy products have already been approved and over two thousand human gene therapy clinical trials have been reported worldwide. These advances raise great hope to treat devastating rare and inherited diseases as well as incurable illnesses. Understanding of the precise pathomechanisms of diseases as well as the development of efficient and specific gene targeting and delivery tools are revolutionizing the global market. Currently, human cancers and monogenic disorders are indications number one. The elevated prevalence of genetic disorders and cancers, clear gene manipulation guidelines and increasing financial support for gene therapy in clinical trials are major trends. Gene therapy is presently starting to become commercially profitable as a number of gene and cell-based gene therapy products have entered the market and the clinic. This article reviews the history and development of twenty approved human gene and cell-based gene therapy products that have been approved up-to-now in clinic and markets of mainly North America, Europe and Asia.

Organ-specific metastasis of breast cancer: molecular and cellular mechanisms underlying lung metastasis.

BACKGROUND: Breast cancer (BC) is the most common type of cancer in women and the second cause of cancer-related mortality world-wide. The majority of BC-related deaths is due to metastasis. Bone, lung, brain and liver are the primary target sites of BC metastasis. The clinical implications and mechanisms underlying bone metastasis have been reviewed before. Given the fact that BC lung metastasis (BCLM) usually produces symptoms only after the lungs have been vastly occupied with metastatic tumor masses, it is of paramount importance for diagnostic and prognostic, as well as therapeutic purposes to comprehend the molecular and cellular mechanisms underlying BCLM. Here, we review current insights into the organ-specificity of BC metastasis, including the role of cancer stem cells in triggering BC spread, the traveling of tumor cells in the blood stream and their migration across endothelial barriers, their adaptation to the lung microenvironment and the initiation of metastatic colonization within the lung. CONCLUSIONS: Detailed understanding of the mechanisms underlying BCLM will shed a new light on the identification of novel molecular targets to impede daunting pulmonary metastases in patients with breast cancer.

Alternative Splicing Generates Different 5' UTRs in OCT4B Variants.

BACKGROUND: The human OCT4 gene, responsible for pluripotency and self-renewal of Embryonic Stem (ES) and Embryonic Carcinoma (EC) cells, can generate several transcripts (OCT4A, OCT4B-variant 2, OCT4B-variant 3, OCT4B-variant 5, OCT4B1, OCT4 B2 and OCT4B3) by alternative splicing and alternative promoters. OCT4A that is responsible for ES and EC cell stemness properties is transcribed from a promoter upstream of Exon1a in those cells. The OCT4B group variants (OCT4B-variant2, OCT4B-variant3, OCT4B-variant5, OCT4B1, OCT4B2 and OCT4B3) are transcribed from a different promoter located in intron 1 and some of them respond to the cell stresses, but cannot sustain the ES/EC cell self-renewal. However, the exact function of OCT4B group variants is still unclear. METHODS: In the present study, we employed RT-PCR and sequencing approaches to explore different forms of OCT4 transcripts. RESULTS: Our data revealed that the OCT4B group variants (OCT4B-variant2, OCT4 B-variant3, OCT4B1, OCT4B2 and OCT4B3) have longer 5' UTR in the human bladder carcinoma cell line of 5637. CONCLUSION: These OCT4 variants undergo alternative splicing in their 5' UTR which might exert regulatory roles in transcription and translation mechanisms.

Linc-ROR and its spliced variants 2 and 4 are significantly up-regulated in esophageal squamous cell carcinoma.

OBJECTIVES: Similar characteristics of molecular pathways between cellular reprogramming events and tumorigenesis have been accentuated in recent years. Reprogramming-related transcription factors, also known as Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC), are also well-known oncogenes promoting cancer initiation, progression, and cellular transformation into cancer stem cells. Long non-coding RNAs (lncRNAs) are a major class of RNA molecules with emerging roles in stem cell pluripotency, cellular reprogramming, cellular transformation, and tumorigenesis. The long intergenic non-coding RNA ROR (lincRNA-ROR, linc-ROR) acts as a regulator of cellular reprograming through sponging miR-145 that normally negatively regulates the expression of the stemness factors NANOG, OCT4, and SOX2. MATERIALS AND METHODS: Here, we employed a real-time PCR approach to determine the expression patterns of linc-ROR and its two novel spliced variants (variants 2 and 4) in esophageal squamous cell carcinoma (ESCC). RESULTS: The quantitative real-time RT-PCR results revealed a significant up-regulation of linc-ROR (P=0.0098) and its variants 2 (P=0.0250) and 4 (P=0.0002) in tumor samples of ESCC, compared to their matched non-tumor tissues obtained from the margin of same tumors. Our data also demonstrated a significant up-regulation of variant 4 in high-grade tumor samples, in comparison to the low-grade ones (P=0.04). Moreover, the ROC curve analysis demonstrated that the variant 4 of ROR has a potential to discriminate between tumor and non-tumor samples (AUC=0.66, P<0.05). CONCLUSION: Our data suggest a significant up-regulation of linc-ROR and its variants 2 and 4 in ESCC tissue samples.

Long non-coding RNA SOX2OT: expression signature, splicing patterns, and emerging roles in pluripotency and tumorigenesis.

SOX2 overlapping transcript (SOX2OT) is a long non-coding RNA which harbors one of the major regulators of pluripotency, SOX2 gene, in its intronic region. SOX2OT gene is mapped to human chromosome 3q26.3 (Chr3q26.3) locus and is extended in a high conserved region of over 700 kb. Little is known about the exact role of SOX2OT; however, recent studies have demonstrated a positive role for it in transcription regulation of SOX2 gene. Similar to SOX2, SOX2OT is highly expressed in embryonic stem cells and down-regulated upon the induction of differentiation. SOX2OT is dynamically regulated during the embryogenesis of vertebrates, and delimited to the brain in adult mice and human. Recently, the disregulation of SOX2OT expression and its concomitant expression with SOX2 have become highlighted in some somatic cancers including esophageal squamous cell carcinoma, lung squamous cell carcinoma, and breast cancer. Interestingly, SOX2OT is differentially spliced into multiple mRNA-like transcripts in stem and cancer cells. In this review, we are describing the structural and functional features of SOX2OT, with an emphasis on its expression signature, its splicing patterns and its critical function in the regulation of SOX2 expression during development and tumorigenesis.