Corrigendum to 'Modeling colorectal tumorigenesis using the organoids derived from conditionally immortalized mouse intestinal crypt cells (ciMICs)' [Genes Dis 8 (2021) 814-826].

[This corrects the article DOI: 10.1016/j.gendis.2021.01.004.].

Janus wireframe DNA cube-based 3D nanomachine for rapid and stable fluorescence detection of exosomal microRNA.

Exosomal microRNAs (miRNAs) have been demonstrated to be credible biomarkers for the diagnosis and monitoring of tumors. Nevertheless, developing simple, rapid, and stable biosensing strategies that are capable of accurately detecting exosomal miRNAs remains a challenge. Herein, an accelerated and biostable three-dimensional (3D) nanomachine based on Janus wireframe DNA cube was constructed for sensitive fluorescence measurement of exosomal miRNA. The Janus wireframe DNA cube could propel target exosomal miRNA-21 rapid movement on its surface by catalytic hairpin assembly (CHA), releasing a massive fluorescence signal. Benefiting from the Janus wireframe DNA cube, the developed 3D nanomachine exhibited significantly improved reaction rate and enhanced biostability in complex biofluids compared to conventional CHA. As a result, this fluorescence biosensing strategy achieved rapid, stable, and single-step detection of exosomal miRNA-21 with the detection limit down to the picomole level. Therefore, this work offers a brief sensing tool for nucleic acid biomarkers detection, which has great application potential in tumor diagnosis.

Modeling colorectal tumorigenesis using the organoids derived from conditionally immortalized mouse intestinal crypt cells (ciMICs).

Intestinal cancers are developed from intestinal epithelial stem cells (ISCs) in intestinal crypts through a multi-step process involved in genetic mutations of oncogenes and tumor suppressor genes. ISCs play a key role in maintaining the homeostasis of gut epithelium. In 2009, Sato et al established a three-dimensional culture system, which mimicked the niche microenvironment by employing the niche factors, and successfully grew crypt ISCs into organoids or Mini-guts in vitro. Since then, the intestinal organoid technology has been used to delineate cellular signaling in ISC biology. However, the cultured organoids consist of heterogeneous cell populations, and it was technically challenging to introduce genomic changes into three-dimensional organoids. Thus, there was a technical necessity to develop a two-dimensional ISC culture system for effective genomic manipulations. In this study, we established a conditionally immortalized mouse intestinal crypt (ciMIC) cell line by using a piggyBac transposon-based SV40 T antigen expression system. We showed that the ciMICs maintained long-term proliferative activity under two-dimensional niche factor-containing culture condition, retained the biological characteristics of intestinal epithelial stem cells, and could form intestinal organoids in three-dimensional culture. While in vivo cell implantation tests indicated that the ciMICs were non-tumorigenic, the ciMICs overexpressing oncogenic ß-catenin and/or KRAS exhibited high proliferative activity and developed intestinal adenoma-like pathological features in vivo. Collectively, these findings strongly suggested that the engineered ciMICs should be used as a valuable tool cell line to dissect the genetic and/or epigenetic underpinnings of intestinal tumorigenesis.

Notch signaling: Its essential roles in bone and craniofacial development.

Notch is a cell-cell signaling pathway that is involved in a host of activities including development, oncogenesis, skeletal homeostasis, and much more. More specifically, recent research has demonstrated the importance of Notch signaling in osteogenic differentiation, bone healing, and in the development of the skeleton. The craniofacial skeleton is complex and understanding its development has remained an important focus in biology. In this review we briefly summarize what recent research has revealed about Notch signaling and the current understanding of how the skeleton, skull, and face develop. We then discuss the crucial role that Notch plays in both craniofacial development and the skeletal system, and what importance it may play in the future.

Identification of a prognostic signature based on copy number variations (CNVs) and CNV-modulated gene expression in acute myeloid leukemia.

OBJECTIVES: Acute myeloid leukemia (AML) is caused by multiple genetic alterations in hematopoietic progenitors, and molecular genetic analyses have provided useful information for AML diagnosis and prognostication. This study aimed to integratively understand the prognostic value of specific copy number variation (CNV) patterns and CNV-modulated gene expression in AML. METHODS: We conducted integrative CNV profiling and gene expression analysis using data from the Therapeutically Applicable Research To Generate Effective Treatments (TARGET) and The Cancer Genome Atlas (TCGA) AML cohorts. CNV-related genes associated with survival were identified using the TARGET AML cohort and validated using the TCGA AML cohort. Genes whose CNV-modulated expression was associated with survival were also identified using the TARGET AML cohort and validated using the TCGA AML cohort, and patient bone marrow samples were then used to further validate the effects of CNV-modulated gene expression on survival. CNV and mRNA survival analyses were conducted using proportional hazards regression models (Cox regression) and the "survminer" and "survival" packages of the R Project for Statistical Computing. Genes belonging to the Kyoto Encyclopedia of Genes and Genomes (KEGG) cancer panel were extracted from KEGG cancer-related pathways. RESULTS: One hundred two CNV-related genes (located at 7q31-34, 16q24) associated with patient survival were identified using the TARGET cohort and validated with the TCGA AML cohort. Among these 102 validated genes, three miRNA genes (MIR29A, MIR183, and MIR335) were included in the KEGG cancer panel. Five genes (SEMA4D, CBFB, CHAF1B, SAE1, and DNMT1) whose expression was modulated by CNVs and significantly associated with clinical outcomes were identified, and the deletion of SEMA4D and CBFB was found to potentially exert protective effects against AML. The results of these five genes were also validated using patient marrow samples. Additionally, the distribution of CNVs affecting these five CNV-modulated genes was independent of the risk group (favorable-, intermediate-, and adverse-risk groups). CONCLUSIONS: Overall, this study identified 102 CNV-related genes associated with patient survival and identified five genes whose expression was modulated by CNVs and associated with patient survival. Our findings are crucial for the development of new modes of prognosis evaluation and targeted therapy for AML.

Stem Cell-Friendly Scaffold Biomaterials: Applications for Bone Tissue Engineering and Regenerative Medicine.

Bone is a dynamic organ with high regenerative potential and provides essential biological functions in the body, such as providing body mobility and protection of internal organs, regulating hematopoietic cell homeostasis, and serving as important mineral reservoir. Bone defects, which can be caused by trauma, cancer and bone disorders, pose formidable public health burdens. Even though autologous bone grafts, allografts, or xenografts have been used clinically, repairing large bone defects remains as a significant clinical challenge. Bone tissue engineering (BTE) emerged as a promising solution to overcome the limitations of autografts and allografts. Ideal bone tissue engineering is to induce bone regeneration through the synergistic integration of biomaterial scaffolds, bone progenitor cells, and bone-forming factors. Successful stem cell-based BTE requires a combination of abundant mesenchymal progenitors with osteogenic potential, suitable biofactors to drive osteogenic differentiation, and cell-friendly scaffold biomaterials. Thus, the crux of BTE lies within the use of cell-friendly biomaterials as scaffolds to overcome extensive bone defects. In this review, we focus on the biocompatibility and cell-friendly features of commonly used scaffold materials, including inorganic compound-based ceramics, natural polymers, synthetic polymers, decellularized extracellular matrix, and in many cases, composite scaffolds using the above existing biomaterials. It is conceivable that combinations of bioactive materials, progenitor cells, growth factors, functionalization techniques, and biomimetic scaffold designs, along with 3D bioprinting technology, will unleash a new era of complex BTE scaffolds tailored to patient-specific applications.

A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries.

RNA sequencing (RNA-seq)-based whole transcriptome analysis (WTA) using ever-evolving next-generation sequencing technologies has become a primary tool for coding and/or noncoding transcriptome profiling. As WTA requires RNA-seq data for both coding and noncoding RNAs, one key step for obtaining high-quality RNA-seq data is to remove ribosomal RNAs, which can be accomplished by using various commercial kits. Nonetheless, an ideal rRNA removal method should be efficient, user-friendly and cost-effective so it can be adapted for homemade RNA-seq library construction. Here, we developed a novel reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) method. We demonstrated that RTR2D was simple and efficient, and depleted human or mouse rRNAs with high specificity without affecting coding and noncoding transcripts. RNA-seq data analysis indicated that RTR2D yielded highly correlative transcriptome landscape with that of NEBNext rRNA Depletion Kit at both mRNA and lncRNA levels. In a proof-of-principle study, we found that RNA-seq dataset from RTR2D-depleted rRNA samples identified more differentially expressed mRNAs and lncRNAs regulated by Nutlin3A in human osteosarcoma cells than that from NEBNext rRNA Depletion samples, suggesting that RTR2D may have lower off-target depletion of non-rRNA transcripts. Collectively, our results have demonstrated that the RTR2D methodology should be a valuable tool for rRNA depletion.

Highly expressed BMP9/GDF2 in postnatal mouse liver and lungs may account for its pleiotropic effects on stem cell differentiation, angiogenesis, tumor growth and metabolism.

Bone morphogenetic protein 9 (BMP9) (or GDF2) was originally identified from fetal mouse liver cDNA libraries. Emerging evidence indicates BMP9 exerts diverse and pleiotropic functions during postnatal development and in maintaining tissue homeostasis. However, the expression landscape of BMP9 signaling during development and/or in adult tissues remains to be analyzed. Here, we conducted a comprehensive analysis of the expression landscape of BMP9 and its signaling mediators in postnatal mice. By analyzing mouse ENCODE transcriptome datasets we found Bmp9 was highly expressed in the liver and detectable in embryonic brain, adult lungs and adult placenta. We next conducted a comprehensive qPCR analysis of RNAs isolated from major mouse tissues/organs at various ages. We found that Bmp9 was highly expressed in the liver and lung tissues of young adult mice, but decreased in older mice. Interestingly, Bmp9 was only expressed at low to modest levels in developing bones. BMP9-associated TGFß/BMPR type I receptor Alk1 was highly expressed in the adult lungs. Furthermore, the feedback inhibitor Smads Smad6 and Smad7 were widely expressed in mouse postnatal tissues. However, the BMP signaling antagonist noggin was highly expressed in fat and heart in the older age groups, as well as in kidney, liver and lungs in a biphasic fashion. Thus, our findings indicate that the circulating BMP9 produced in liver and lungs may account for its pleiotropic effects on postnatal tissues/organs although possible roles of BMP9 signaling in liver and lungs remain to be fully understood.

Leptin Potentiates BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells Through the Activation of JAK/STAT Signaling.

Mesenchymal stem cells (MSCs) are multipotent progenitors that have the ability to differentiate into multiple lineages, including bone, cartilage, and fat. We previously demonstrated that the least known bone morphogenetic protein (BMP)9 (also known as growth differentiation factor 2) is one of the potent osteogenic factors that can induce both osteogenic and adipogenic differentiation of MSCs. Nonetheless, the molecular mechanism underlying BMP9 action remains to be fully understood. Leptin is an adipocyte-derived hormone in direct proportion to the amount of body fat, and exerts pleiotropic functions, such as regulating energy metabolism, bone mass, and mineral density. In this study, we investigate the potential effect of leptin signaling on BMP9-induced osteogenic differentiation of MSCs. We found that exogenous leptin potentiated BMP9-induced osteogenic differentiation of MSCs both in vitro and in vivo, while inhibiting BMP9-induced adipogenic differentiation. BMP9 was shown to induce the expression of leptin and leptin receptor in MSCs, while exogenous leptin upregulated BMP9 expression in less differentiated MSCs. Mechanistically, we demonstrated that a blockade of JAK signaling effectively blunted leptin-potentiated osteogenic differentiation induced by BMP9. Taken together, our results strongly suggest that leptin may potentiate BMP9-induced osteogenesis by cross-regulating BMP9 signaling through the JAK/STAT signaling pathway in MSCs. Thus, it is conceivable that a combined use of BMP9 and leptin may be explored as a novel approach to enhancing efficacious bone regeneration and fracture healing.

lncRNA Rmst acts as an important mediator of BMP9-induced osteogenic differentiation of mesenchymal stem cells (MSCs) by antagonizing Notch-targeting microRNAs.

Understanding the bone and musculoskeletal system is essential to maintain the health and quality of life of our aging society. Mesenchymal stem cells (MSCs) can undergo self-renewal and differentiate into multiple tissue types including bone. We demonstrated that BMP9 is the most potent osteogenic factors although molecular mechanism underlying BMP9 action is not fully understood. Long noncoding RNAs (lncRNAs) play important regulatory roles in many physiological and/or pathologic processes. Here, we investigated the role of lncRNA Rmst in BMP9-induced osteogenic differentiation of MSCs. We found that Rmst was induced by BMP9 through Smad signaling in MSCs. Rmst knockdown diminished BMP9-induced osteogenic, chondrogenic and adipogenic differentiation in vitro, and attenuated BMP9-induced ectopic bone formation. Silencing Rmst decreased the expression of Notch receptors and ligands. Bioinformatic analysis predicted Rmst could directly bind to eight Notch-targeting miRNAs, six of which were downregulated by BMP9. Silencing Rmst restored the expression of four microRNAs (miRNAs). Furthermore, an activating Notch mutant NICD1 effectively rescued the decreased ALP activity caused by Rmst silencing. Collectively, our results strongly suggest that the Rmst-miRNA-Notch regulatory axis may play an important role in mediating BMP9-induced osteogenic differentiation of MSCs.

A Novel Electrochemiluminescent Immunoassay Based on Target Transformation Assisted with Catalyzed Hairpin Assembly Amplification for the Ultrasensitive Bioassay.

In this work, we constructed a novel electrochemiluminescent (ECL) strategy based on sandwich immunoassay-induced target transformation assisted with catalyzed hairpin assembly (CHA) amplification for ultrasensitive bioassay with cysteine-rich protein 61 (CCN1) as a model. First, the target CCN1 could be equally transformed into the specific oligonucleotide (initiator I) labeled on the detection antibody based on the specific sandwich immunoassay. In addition, the initiator I triggered an efficient nonenzymatic CHA amplification in the presence of ferrocene-labeled hairpin 1 (Fc-H1) and hairpin 2 (H2) to produce massive hybrids (Fc-H1-H2) containing a sticky end labeled with ferrocene. Finally, Fc-H1-H2 could be immobilized on the capture probe single-stranded DNA (ssDNA)-modified electrode through the hybridization between the sticky end of Fc-H1-H2 and ssDNA, and a significantly quenched ECL signal could be obtained due to the efficient quench effect between ferrocene and the ECL indicator, ruthenium(II) tris(4,4'-dicarboxylicacid-2,2'-bipyridyl) [Ru(dcbpy)32+], immobilized on the surface of the electrode, which was related to the concentration of target CCN1. As expected, the proposed ECL biosensor exhibited a relatively low detection limit of 3.9 fg/mL in a linear range from 10 fg/mL to 100 ng/mL. This ECL strategy inspired the clinical examination of the biomarker CCN1, providing potential application in early diagnosis and malignant monitoring of cancer.

Polymorphisms of the CYR61 gene in patients with acute myeloid leukemia in a Han Chinese population.

It was demonstrated in previous studies that cysteine-rich angiogenic inducer 61 (Cyr61) plays vital roles in hematological disorders, and we have already reported that the Cyr61 protein is a tumor promoter in acute myeloid leukemia (AML). Here, we investigated the association between CYR61 gene polymorphisms and susceptibility to AML.We genotyped 2 single-nucleotide polymorphisms (rs2297141 and rs6576776) in the region of the CYR61 gene by improved multiplex ligase detection reaction genotyping assays in a total of 275 samples, including samples from 137 AML patients and 138 healthy controls. Chi-squared tests and logistic regression analysis were performed to compare the different distributions of the genotypes and alleles between patients and healthy controls.The rs2297141 A allele was associated with lower risk of AML compared with the G allele (odds ratio [OR] = 0.704, 95% confidence interval [CI] = 0.503-0.985, P = .04) in both the dominant (OR = 0.447, 95% CI = 0.22-0.909, P = .025, AA vs GG) and recessive inheritance models (OR = 0.419, 95% CI = 0.23-0.763, P = .004, AA vs GA + GG). Although the distribution of the rs6576776 alleles was not different between patients with AML and normal controls, the CC genotype significantly increased the risk of AML in the dominant inheritance model (OR = 6.064, 95% CI = 1.303-28.216, P = .01, CC vs GG) and the recessive inheritance model (OR = 5.937, 95% CI = 1.291-27.306, P = .01, CC vs GC + GG). Additionally, it was shown that the rs2297141 and rs6576776 genotypes were associated with AML-M5 and AML-M2, respectively.Our findings indicated that genetic polymorphisms in the CYR61 gene may be considered potential AML risk factors in the Han Chinese population.

Inhibiting CCN1 blocks AML cell growth by disrupting the MEK/ERK pathway.

BACKGROUND: CCN1 plays distinct roles in various tumor types, but little is known regarding the role of CCN1 in leukemia. METHODS: We analyzed CCN1 protein expression in leukemia cell lines and in AML bone marrow samples. We also evaluated the effects of antibody- or siRNA-mediated inhibition of CCN1 on the growth of two AML cell lines (U937 and Kasumi-1 cells) and on the MEK/ERK pathway, ß-catenin and other related genes. RESULTS: U937 and Kasumi-1 cells had markedly higher CCN1 expression than the 5 other leukemia cell lines, and CCN1 protein expression was higher in the AML bone marrow samples than in the normal bone marrow samples. Blocking CCN1 with an antibody in U937 and Kasumi-1 cells suppressed proliferation, increased apoptosis, down-regulated Bcl-xL and c-Myc expression, up-regulated Bax expression, and had no effect on Survivin. siRNA-mediated down-regulation of CCN1 inhibited the proliferation and colony formation of U937 and Kasumi-1 cells and increased cytarabine-induced apoptosis. Furthermore, CCN1 siRNA reduced MEK and ERK phosphorylation without affecting ß-catenin; the CCN1 antibody similarly affected MEK and ERK phosphorylation. These changes in phosphorylation could influence the expression of Bcl-xL, c-Myc and Bax in AML cells. CONCLUSIONS: The data suggested that CCN1 is a tumor promoter in AML that acts through the MEK/ERK pathway to up-regulate c-Myc and Bcl-xL and to down-regulate Bax.

Wnt5a enhances the response of CML cells to Imatinib Mesylate through JNK activation and γ-catenin inhibition.

Imatinib Mesylate is widely used for the treatment of chronic myelogenous leukaemia (CML), and its effects on CML cells are influenced by several signalling proteins. The research is aimed at determining whether Wnt5a affects the effects of Imatinib Mesylate against BCR-ABL positive CML cells (K562 cells and KU812 cells) and which signalling proteins are involved in. The results showed that Wnt5a augmented the effects of Imatinib Mesylate on inhibiting CML cells proliferation and inducing apoptosis in vitro; Wnt5a enhanced the inhibition effect of Imatinib Mesylate on the growth of K562 cells xenograft tumour in an animal model. Furthermore, Wnt5a inhibited ß-catenin and its target gene Survivin, increased the activity of JNK and suppressed γ-catenin expression. When inhibiting the activity of JNK, the influence of Wnt5a on the effects of Imatinib Mesylate was attenuated. Moreover, JNK suppressed ß-catenin and its target gene Survivin, and enhanced the effects of Imatinib Mesylate. These results suggest that Wnt5a can enhance the efficacy of Imatinib Mesylate through JNK/ß-catenin/Survivin and γ-catenin/ß-catenin/Survivin pathways.

Downregulation of γ-catenin inhibits CML cell growth and potentiates the response of CML cells to imatinib through ß-catenin inhibition.

γ-catenin plays different roles in different types of tumors, and its role in chronic myeloid leukemia (CML) cells has yet to be identified. In our study, two CML cell lines (K562, KU812) had higher γ-catenin expression levels compared to five types of BCR-ABL-negative leukemia cells. Knockdown of the expression of BCR-ABL resulted in downregulation of γ-catenin. Furthermore, downregulation of γ-catenin by siRNA inhibited the proliferation and colony formation of CML cells and the expression of the c-Myc and cyclin D1 genes; downregulation of γ-catenin also potentiated the effects of imatinib (inhibiting CML cell proliferation and inducing apoptosis) and suppressed the anti-apoptotic genes Bcl-xL and survivin. We also showed that downregulation of γ-catenin suppressed the phosphorylation of STAT5, promoted the phosphorylation of ß-catenin and reduced the translocation of ß-catenin into the nucleus, although there were no effects on the total level of ß-catenin expression in the whole cells. Furthermore, downregulation of γ-catenin was found to promote glycogen synthase kinase-3ß (GSK3ß) and inhibit its phosphorylation. Collectively, our results suggest that γ-catenin is an oncogene protein in CML that can be regulated by BCR-ABL and that suppression of γ-catenin inhibits CML cell growth and potentiates the effects of imatinib on CML cells through inhibition of the activation of STAT5 and suppression of ß-catenin by activating GSK3ß.