Highly sensitive and accurate detection of ALK-TKI resistance mutations by oligoribonucleotide interference-PCR (ORNi-PCR)-based methods.

BACKGROUND: Patients with anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) are treated with ALK tyrosine kinase inhibitors (TKIs). Although most patients benefit from ALK-TKIs, the development of resistance mutations is common and results in NSCLC recurrence. To identify ALK-TKI-resistant NSCLC at the early recurrent phase, highly sensitive and accurate methods for the detection of mutations are essential. OBJECTIVE: The aim of this study was to establish highly sensitive, accurate, cost-effective, and clinically practical methods for the detection of two frequent ALK-TKI resistance mutations, ALK G1202R and L1196M, by liquid biopsy. METHODS: The efficacy of oligoribonucleotide interference-PCR (ORNi-PCR) was examined by first optimizing experimental conditions to specifically amplify the ALK-TKI resistance mutant DNA corresponding to ALK G1202R and L1196M mutations. ORNi-PCR was then combined with droplet digital PCR (ddPCR) or real-time PCR to detect these mutations in cell-free DNA (cfDNA) extracted from NSCLC patients. RESULTS: ORNi-PCR followed by ddPCR/real-time PCR detected 1-10 copy(s) of G1202R and L1196M DNA in model cfDNA. These mutations in patients' cfDNA were identified using ORNi-PCR-based methods, whereas conventional ddPCR failed to detect them. CONCLUSION: ORNi-PCR followed by ddPCR/real-time PCR enables highly sensitive and accurate detection of ALK mutations by liquid biopsy. Although the clinical data are limited, our results show that these methods are potentially useful for identifying ALK-TKI-resistant NSCLC at the early recurrent phase.

Oligoribonucleotide interference-PCR-based methods for the sensitive and accurate detection of KRAS mutations.

Pancreatic cancer is an aggressive malignancy with a poor prognosis. Single-nucleotide mutations in the KRAS gene are detected in the majority of patients with pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer. Identifying KRAS mutations by liquid biopsy could be effective for detecting de novo and recurrent PDAC; however, sensitive and accurate detection remains challenging. We examined the utility of oligoribonucleotide interference-PCR (ORNi-PCR) followed by real-time PCR or droplet digital PCR (ddPCR) for detecting KRAS single-nucleotide mutations by liquid biopsy. A model of cell-free DNA was used to demonstrate that the ORNi-PCR-based methods are more sensitive and accurate for detecting KRAS mutant DNA than conventional real-time PCR or ddPCR. ORNi-PCR-based methods could be useful for early detection of de novo and recurrent PDAC by liquid biopsy for cancer diagnosis.

Real-time MBDi-RPA using methyl-CpG binding protein 2: A real-time detection method for simple and rapid estimation of CpG methylation status.

BACKGROUND: Analysis of CpG methylation is informative for cancer diagnosis. Previously, we developed a novel method to discriminate CpG methylation status in target DNA by blocking recombinase polymerase amplification (RPA), an isothermal DNA amplification technique, using methyl-CpG binding domain (MBD) protein 2 (MBD2). The method was named MBD protein interference-RPA (MBDi-RPA). In this study, MBDi-RPA was performed using methyl-CpG binding protein 2 (MeCP2), another MBD family protein, as the blocking agent. RESULTS: MBDi-RPA using MeCP2 detected low levels of CpG methylation, showing that it had higher sensitivity than MBDi-RPA using MBD2. We also developed real-time RPA, which enabled rapid analysis of DNA amplification without the need for laborious agarose gel electrophoresis and used it in combination with MBDi-RPA. We termed this method real-time MBDi-RPA. The method using MeCP2 could determine the abundance ratio of CpG-methylated target DNA simply and rapidly, although highly sensitive detection was challenging. SIGNIFICANCE AND NOVELTY: Real-time MBDi-RPA using MeCP2 could be potentially useful for estimating CpG methylation status in target DNA prior to more detailed analyses.

iChIP-SILAC analysis identifies epigenetic regulators of CpG methylation of the p16INK4A gene.

Allele-specific epigenetic events regulate the expression of specific genes such as tumor suppressor genes. Methods to biochemically identify epigenetic regulators remain limited. Here, we used insertional chromatin immunoprecipitation (iChIP) to address this issue. iChIP combined with quantitative mass spectrometry identified DNA methyltransferase 1 (DNMT1) and epigenetic regulators as proteins that potentially interact with a region of the p16INK4A gene that is CpG-methylated in one allele in HCT116 cells. Some of the identified proteins are involved in the CpG methylation of this region, and of these, DEAD-box helicase 24 (DDX24) contributes to CpG methylation by regulating the protein levels of DNMT1. Thus, iChIP is a useful method to identify proteins which bind to a target locus of interest.

GARP on hepatic stellate cells is essential for the development of liver fibrosis.

BACKGROUND & AIMS: Glycoprotein A repetitions predominant (GARP) is a membrane protein that functions as a latent TGF-ß docking molecule. While the immune regulatory properties of GARP on blood cells have been studied, the function of GARP on tissue stromal cells remains unclear. Here, we investigate the role of GARP expressed on hepatic stellate cells (HSCs) in the development of liver fibrosis. METHODS: The function of GARP on HSCs was explored in toxin-induced and metabolic liver fibrosis models, using conditional GARP-deficient mice or a newly generated inducible system for HSC-specific gene ablation. Primary mouse and human HSCs were isolated to evaluate the contribution of GARP to the activation of latent TGF-ß. Moreover, cell contraction of HSCs in the context of TGF-ß activation was tested in a GARP-dependent fashion. RESULTS: Mice lacking GARP in HSCs were protected from developing liver fibrosis. Therapeutically deleting GARP on HSCs alleviated the fibrotic process in established disease. Furthermore, natural killer T cells exacerbated hepatic fibrosis by inducing GARP expression on HSCs through IL-4 production. Mechanistically, GARP facilitated fibrogenesis by activating TGF-ß and enhancing endothelin-1-mediated HSC contraction. Functional GARP was expressed on human HSCs and significantly upregulated in the livers of patients with fibrosis. Lastly, deletion of GARP on HSCs did not augment inflammation or liver damage. CONCLUSIONS: GARP expressed on HSCs drives the development of liver fibrosis via cell contraction-mediated activation of latent TGF-ß. Considering that systemic blockade of TGF-ß has major side effects, we highlight a therapeutic niche provided by GARP and surface-mediated TGF-ß activation. Thus, our findings suggest an important role of GARP on HSCs as a promising target for the treatment of liver fibrosis. IMPACT AND IMPLICATIONS: Liver fibrosis represents a substantial and increasing public health burden globally, for which specific treatments are not available. Glycoprotein A repetitions predominant (GARP) is a membrane protein that functions as a latent TGF-ß docking molecule. Here, we show that GARP expressed on hepatic stellate cells drives the development of liver fibrosis. Our findings suggest GARP as a novel target for the treatment of fibrotic disease.

Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences.

Blocking PCR is a method that inhibits amplification of DNA possessing a nucleotide sequence complementary to that of a blocker; the method can be used to suppress amplification of target wild-type DNA while amplifying mutated DNA. Previously, we demonstrated that an oligoribonucleotide (ORN) functions as a cost-effective and sequence-specific blocker. This blocking PCR system, named ORN interference-PCR (ORNi-PCR), is compatible with DNA polymerases lacking 5'-3' exonuclease activity but not with those possessing the activity (e.g., Taq DNA polymerase), which can remove a hybridized ORN during DNA extension. Here, we demonstrate that under specific experimental conditions, an intact or phosphorothioated ORN strongly suppresses extension of target DNA by Taq DNA polymerases. This method was applied successfully to real-time ORNi-PCR and one-step real-time reverse transcription-ORNi-PCR using a dual-labeled fluorescent probe to detect a single-nucleotide mutation in DNA and RNA in a sequence-specific manner. The results reaffirm the utility of blocking PCR and provide technical hints for its improvement.

Development of transgenic mice overexpressing mouse carbonyl reductase 1.

BACKGROUND: Carbonyl reductase 1 (CBR1) is a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase with broad substrate specificity. CBR1 catalyzes the reduction of numerous carbonyl compounds, including quinones, prostaglandins, menadione, and multiple xenobiotics, while also participating in various cellular processes, such as carcinogenesis, apoptosis, signal transduction, and drug resistance. In this study, we aimed to generate transgenic mice overexpressing mouse Cbr1 (mCbr1), characterize the mCbr1 expression in different organs, and identify changes in protein expression patterns. METHODS AND RESULTS: To facilitate a deeper understanding of the functions of CBR1, we generated transgenic mice overexpressing CBR1 throughout the body. These transgenic mice overexpress 3xFLAG-tagged mCbr1 (3xFLAG-mCbr1) under the CAG promoter. Two lines of transgenic mice were generated, one with 3xFLAG-mCbr1 expression in multiple tissues, and the other, with specific expression of 3xFLAG-mCbr1 in the heart. Pathway and network analysis using transgenic mouse hearts identified 73 proteins with levels of expression correlating with mCbr1 overexpression. The expression of voltage-gated anion channels, which may be directly related to calcium ion-related myocardial contraction, was also upregulated. CONCLUSION: mCbr1 transgenic mice may be useful for further in vivo analyses of the molecular mechanisms regulated by Cbr1; such analyses will provide a better understanding of its effects on carcinogenesis and cardiotoxicity of certain cancer drugs.

An enChIP system for the analysis of genome functions in budding yeast.

The identification of molecules associated with a specific genomic region is essential for elucidating the molecular mechanisms underlying genome functions such as transcription. Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) is a technology that enables the purification of specific genomic regions and the subsequent identification of their associated molecules. In enChIP, the target genomic region is tagged with engineered DNA-binding molecules, such as variants of the clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a catalytically inactive form of Cas9 (dCas9) and a guide RNA. This article describes the generation of a plasmid expressing Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-Sp-dCas9) and its successful expression in the budding yeast, Saccharomyces cerevisiae. Furthermore, we showed that this plasmid can be used for enChIP analysis in budding yeast. In addition, the plasmid may also be a useful tool for researchers analyzing genome functions such as transcription and for CRISPR interference experiments in budding yeasts.

Circular RNAs hsa_circ_0001438 and hsa_circ_0000417 are downregulated and upregulated, respectively, in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most predominant type of liver cancer and is frequently fatal. Alpha-fetoprotein, alpha-fetoprotein-L3, and protein induced by vitamin K absence or antagonist-II are used as biomarkers to diagnose HCC. However, these biomarkers are not highly specific, especially for early-stage HCC diagnosis; therefore, more specific biomarkers are needed. Recently, circular RNA (circRNA) biomarkers have been used to diagnose several intractable diseases. In this study, we sought to identify circRNA biomarkers for the specific diagnosis of HCC. To this end, we compared the expression levels of circRNAs in primary HCC and normal tissues using publicly available RNA-seq data. Our analysis revealed that the expression levels of eight circRNAs were altered in primary HCC tissues compared with normal tissues. To confirm our findings, we examined the expression levels of selected circRNAs in HCC cell lines and normal hepatocytes. The expression level of hsa_circ_0001438, a circRNA that was downregulated in primary HCC, was lower in poorly and well-differentiated HCC cell lines than in normal hepatocytes. By contrast, the expression level of hsa_circ_0000417, which was increased in primary HCC, was strongly upregulated in a well-differentiated HCC cell line compared with normal hepatocytes. Thus, hsa_circ_0001438 and hsa_circ_0000417 might be potential biomarkers for the specific diagnosis of HCC. The experimental strategy described here, using publicly available RNA-seq data, is a useful and cost-effective method of identifying circRNA biomarkers.

Oligoribonucleotide interference-PCR: principles and applications.

Polymerase chain reaction (PCR) amplification of multiple templates using common primers is used widely for molecular biological research and clinical diagnosis. However, amplifying a specific DNA sequence harboring a mutation that is present in a small number of mutant cells within a large population of normal cells (e.g., as in cancer) in a tissue is difficult using the original PCR protocol. Thus, some measures are necessary to suppress amplification of background signals. To achieve this, we developed the oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) technology in which an ORN (short RNA) hybridizes with a complementary DNA sequence to inhibit PCR amplification across the specific target sequence. ORNs can be prepared inexpensively, and ORNi-PCR can be carried out easily by adding ORNs to the PCR reaction mixture. Suppressing amplification of target sequences by ORNi-PCR is useful for detecting target sequence mutations. We showed that ORNi-PCR can discriminate single-nucleotide mutations in cancer cells and indel mutations introduced by genome editing. We also showed that ORNi-PCR can identify the CpG methylation status of a target sequence within bisulfite-treated DNA, and can enrich DNA sequences of interest from a DNA mixture by suppressing amplification of unwanted sequences. Thus, ORNi-PCR has many potential applications in various fields, including medical diagnosis and molecular biology. In this review, we outline the principles of the ORNi-PCR method and its use to detect nucleotide mutations in a variety of specimens.

IL-3-Induced Immediate Expression of c-fos and c-jun Is Modulated by the IKK2-JNK Axis.

Interleukin (IL)-3 is a pleiotropic cytokine that regulates the survival, proliferation, and differentiation of hematopoietic cells. The binding of IL-3 to its receptor activates intracellular signaling, inducing transcription of immediate early genes (IEGs) such as c-fos, c-jun, and c-myc; however, transcriptional regulation under IL-3 signaling is not fully understood. This study assessed the role of the inhibitor of nuclear factor-κB kinases (IKKs) in inducing IL-3-mediated expression of IEGs. We show that IKK1 and IKK2 are required for the IL-3-induced immediate expression of c-fos and c-jun in murine hematopoietic Ba/F3 cells. Although IKK2 is well-known for its pivotal role as a regulator of the canonical nuclear factor-κB (NF-κB) pathway, activation of IKKs did not induce the nuclear translocation of the NF-κB transcription factor. We further revealed the important role of IKK2 in the activation of c-Jun N-terminal kinase (JNK), which mediates the IL-3-induced expression of c-fos and c-jun. These findings indicate that the IKK2-JNK axis modulates the IL-3-induced expression of IEGs in a canonical NF-κB-independent manner.

enChIP-Seq Analyzer: A Software Program to Analyze and Interpret enChIP-Seq Data for the Detection of Physical Interactions between Genomic Regions.

Accumulating evidence suggests that the physical interactions between genomic regions play critical roles in the regulation of genome functions, such as transcription and epigenetic regulation. Various methods to detect the physical interactions between genomic regions have been developed. We recently developed a method to search for genomic regions interacting with a locus of interest in a non-biased manner that combines pull-down of the locus using engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) and next-generation sequencing (NGS) analysis (enChIP-Seq). The clustered regularly interspaced short palindromic repeats (CRISPR) system, consisting of a nuclease-dead form of Cas9 (dCas9) and a guide RNA (gRNA), or transcription activator-like (TAL) proteins, can be used for enChIP. In enChIP-Seq, it is necessary to compare multiple datasets of enChIP-Seq data to unambiguously detect specific interactions. However, it is not always easy to analyze enChIP-Seq datasets to subtract non-specific interactions or identify common interactions. To facilitate such analysis, we developed the enChIP-Seq analyzer software. It enables easy extraction of common signals as well as subtraction of non-specific signals observed in negative control samples, thereby streamlining extraction of specific enChIP-Seq signals. enChIP-Seq analyzer will help users analyze enChIP-Seq data and identify physical interactions between genomic regions.

A stem cell marker KLF5 regulates CCAT1 via three-dimensional genome structure in colorectal cancer cells.

BACKGROUND: KLF5 plays a crucial role in stem cells of colorectum in cooperation with Lgr5 gene. In this study, we aimed to explicate a regulatory mechanism of the KLF5 gene product from a view of three-dimensional genome structure in colorectal cancer (CRC). METHODS: In vitro engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)-seq method was used to identify the regions that bind to the KLF5 promoter. RESULTS: We revealed that the KLF5 promoter region interacted with the KLF5 enhancer region as well as the transcription start site (TSS) region of the Colon Cancer Associated Transcript 1 (CCAT1) gene. Notably, the heterodeletion mutants of KLF5 enhancer impaired the cancer stem-like properties of CRC cells. The KLF5 protein participated in the core-regulatory circuitry together with co-factors (BRD4, MED1, and RAD21), which constructs the three-dimensional genome structures consisting of KLF5 promoter, enhancer and CCAT1 TSS region. In vitro analysis indicated that KLF5 regulated CCAT1 expression and we found that CCAT1 expression was highly correlated with KLF5 expression in CRC clinical samples. CONCLUSIONS: Our data propose the mechanistic insight that the KLF5 protein constructs the core-regulatory circuitry with co-factors in the three-dimensional genome structure and coordinately regulates KLF5 and CCAT1 expression in CRC.

pSIR-bsr, a self-inactivating retrovirus vector expressing the blasticidin S-resistance gene.

Self-inactivating retrovirus vectors are useful tools for generating stable cell lines harbouring designed exogenous sequences but lacking the constitutive transcriptional activity of the long terminal repeats that are usually retained by non-self-inactivating retrovirus vectors. Thus, self-inactivating retrovirus vectors are ideal vehicles for integrated transgenes comprising transcriptional regulatory sequences, and for the genes expressed by these regulatory sequences. This article describes the development of a self-inactivating retrovirus vector retaining a blasticidin S-resistance (bsr) gene. The vector, named pSIR-bsr, would be useful for transducing multiple expression vectors with different selection markers.

MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis.

Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) is a technology for purifying specific genomic regions to facilitate identification of their associated molecules, including proteins, RNAs, and other genomic regions. In enChIP, the target genomic region is tagged with engineered DNA-binding molecules, for example, a variant of the clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). In this study, to increase the flexibility of enChIP and expand the range of target cells, we generated murine stem cell virus (MSCV)-based retroviral plasmids for expressing dCas9. We constructed MSCV-based retroviral plasmids expressing Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-Sp-dCas9) and various drug resistance genes. We showed that by using these plasmids, it is feasible to purify target genomic regions with yields comparable to those reported using other systems. These systems might give enChIP users greater flexibility in choosing optimal systems for drug selection of transduced cells.

Protein or ribonucleoprotein-mediated blocking of recombinase polymerase amplification enables the discrimination of nucleotide and epigenetic differences between cell populations.

Isothermal DNA amplification, such as recombinase polymerase amplification (RPA), is well suited for point-of-care testing (POCT) as it does not require lengthy thermal cycling. By exploiting DNA amplification at low temperatures that do not denature heat-sensitive molecules such as proteins, we have developed a blocking RPA method to detect gene mutations and examine the epigenetic status of DNA. We found that both nucleic acid blockers and nuclease-dead clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoproteins suppress RPA reactions by blocking elongation by DNA polymerases in a sequence-specific manner. By examining these suppression events, we are able to discriminate single-nucleotide mutations in cancer cells and evaluate genome-editing events. Methyl-CpG binding proteins similarly inhibit elongation by DNA polymerases on CpG-methylated template DNA in our RPA reactions, allowing for the detection of methylated CpG islands. Thus, the use of heat-sensitive molecules such as proteins and ribonucleoprotein complexes as blockers in low-temperature isothermal DNA amplification reactions markedly expands the utility and application of these methods.

Sequence-specific inhibition of reverse transcription by recombinant CRISPR/dCas13a ribonucleoprotein complexes in vitro.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for genome editing because of its ability to cleave specific DNA sequences. Recently, RNA-specific CRISPR systems have been reported. CRISPR systems, consisting of a guide RNA (gRNA) and a nuclease-dead form of Cas13a (dCas13a), can be used for RNA editing and visualization of target RNA. In this study, we examined whether a recombinant CRISPR/dCas13a ribonucleoprotein (RNP) complex could be used to inhibit reverse transcription (RT) in a sequence-specific manner in vitro. Recombinant Leptotrichia wadei dCas13a was expressed using the silkworm-baculovirus expression system and affinity-purified. We found that the CRISPR/dCas13a RNP complex, combined with a chemically synthesized gRNA sequence, could specifically inhibit RT of EGFR and NEAT1, but not nonspecific RNA. Thus, the CRISPR/dCas13a RNP complex can inhibit RT reactions in a sequence-specific manner. RT inhibition by the CRISPR/dCas13a system may be useful to assess target binding activity, to discriminate RNA species retaining target sequences of gRNA, or to suppress RT from undesirable RNA species.

Locus-Specific Genomic DNA Purification Using the CRISPR System: Methods and Applications.

A multitude of molecular interactions with chromatin governs various chromosomal functions in cells. Insights into the molecular compositions at specific genomic regions are pivotal to deepen our understanding of regulatory mechanisms and the pathogenesis of disorders caused by the abnormal regulation of genes. The locus-specific purification of genomic DNA using the clustered regularly interspaced short palindromic repeats (CRISPR) system enables the isolation of target genomic regions for identification of bound interacting molecules. This CRISPR-based DNA purification method has many applications. In this study, we present an overview of the CRISPR-based DNA purification methodologies as well as recent applications.

Discrimination of CpG Methylation Status and Nucleotide Differences in Tissue Specimen DNA by Oligoribonucleotide Interference-PCR.

Oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) is a method in which PCR amplification of a target sequence is inhibited in a sequence-specific manner by the hybridization of an ORN with the target sequence. Previously, we reported that ORNi-PCR could detect nucleotide mutations in DNA purified from cultured cancer cell lines or genome-edited cells. In this study, we investigated whether ORNi-PCR can discriminate nucleotide differences and CpG methylation status in damaged DNA, such as tissue specimen DNA and bisulfite-treated DNA. First, we showed that ORNi-PCR could discriminate nucleotide differences in DNA extracted from acetone-fixed paraffin-embedded rat liver specimens or formalin-fixed paraffin-embedded human specimens. Rat whole blood specimens were compatible with ORNi-PCR for the same purpose. Next, we showed that ORNi-PCR could discriminate CpG methylation status in bisulfite-treated DNA. These results demonstrate that ORNi-PCR can discriminate nucleotide differences and CpG methylation status in multiple types of DNA samples. Thus, ORNi-PCR is potentially useful in a wide range of fields, including molecular biology and medical diagnosis.