Virus Integration Site Research Articles

Dynamic mRNA Expression Analysis of the Secondary Palatal Morphogenesis in Miniature Pigs.

Normal mammalian palatogenesis is a complex process that requires the occurrence of a tightly regulated series of specific and sequentially regulated cellular events. Cleft lip/palate (CLP), the most frequent craniofacial malformation birth defects, may occur if any of these events undergo abnormal interference. Such defects not only affect the patients, but also pose a financial risk for the families. In our recent study, the miniature pig was shown to be a valuable alternative large animal model for exploring human palate development by histology. However, few reports exist in the literature to document gene expression and function during swine palatogenesis. To better understand the genetic regulation of palate development, an mRNA expression profiling analysis was performed on miniature pigs, Sus scrofa. Five key developmental stages of miniature pigs from embryonic days (E) 30–50 were selected for transcriptome sequencing. Gene expression profiles in different palate development stages of miniature pigs were identified. Nine hundred twenty significant differentially expressed genes were identified, and the functional characteristics of these genes were determined by gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Some of these genes were associated with HH (hedgehog), WNT (wingless-type mouse mammary tumor virus integration site family), and MAPK (mitogen-activated protein kinase) signaling, etc., which were shown in the literature to affect palate development, while some genes, such as HIP (hedgehog interacting protein), WNT16, MAPK10, and LAMC2 (laminin subunit gamma 2), were additions to the current understanding of palate development. The present study provided a comprehensive analysis for understanding the dynamic gene regulation during palate development and provided potential ideas and resources to further study normal palate development and the etiology of cleft palate.

HIV-1 DNA-capture-seq is a useful tool for the comprehensive characterization of HIV-1 provirus

Regardless of recent advances in the development of anti-retroviral drugs, it is still extremely difficult to eradicate HIV-1 from infected individuals. The characterization of the HIV-1 provirus, a type of viral reservoir, with a high resolution is key to HIV-1 cure research. Here, we demonstrate that DNA-capture-seq is a powerful tool to obtain comprehensive information on the HIV-1 provirus. We use biotinylated DNA probes targeting the entire HIV-1 sequence to capture fragments containing HIV-1 sequences from DNA-seq libraries prepared for high throughput sequencing. We demonstrate that the protocol provided the entire proviral sequence from the beginning of the 5′ LTR to the end of the 3′ LTR. Since HIV-1 DNA-probes can hybridize not only viral fragments but also virus-host chimeric ones, the viral integration site information can also be obtained. We verify the efficiency of the protocol by using latently infected cell lines, such as ACH-2 and J1.1, and newly generated ones. The results reveal that the 2 new clones that we analyse harbour one copy of replication-competent provirus, suggesting that latency is not caused by genetic mutations or deletions of the provirus. In conclusion, HIV-1 DNA-capture-seq is a powerful tool to characterize the HIV-1 provirus at a single nucleotide resolution and therefore might be useful for various experiments aiming for an HIV-1 cure.

Downregulation of hsa_circ_0007534 restricts the proliferation and invasion of cervical cancer through regulating miR-498/BMI-1 signaling

AimsHsa_circ_0007534 has been reported to be a novel cancer-related circRNA affecting multiple cancers. However, little is known about the role of hsa_circ_0007534 in cervical cancer specifically. In the current study, we aimed to explore the expression and function of hsa_circ_0007534 in cervical cancer. Main methodsThe expression of circRNA, miRNA and mRNA was measured using real-time quantitative polymerase chain reactions. Protein expression was assessed by Western blot. Knockdown of has_circ_0007534 was achieved by siRNA-mediated gene silencing. Cell proliferation was determined using a cell counting kit-8 and colony formation assays. Cell invasion was assessed using a Transwell invasion assay. RNA interactions were measured using an RNA pull-down and dual-luciferase reporter assays. Key findingsThe expression of hsa_circ_0007534 was upregulated in cervical cancer tissues and cell lines. Depletion of hsa_circ_0007534 decreased both the proliferation and invasion of cervical cancer cells. MicroRNA-498 (miR-498) was identified as a target of the miRNA encoded by hsa_circ_0007534. Levels of miR-498 were decreased in cervical cancer tissues, a finding that was inversely correlated with hsa_circ_0007534 expression. miR-498 overexpression repressed the proliferation and invasion of cervical cancer cells. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) was verified as a target gene of miR-498. BMI-1 overexpression reversed the effects of hsa_circ_0007534 depletion or miR-498 overexpression on cervical cancer cell proliferation and invasion. SignificanceOur study demonstrates that downregulation of hsa_circ_0007534 represses the proliferation and invasion of cervical cancer through regulating the miR-498/BMI-1 axis, suggesting the hsa_circ_0007534/miR-498/BMI-1 axis as a potential therapeutic target to treat cervical cancer.

Chronic iron exposure and c-Myc/H-ras-mediated transformation in fallopian tube cells alter the expression of EVI1, amplified at 3q26.2 in ovarian cancer

Mechanisms underlying the pathogenesis of high-grade serous epithelial ovarian cancers (HGSOC) are not yet well defined although key precursor cells have been identified (including fimbriated fallopian tube epithelium, FTSECs). Since iron is elevated in endometriotic cysts and the pelvic cavity, it is suggested that this source of redox-active iron may contribute to ovarian cancer pathogenesis. Specifically, sources of nontransferrin-bound iron (NTBI) within the pelvic cavity could arise from ovulation, retrograde menstruation, follicular fluid, or iron overload conditions (i.e., hemochromatosis). Herein, we investigated the cellular response of p53-inactivated and telomerase-expressing (immortalized) FTSECs (Pax8+/FoxJ1−) to NTBI (presented as ferric ammonium citrate (FAC), supplemented in media for >2 months) in order to assess its ability to promote the transition to a tumor-like phenotype; this cellular response was compared with immortalized FTSECs transformed with H-RasV12A and c-MycT58A. Both approaches resulted in increased cell numbers and expression of the oncogenic transcriptional regulator, ecotropic virus integration site 1 (EVI1, a gene most frequently amplified at 3q26.2 in HGSOC, represented by multiple variants), along with other oncogenic gene products. In contrast to the transformed cells, FAC-exposed FTSECs elicited elevated migratory capacity (and epithelial–mesenchymal transition mRNA profile) along with increased expression of DNA damage response proteins (i.e., FANCD2) and hTERT mRNA relative to controls. Interestingly, in FAC-exposed FTSECs, EVI1 siRNA attenuated hTERT mRNA expression, whereas siRNAs targeting β-catenin and BMI1 (both elevated with chronic iron exposure) reduced Myc and Cyclin D1 proteins. Collectively, our novel findings provide strong foundational evidence for potential iron-induced initiation events, including EVI1 alterations, in the pathogenesis of HGSOC, warranting further in depth investigations. Thus, these findings will substantially advance our understanding of the contribution of iron enriched within the pelvic cavity, which may identify patients at risk of developing this deadly disease.

Targeted Integration and High-Level Transgene Expression in AAVS1 Transgenic Mice after In Vivo HSC Transduction with HDAd5/35++ Vectors

Our goal is the development of invivo hematopoietic stem cell (HSC) transduction technology with targeted integration. To achieve this, we modified helper-dependent HDAd5/35++ vectors to express a CRISPR/Cas9 specific to the "safe harbor" adeno-associated virus integration site 1 (AAVS1) locus and to provide a donor template for targeted integration through homology-dependent repair. We tested the HDAd-CRISPR+ HDAd-donor vector system in AAVS1 transgenic mice using a standard exvivo HSC gene therapy approach as well as a new invivo HSC transduction approach that involves HSC mobilization and intravenous HDAd5/35++ injections. In both settings, the majority of treated mice had transgenes (GFP or human γ-globin) integrated into the AAVS1 locus. On average, >60% of peripheral blood cells expressed the transgene after invivo selection with low-dose O6BG/bis-chloroethylnitrosourea (BCNU). Exvivo and invivo HSC transduction and selection studies with HDAd-CRISPR+ HDAd-globin-donor resulted in stable γ-globin expression at levels that were significantly higher (>20% γ-globin of adult mouse globin) than those achieved in previous studies with a SB100x-transposase-based HDAd5/35++ system that mediates random integration. The ability to achieve therapeutically relevant transgene expression levels after invivo HSC transduction and selection and targeted integration make our HDAd5/35++-based vector system a new tool in HSC gene therapy.

Low-density lipoprotein receptor-deficient hepatocytes differentiated from induced pluripotent stem cells allow familial hypercholesterolemia modeling, CRISPR/Cas-mediated genetic correction, and productive hepatitis C virus infection

BackgroundFamilial hypercholesterolemia type IIA (FH) is due to mutations in the low-density lipoprotein receptor (LDLR) resulting in elevated levels of low-density lipoprotein cholesterol (LDL-c) in plasma and in premature cardiovascular diseases. As hepatocytes are the only cells capable of metabolizing cholesterol, they are therefore the target cells for cell/gene therapy approaches in the treatment of lipid metabolism disorders. Furthermore, the LDLR has been reported to be involved in hepatitis C virus (HCV) entry into hepatocytes; however, its role in the virus infection cycle is still disputed.MethodsWe generated induced pluripotent stem cells (iPSCs) from a homozygous LDLR-null FH-patient (FH-iPSCs). We constructed a correction cassette bearing LDLR cDNA under the control of human hepatic apolipoprotein A2 promoter that targets the adeno-associated virus integration site AAVS1. We differentiated both FH-iPSCs and corrected FH-iPSCs (corr-FH-iPSCs) into hepatocytes to study statin-mediated regulation of genes involved in cholesterol metabolism. Upon HCV particle inoculation, viral replication and production were quantified in these cells.ResultsWe showed that FH-iPSCs displayed the disease phenotype. Using homologous recombination mediated by the CRISPR/Cas9 system, FH-iPSCs were genetically corrected by the targeted integration of a correction cassette at the AAVS1 locus. Both FH-iPSCs and corr-FH-iPSCs were then differentiated into functional polarized hepatocytes using a stepwise differentiation approach (FH-iHeps and corr-FH-iHeps). The correct insertion and expression of the correction cassette resulted in restoration of LDLR expression and function (LDL-c uptake) in corr-FH-iHeps. We next demonstrated that pravastatin treatment increased the expression of genes involved in cholesterol metabolism in both cell models. Moreover, LDLR expression and function were also enhanced in corr-FH-iHeps after pravastatin treatment. Finally, we demonstrated that both FH-iHeps and corr-FH-iHeps were as permissive to viral infection as primary human hepatocytes but that virus production in FH-iHeps was significantly decreased compared to corr-FH-iHeps, suggesting a role of the LDLR in HCV morphogenesis.ConclusionsOur work provides the first LDLR-null FH cell model and its corrected counterpart to study the regulation of cholesterol metabolism and host determinants of HCV life cycle, and a platform to screen drugs for treating dyslipidemia and HCV infection.

Serum sclerostin levels are positively related to bone mineral density in peritoneal dialysis patients: a cross-sectional study

BackgroundSclerostin, an antagonist of the Wingless-type mouse mammary tumor virus integration site (Wnt) pathway that regulates bone metabolism, is a potential contributor of chronic kidney disease (CKD)–mineral and bone disorder (MBD), which has various forms of presentation, from osteoporosis to vascular calcification. The positive association of sclerostin with bone mineral density (BMD) has been demonstrated in CKD and hemodialysis (HD) patients but not in peritoneal dialysis (PD) patients. This study assessed the association between sclerostin and BMD in PD patients.MethodsEighty-nine PD patients were enrolled; their sera were collected for measurement of sclerostin and other CKD–MBD-related markers. BMD was also assessed simultaneously. We examined the relationship between sclerostin and each parameter through Spearman correlation analysis and by comparing group data between patients with above- and below-median sclerostin levels. Univariate and multiple logistic regression models were employed to define the most predictive of sclerostin levels in the above-median category.ResultsBivariate analysis revealed that sclerostin was correlated with spine BMD (r = 0.271, P = 0.011), spine BMD T-score (r = 0.274, P = 0.010), spine BMD Z-score (r = 0.237, P = 0.027), and intact parathyroid hormone (PTH; r = − 0.357, P < 0.001) after adjustments for age and sex. High BMD, old age, male sex, increased weight and height, diabetes, and high osteocalcin and uric acid levels were observed in patients with high serum sclerostin levels and an inverse relation was noticed between PTH and sclerostin. Univariate logistic regression analysis demonstrated that BMD is positively correlated with above-median sclerostin levels (odds ratio [OR] = 65.61, P = 0.002); the correlation was retained even after multivariate adjustment (OR = 121.5, P = 0.007).ConclusionsFor the first time, this study demonstrated a positive association between serum sclerostin levels and BMD in the PD population.

A Review of the Hereditary Component of Triple Negative Breast Cancer: High- and Moderate-Penetrance Breast Cancer Genes, Low-Penetrance Loci, and the Role of Nontraditional Genetic Elements.

Triple negative breast cancer (TNBC), representing 10-15% of breast tumors diagnosed each year, is a clinically defined subtype of breast cancer associated with poor prognosis. The higher incidence of TNBC in certain populations such as young women and/or women of African ancestry and a unique pathological phenotype shared between TNBC and BRCA1-deficient tumors suggest that TNBC may be inherited through germline mutations. In this article, we describe genes and genetic elements, beyond BRCA1 and BRCA2, which have been associated with increased risk of TNBC. Multigene panel testing has identified high- and moderate-penetrance cancer predisposition genes associated with increased risk for TNBC. Development of large-scale genome-wide SNP assays coupled with genome-wide association studies (GWAS) has led to the discovery of low-penetrance TNBC-associated loci. Next-generation sequencing has identified variants in noncoding RNAs, viral integration sites, and genes in underexplored regions of the human genome that may contribute to the genetic underpinnings of TNBC. Advances in our understanding of the genetics of TNBC are driving improvements in risk assessment and patient management.

Use of syngeneic cells expressing membrane-bound GM-CSF as an adjuvant to induce antibodies against native multi-pass transmembrane protein

Membrane antigens (mAgs) are important targets for the development of antibody (Ab) drugs. However, native mAgs are not easily prepared, causing difficulties in acquiring functional Abs. In this study, we present a platform in which human mAgs were expressed in native form on cell adjuvants made with membrane-bound cytokines that were then used immunize syngeneic mice directly. The membrane-bound cytokines were used as immune stimulators to enhance specific Ab responses against the desired mAgs. Then, mAgs-expressing xenogeneic cells were used for Ab characterization to reduce non-specific binding. We established cell adjuvants by expressing membrane-bound cytokines (mIL-2, mIL-18, or mGM-CSF) on BALB/3T3 cells, which were effective in stimulating splenocyte proliferation in vitro. We then transiently expressed ecotropic viral integration site 2B (EVI2B) on the adjuvants and used them to directly immunize BALB/c mice. We found that 3T3/mGM-CSF cells stimulated higher specific anti-EVI2B Ab response in the immunized mice than the other cell adjuvants. A G-protein coupled receptor (GPCR), CXCR2, was then transiently expressed on 3T3/mGM-CSF cell adjuvant to immunize mice. The immune serum exhibited relatively higher binding to xenogeneic 293 A/CXCR2 cells than 293 A cells (~3.5-fold). Several hybridoma clones also exhibited selective binding to 293 A/CXCR2 cells. Therefore, the cell adjuvant could preserve the native conformation of mAgs and exhibit anti-mAg Ab stimulatory ability, providing a more convenient and effective method to generate functional Abs, thus possibly accelerating Ab drug development.

Increased expression of ecotropic viral integration site 2A indicates a poor prognosis and promotes osteosarcoma evolution through activating MEK/ERK pathway

Although ecotropic viral integration site 2 A (EVI2A) plays key roles in several cancers, the expression and function of EVI2A in osteosarcoma (OS) have not been investigated. Hence, we explored the expression of EVI2A and its clinical significance of EVI2A of OS. Firstly, we investigated the expression of EVI2A in OS tissues. The relationship of EVI2A expression and survival time was analyzed using Kaplan–Meier plotter. Then, we used quantitative reverse transcription PCR (qRT-PCR) to confirm the expression level of EVI2A in OS cell lines. Cell proliferation, and wound-healing experiments were used to identify the biological function of EVI2A. Moreover, EVI2A-mediated MEK/ERK signaling pathway was evaluated using western blotting. Data suggested that EVI2A was highly expressed in OS tissues, and high-expression of EVI2A was associated with worse overall survival in OS patients. Moreover, the up-regulation of it was observed in OS cell lines (Saos2, and MG63). Knockdown of EVI2A suppressed cell proliferation and migration of OS. Western blotting revealed that the inactivation of MEK/ERK pathway was found in OS cells after EVI2A knockdown. Our data implicated the crucial role of EVI2A in the progression of OS, demonstrating that expression of EVI2A may offer an attractive novel prognostic signature for OS.

Abstract 2611: Activated pentose phosphate pathway mediated by FBP1 upregulation supports progression of acute myeloid leukemia with high EVI1 expression

Abstract EVI1 (ecotropic viral integration site 1) is a transcription factor essential for the development and progression of acute myeloid leukemia (AML). Since high EVI1 expression, seen in approximately 10% of AML patients (EVI1high AML), is associated with poor clinical outcome, development of a novel therapeutic strategy is required. In this study, we investigated key molecules that are induced by aberrant EVI1 expression and modulate the metabolomics of leukemia cells. We conducted whole transcriptome analysis by RNA-sequencing in murine leukemia cells induced by retroviral transduction with EVI1 followed by transplantation into irradiated mice. We identified 45 genes with more than 10-fold expression in EVI1-transduced bone marrow cells compared to those with the mock vector at both pre-leukemic phase and leukemia phase. Intriguingly, the fructose bisphosphatase 1 gene (Fbp1), which encodes a key enzyme of gluconeogenesis was highly upregulated in EVI1-induced AML cells. We confirmed that FBP1 mRNA expression is quickly upregulated by ectopic expression of EVI1 in murine LSK (Linneg, c-Kitpos, Sca-1pos) cells. Moreover, we observed an enrichment of EVI1 in the promoter and enhancer region of Fbp1 by chromatin immunoprecipitation followed by qPCR analysis in murine hematopoietic cells, suggesting that Fbp1 expression is directly regulated by EVI1. Although high FBP1 expression suppresses glycolytic metabolism and negatively affects cellular proliferation in various types of solid tumor, its role in AML has not been investigated. In contrast to the results previously reported in other types of cancers, pharmacologic inhibition or short hairpin RNA (shRNA)-mediated knockdown of FBP1 significantly decreased colony-forming cell (CFC) capacity in EVI1-transduced murine LSK cells and significantly delayed progression of leukemia in the mice secondarily transplanted with EVI1-induced AML cells in vivo. These results suggest that aberrant expression of FBP1 is important for progression of EVI1high AML. While high FBP1 enzymatic activity negatively affects the glycolytic pathway, it in turn enhances the pentose phosphate pathway (PPP) through elevating the fructose-6-phosphate level. Activated PPP results in enhanced nucleotide synthesis essential for rapidly dividing cells. Importantly, individual knockdown of main PPP enzymes (G6pd, Pgd, Rpia) by shRNA transduction significantly decreased the CFC capacity of EVI1 transduced murine LSK cells, further confirming an important role of PPP in driving proliferation of EVI1high leukemia cells. Collectively, these results indicate that the activated PPP through transcriptional upregulation of FBP1 is crucial for progression of EVI1-driven leukemia. Since inhibition of FBP-1 did not compromise normal hematopoiesis, targeting the enzyme can be a promising therapeutic approach for the EVI1high AML. Citation Format: Hideaki Mizuno, Yuki Kagoya, Akira Chiba, Junji Koya, Yosuke Masamoto, Mineo Kurokawa. Activated pentose phosphate pathway mediated by FBP1 upregulation supports progression of acute myeloid leukemia with high EVI1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2611.

Abstract 3727: An orthotopic xenograft model for studying reirradiation and glioblastoma evolution

Abstract Glioblastoma (GBM), the most common and malignant primary adult brain cancer, has a median survival of 15 months despite multimodal treatment involving surgical resection and chemo-radiotherapy. Ongoing clinical trials seek to examine the safety and benefit of retreatment with radiation therapy for recurrent glioblastoma. By adding a reirradiation protocol to a previously described glioma stem-like cell (GSC) initiated orthotopic xenograft model, this study seeks to better understand the impact of radiotherapy on both primary and recurrent GBM evolution and to establish an in vivo model for studying reirradiation. Therefore, we intracranially implanted CD133+ NSC11 cells, and other GSC lines, into nude mice. After 21 days, bioluminescence imaging was performed to confirm the presence of tumor prior to randomization into control and radiation therapy groups (3x5Gy). After treatment tumors were imaged weekly to track changes in BLI ratios. Once the average BLI ratio for the treated mice was found to be between 1 and 10, the mice were rerandomized into control (3x5Gy-Control) and radiation therapy groups (3x5Gy-3x5Gy). Following treatment, brain samples were collected at various time points out to morbidity to investigate changes in tumor morphology and histology. Further, tumors from morbid mice were collected for viral integration site analysis (VISA), whole-exome sequencing (WES), and NanoString gene expression analysis. Survival analysis demonstrated a significant survival advantage for mice undergoing radiation therapy (+34.2 days) compared to controls. A further survival advantage was found for mice undergoing reirradiation (+30.0 days) compared to mice receiving only one course of radiation. On gross examination of morphology and H&amp;E/SOX2 staining, brains bearing irradiated tumors and reirradiated tumors contained tumor tissue that was more likely to efface olfactory bulb(s) and less infiltrative than control tumors. These histological changes were followed up with VISA which revealed that control tumors harbor fewer clones than in vitro lines and that irradiated tumors harbor the fewest clones of all. Gene expression and IPA analyses showed that pathways involved in cell movement, survival, and proliferation are differentially regulated between irradiated and control tumors. WES was performed to compare gene mutation patterns between reirradiated, irradiated, and control samples. Our results demonstrate that radiation, a central component of glioblastoma treatment, can have wide-ranging effects on the evolution of this dynamic tumor after initial presentation and recurrence. We have demonstrated for the first time the utility of a GSC-initiated orthotopic xenograft model for studying recurrent GBM biology and evolution. This reirradiation model may provide the opportunity to design and test more effective recurrent GBM treatment strategies that are centered around recurrent biology. Citation Format: Joseph H. McAbee, Barbara H. Rath, Xiaolin Wu, Uma Shankavaram, Kevin Camphausen, Philip J. Tofilon. An orthotopic xenograft model for studying reirradiation and glioblastoma evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3727.

Viral integration drives multifocal HCC during the occult HBV infection

Background & AimsAlthough the prognosis of patients with occult hepatitis B virus (HBV) infection (OBI) is usually benign, a small portion may undergo cirrhosis and subsequently hepatocellular carcinoma (HCC). We studied the mechanism of life-long Integration of virus DNA into OBI host’s genome, of which may induce hepatocyte transformation.MethodsWe applied HBV capture sequencing on single cells from an OBI patient who, developed multiple HCC tumors and underwent liver resection in May 2013 at Tongji Hospital in China. Despite with the undetectable virus DNA in serum, we determined the pattern of viral integration in tumor cells and adjacent non-tumor cells and obtained the details of the viral arrangement in host genome, and furthermore the HBV integrated region in cancer genome.ResultsHBV captured sequencing of tissues and individual cells revealed that samples from multiple tumors shared two viral integration sites that could affect three host genes, including CSMD2 on chr1 and MED30/EXT1 on chr8. Whole genome sequencing further indicated one hybrid chromosome formed by HBV integrations between chr1 and chr8 that was shared by multiple tumors. Additional 50 poorly differentiated liver tumors and the paired adjacent non-tumors were evaluated and functional studies suggested up-regulated EXT1 expression promoted HCC growth. We further observed that the most somatic mutations within the tumor cell genome were common among the multiple tumors, suggesting that HBV associated, multifocal HCC is monoclonal in origin.ConclusionThrough analyzing the HBV integration sites in multifocal HCC, our data suggested that the tumor cells were monoclonal in origin and formed in the absence of active viral replication, whereas the affected host genes may subsequently contribute to carcinogenesis.

Ecotropic virus integration-1 and calreticulin as novel prognostic markers in triple-negative breast cancer: A retrospective cohort study.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer, for which no specific targete d therapy is currently available. The present study aimed to examine the associations of ecotropic virus integration site 1 (EVI-1) and calreticulin (CRT) with other clinicopathological variables and the prognosis of patients with TNBC. The present retrospective cohort study reviewed the medical records of patients with TNBC treated in the Affiliated Hospitals of Jinzhou Medical University between January 2010 and June 2015. The protein expression levels of EVI-1 and CRT in tumor samples obtained from the patients were examined by immunohistochemical analysis. Univariate and multivariate regression analyses were used to identify associations between clinical characteristics and disease-free survival (DFS) or overall survival (OS). Kaplan-Meier analysis was performed to observe the survival condition (DFS/OS) according to EVI-1 and CRT expression. A total of 88 TNBC patients were included in the present study. Tumor tissues in 52 (59.1%) patients were EVI-1 positive, and tumor tissues in 64 (72.7%) patients were CRT-positive, and these rates were significantly higher compared with those in the corresponding paracancerous tissues (P<0.05). Multivariate analysis revealed that EVI-1 and CRT expression levels were independent variables associated with OS and DFS, and high expression of both CRT and EVI-1 was significantly associated with decreased OS and DFS compared with the other subgroups (low EVI-1/low CRT expression, low EVI-1/high CRT expression and high EVI-1/low CRT expression) of patients with TNBC. EVI-1 and CRT expression in TNBC was significantly correlated with poor outcome. Evaluation of the EVI-1 and CRT status may provide insight into prognosis prediction for patients with TNBC.

Capsid-CPSF6 interaction: Master regulator of nuclear HIV-1 positioning and integration.

HIV-1 integration favors active chromatin, which is primarily mediated through interactions between the viral capsid and integrase proteins with host factors cleavage and polyadenylation specificity factor 6 (CPSF6) and lens epithelium-derived growth factor/p75, respectively. Previously published image-based studies had suggested that HIV-1 prefers to integrate into chromatin that associates spatially with the nuclear periphery. Here, we re-evaluated previously reported HIV-1 nuclear distance measures across studies and show that HIV-1 prefers peri-nuclear and mid-nuclear zones similarly, with a common preference between studies mapping to the boundary between these two radial areas. We also discuss emerging roles for the capsid-CPSF6 interaction in facilitating HIV-1 pre-integration complex nuclear import and subsequent intranuclear trafficking to preferred sites of viral DNA integration.

WNT10A rs147680216 G>A mutation indicates a higher risk for non-syndromic oral cleft in a northeastern Chinese population

Non-syndromic oral cleft lip and palate is a heterogeneous group of congenital malformations that consist of cleft palate, and cleft lip with or without cleft palate. The members of the wingless type mouse mammary tumour virus (MMTV) integration site family (Wnts) regulate various developmental processes including craniofacial development, and have a role in that of cleft lip and palate. We aimed to identify the potential polymorphisms in the Wnt10a gene, and to explore the association between the variations in the gene and the risk of development of cleft palate. A total of 198 affected patients (cleft lip, n=67; cleft palate, n=48; and both, n=83) together with 187 healthy controls were enrolled (all from the Chinese Han population in NE China). A fragment of 316 bp was amplified from the blood genome of each participant by polymerase chain reaction (PCR) using specific primers that targeted the Homo sapiens Wnt10a gene. By using the restriction enzyme AluI, the population analysed were classified into three genotypes, GG (316 bp), GA (316 bp, 117bp, 199bp) and AA (117bp, 199bp) based on the rs147680216 G/A polymorphism (Gly>Ser mutation at position 213 of Wnt10a protein) of theWnt10a gene. The frequency of allele A in the affected group was significantly higher (14.1% compared with 3.2% in the control group). The allele G with an odds ratio (OR) of 0.201 and 95% CI of 0.445 to 0.091 was not a risk factor for the condition in the affected group. However, the distribution of the genotype did affect its occurrence in the affected group (p<0.001), but not the classification of types (p=0.901). In conclusion we found an rs147680216 G>A mutation that was associated with non-syndromic cleft lip and palate in the Wnt10a gene.

MEIS1 knockdown may promote differentiation of esophageal squamous carcinoma cell line KYSE-30.

Background MEIS1 (Myeloid ecotropic viral integration site 1), as a homeobox (HOX) transcription factor, has a dual function in different types of cancer. Although numerous roles are proposed for MEIS1 in differentiation, stem cell function, gastrointestinal development and tumorigenesis, the involved molecular mechanisms are poor understood. Our aim in this study was to elucidate the functional correlation between MEIS1, as regulator of differentiation process, and the involved genes in cell differentiation in human esophageal squamous carcinoma (ESC) cell line KYSE‐30.MethodsThe KYSE‐30 cells were transduced using recombinant retroviral particles containing specific shRNA sequence against MEIS1 to knockdown MEIS1 gene expression. Following RNA extraction and cDNA synthesis, mRNA expression of MEIS1 and the selected genes including TWIST1, EGF, CDX2, and KRT4 was examined using relative comparative real‐time PCR.ResultsRetroviral transduction caused a significant underexpression of MEIS1 in GFP‐hMEIS1 compared to control GFP cells approximately 5.5‐fold. While knockdown of MEIS1 expression caused a significant decrease in EGF and TWIST1 mRNA expression, nearly ‐8‐ and ‐12‐fold respectively, it caused a significant increase in mRNA expression of differentiation markers including KRT4 and CDX2, approximately 34‐ and 1.14‐fold, correspondingly.Conclusion MEIS1 gene silencing in KYSE‐30 cells increased expression of epithelial markers and decreased expression of epithelial‐mesenchymal transition (EMT) marker TWIST1. It may highlight the role of MEIS1 in differentiation process of KYSE‐30 cells. These results may confirm that MEIS1 silencing promotes differentiation and decreases EMT capability of ESC cell line KYSE‐30.

Myeloid leukemia with high EVI1 expression is sensitive to 5-aza-2'-deoxycytidine by targeting miR-9.

High expression of ecotropic viral integration site 1 (EVI1) has been associated with a poor prognosis in leukemia patients, but the underlying mechanism remains unclear. Aberrant expression of microRNAs plays critical roles in leukemia development. MiR-9 is a putative potential target of EVI1. We have investigated the regulating mechanism of miR-9 by EVI1 in leukemia cells. We first examined the relationship between miR-9 and EVI1 expression levels in nine leukemia cell lines by RT-PCR. Then we forced high expression of EVI1 in UoCM1 and K562 cells to confirm the downregulation of miR-9 by EVI1. Methylation of the miR-9 promoter region was detected by DNA bisulfite sequencing. We treated the EVI1-overexpressing cells with the hypomethylating agent 5-aza-2'-deoxycytidine (5-AZA) to reverse EVI1-induced hypermethylation of miR-9. EVI1 and miR-9 expression was negative related. Forced expression of EVI1 downregulated miR-9 by inducing hypermethylation of the miR-9 promoter. 5-AZA reversed high EVI1-induced hypermethylation of the miR-9 promoter and restored the expression of miR-9. 5-AZA induced extensive apoptosis and inhibited proliferation through cell cycle arrest in EVI1-overexpressing leukemia cells. Our results suggest that EVI1 may be involved in leukemia cell proliferation and apoptosis via the regulation of miR-9 promoter methylation. 5-AZA may represent a promising therapeutic option for EVI1-high leukemia patients.

The regulatory elements of PLZF gene are not conserved as reveled by molecular cloning and functional characterization of PLZF gene promoter of Clarias batrachus

The promyelocytic leukemia zinc finger (Plzf) gene, being a transcriptional repressor, plays a crucial role in self-renewal and maintenance of spermatogonial stem cells (SSCs) across mammalian and non-mammalian species. It is now being considered as a candidate marker of proliferating SSC in teleost. The knowledge on its mechanism of expression is limited. In this study, we intended to clone and characterize Plzf gene promoter of Clarias batrachus, a commercially valuable farmed catfish (also known as magur). A comparative cDNA sequence analysis between Labeo rohita carp and C. batrachus catfish confirmed about perfect homology. We have isolated, sequenced and characterized the regulatory elements of Plzf by genome walking. About 3.6 kb sequence of 5′ upstream region relative to ATG start codon was sequenced. Alignment of genome walking and cDNA sequence data identified transcriptional start site (TSS). The 5′ UTR comprises of non-coding exons (EI and EII) containing a long stretch of intron. Computational analysis revealed several important putative elements like vertebrate TATA binding protein factor (VTBP), ecotropic viral integration site (Evi-1), myoblast determining factors (MyoD), myocyte-specific enhancer binding factor (MEF2), general transcription factor IID initiator (TFIID), etc.; positioned in the upstream sequence above transcriptional start site; indicating as potential promoter/regulatory elements. Dual luciferase reporter assay was performed for various deleted constructs designed to measure their promoter activity in vivo. Its full promoter activity was dependent on upstream region containing a putative Evi-1-like element as enhancer. We also identified the region possibly acting as a transcriptional repressor (Gfi-1). This is the first report of identifying Plzf gene being expressed in spermatogonial cells of C. batrachus. Even though the Plzf cDNA sequence is conserved across species, its regulatory elements are not conserved among human, carps and catfishes.

Tumor Suppressors RB1 and CDKN2a Cooperatively Regulate Cell-Cycle Progression and Differentiation During Cardiomyocyte Development and Repair.

Although rare cardiomyogenesis is reported in the adult mammalian heart, whether this results from differentiation or proliferation of cardiomyogenic cells remains controversial. The tumor suppressor genes RB1 (retinoblastoma) and CDKN2a (cyclin-dependent kinase inhibitor 2a) are critical cell-cycle regulators, but their roles in human cardiomyogenesis remains unclear. We hypothesized that developmental activation of RB1 and CDKN2a cooperatively cause permanent cell-cycle withdrawal of human cardiac precursors (CPCs) driving terminal differentiation into mature cardiomyocytes, and that dual inactivation of these tumor suppressor genes promotes myocyte cell-cycle reentry. Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes revealed that RB1 and CDKN2a are upregulated at the onset of cardiac precursor specification, simultaneously with GATA4 (GATA-binding protein 4) homeobox genes PBX1 (pre-B-cell leukemia transcription factor 1) and MEIS1 (myeloid ecotropic viral integration site 1 homolog), and remain so until terminal cardiomyocyte differentiation. In both GATA4+ hPSC cardiac precursors and postmitotic hPSC-cardiomyocytes, RB1 is hyperphosphorylated and inactivated. Transient, stage-specific, depletion of RB1 during hPSC differentiation enhances cardiomyogenesis at the cardiac precursors stage, but not in terminally differentiated hPSC-cardiomyocytes, by transiently upregulating GATA4 expression through a cell-cycle regulatory pathway involving CDKN2a. Importantly, cytokinesis in postmitotic hPSC-cardiomyocytes can be induced with transient, dual RB1, and CDKN2a silencing. The relevance of this pathway in vivo was suggested by findings in a porcine model of cardiac cell therapy post-MI, whereby dual RB1 and CDKN2a inactivation in adult GATA4+ cells correlates with the degree of scar size reduction and endogenous cardiomyocyte mitosis, particularly in response to combined transendocardial injection of adult human hMSCs (bone marrow-derived mesenchymal stromal cells) and cKit+ cardiac cells. Together these findings reveal an important and coordinated role for RB1 and CDKN2a in regulating cell-cycle progression and differentiation during human cardiomyogenesis. Moreover, transient, dual inactivation of RB1 and CDKN2a in endogenous adult GATA4+ cells and cardiomyocytes mediates, at least in part, the beneficial effects of cell-based therapy in a post-MI large mammalian model, a finding with potential clinical implications.