In situ hybridization: new approaches for the in vivo localization of fibrinolytic components

Abstract Background Accumulating evidence indicates that circRNAs may serve as essential regulators in the progression of several human cancers, but the function and mechanism of circRNAs in intrahepatic cholangiocarcinoma (ICC) are largely unknown. Methods RNA-seq was used to assess differentially expressed circRNAs between 4 ICC and peritumor tissues. Quantitative RT-PCR and in situ hybridization were used to determine the circHMGCS1–016 expression in ICC tissues. The function and mechanism of circHMGCS1–016 were further identified via in vivo experiments. The clinical characteristics and prognostic significance of circHMGCS1–016 were analyzed by a retrospective study. The functions of circHMGCS1–016 were assessed via modifying circRNA expression in ICC cells. Moreover, the molecular mechanisms of circHMGCS1–016 in ICC cells were explored by circRNA precipitation, miRNA immunoprecipitation, SILAC and luciferase reporter assays. Results We identified that compared with peritumor tissues, ICC tissues expressed hsa_circ_0008621 (circHMGCS1–016) high by RNA-seq, which was further identified by qRT-PCR and in situ hybridization. Moreover, the expression of circHMGCS1–016 was revealed to be associated with survival and recurrence of ICC patients. By regulating circHMGCS1–016 expression, we found that elevated circHMGCS1–016 promoted ICC development both in vitro and in vivo. By SILAC and circRNA-pull down, we demonstrated that circHMGCS1–016 induced ICC cell invasion and reshaped the tumor immune microenvironment via the miR-1236-3p/CD73 and GAL-8 axis. In ICC tissues, we uncovered that a high level of circHMGCS1–016 was positively associated with CD73 and GAL-8 expression and negatively related to the CD8+ T cells infiltration, which was further validated by establishing a humanized mouse tumor model. Importantly, we displayed that ICC patients with high levels of circHMGCS1–016 in tumor tissues benefited less from anti-PD1 treatment compared to those with low levels of circHMGCS1–016. Conclusions CircHMGCS1–016 is a forceful contributor in ICC development and immune tolerance via miR-1236-3p/CD73 and GAL-8 axis. CircHMGCS1–016 can be explored as a new potential biomarker and therapeutic target for PD1-resistant ICC.

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Use of cross-species in-situ hybridization (ZOO-FISH) to assess chromosome abnormalities in day-6 in-vivo- or in-vitro-produced sheep embryos

Chromosome Research ◽

10.1007/s10577-007-1125-2 ◽

2007 ◽

Vol 15 (3) ◽

pp. 399-408 ◽

Cited By ~ 11

Author(s):

Gianfranco Coppola ◽

Basil Alexander ◽

Dino Di Berardino ◽

Elizabeth St John ◽

Parvathi K. Basrur ◽

...

Keyword(s):

In Situ Hybridization ◽

Chromosome Abnormalities

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Interactions of human cytomegalovirus with leukocytes in vivo: analysis by in situ hybridization

Microbial Pathogenesis ◽

10.1016/0882-4010(87)90062-3 ◽

1987 ◽

Vol 3 (4) ◽

pp. 287-297 ◽

Cited By ~ 28

Author(s):

Lloyd W. Turtinen ◽

Robin Saltzman ◽

M.Colin Jordan ◽

Ashley T. Haase

Keyword(s):

In Situ Hybridization ◽

Human Cytomegalovirus ◽

In Vivo Analysis

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Fluorescent In Situ Hybridization for Monosomy 3 via 30-Gauge Fine-Needle Aspiration Biopsy of Choroidal Melanoma In Vivo

Ophthalmology ◽

10.1016/j.ophtha.2006.06.040 ◽

2007 ◽

Vol 114 (1) ◽

pp. 142-146 ◽

Cited By ~ 45

Author(s):

Tara A. Young ◽

Nagesh P. Rao ◽

Ben J. Glasgow ◽

Joel N. Moral ◽

Bradley R. Straatsma

Keyword(s):

In Situ Hybridization ◽

Fine Needle Aspiration ◽

Fluorescent In Situ Hybridization ◽

Fine Needle Aspiration Biopsy ◽

Choroidal Melanoma ◽

Needle Aspiration ◽

Fine Needle ◽

Monosomy 3

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Analysis of HIV-1 expression in vivo with in situ hybridization and the polymerase chain reaction

Molecular and Cellular Probes ◽

10.1016/0890-8508(92)90019-t ◽

1992 ◽

Vol 6 (3) ◽

pp. 215-221 ◽

Cited By ~ 2

Author(s):

B. Delord ◽

M. Ottmann ◽

M.-H. Schrive ◽

J.-M. Ragnaud ◽

J.-M. Seigneurin ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

In Situ Hybridization ◽

Chain Reaction ◽

Polymerase Chain ◽

Hiv 1

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Nonrandom Transduction of Recombinant Adeno-Associated Virus Vectors in Mouse Hepatocytes In Vivo: Cell Cycling Does Not Influence Hepatocyte Transduction

Journal of Virology ◽

10.1128/jvi.74.8.3793-3803.2000 ◽

2000 ◽

Vol 74 (8) ◽

pp. 3793-3803 ◽

Cited By ~ 101

Author(s):

Carol H. Miao ◽

Hiroyuki Nakai ◽

Arthur R. Thompson ◽

Theresa A. Storm ◽

Winnie Chiu ◽

...

Keyword(s):

In Situ Hybridization ◽

Gene Transfer ◽

Genetic Diseases ◽

Critical Event ◽

Dimer Formation ◽

Adeno Associated Virus ◽

Virus Vectors ◽

Preclinical Trials

ABSTRACT Recombinant adeno-associated virus vectors (rAAV) show promise in preclinical trials for the treatment of genetic diseases including hemophilia. Liver-directed gene transfer results in a slow rise in transgene expression, reaching steady-state levels over a period of 5 weeks concomitant with the conversion of the single-stranded rAAV molecules into high-molecular-weight concatemers in about 5% of hepatocytes. Immunohistochemistry and RNA in situ hybridization show that the transgene product is made in about ∼5% of hepatocytes, suggesting that most rAAV-mediated gene expression occurs in hepatocytes containing the double-stranded concatemers. In this study, the mechanism(s) involved in stable transduction in vivo was evaluated. While only ∼5% of hepatocytes are stably transduced, in situ hybridization experiments demonstrated that the vast majority of the hepatocytes take up AAV-DNA genomes after portal vein infusion of the vector. Two different vectors were infused together or staggered by 1, 3, or 5 weeks, and two-color fluorescent in situ hybridization and molecular analyses were performed 5 weeks after the infusion of the second vector. These experiments revealed that a small but changing subpopulation of hepatocytes were permissive to stable transduction. Furthermore, in animals that received a single infusion of two vectors, about one-third of the transduced cells contained heteroconcatemers, suggesting that dimer formation was a critical event in the process of concatemer formation. To determine if the progression through the cell cycle was important for rAAV transduction, animals were continuously infused with 5′-bromo-2′-deoxyuridine (BrdU), starting at the time of administration of a rAAV vector that expressed cytoplasmic β-galactosidase. Colabeling for β-galactosidase and BrdU revealed that there was no preference for transduction of cycling cells. This was further confirmed by demonstrating no increase in rAAV transduction efficiencies in animals whose livers were induced to cycle at the time of or after vector administration. Taken together, our studies suggest that while virtually all hepatocytes take up vector, unknown cellular factors are required for stable transduction, and that dimer formation is a critical event in the transduction pathway. These studies have important implications for understanding the mechanism of integration and may be useful for improving liver gene transfer in vivo.

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Developmental expression of 2ar (osteopontin) and SPARC (osteonectin) RNA as revealed by in situ hybridization

The Journal of Cell Biology ◽

10.1083/jcb.106.2.441 ◽

1988 ◽

Vol 106 (2) ◽

pp. 441-450 ◽

Cited By ~ 354

Author(s):

S Nomura ◽

AJ Wills ◽

DR Edwards ◽

JK Heath ◽

BL Hogan

Keyword(s):

In Situ Hybridization ◽

Bone Development ◽

Mouse Cell ◽

Sensory Epithelium ◽

Developmental Expression ◽

Granulated Metrial Gland Cells ◽

Metrial Gland ◽

Temporal And Spatial

2ar has been identified as a gene inducible by tumor promoters and growth factors in a variety of cultured mouse cell lines (Smith, J. H., and D. T. Denhardt. 1987. J. Cell. Biochem. 34:13-22). Sequence analysis shows that it codes for mouse osteopontin, an RGDS-containing, phosphorylated, sialic acid-rich Ca++-binding protein originally isolated from bone (Oldberg, A., A. Franzen, and D. Heinegard. 1986. Proc. Natl. Acad. Sci. USA. 83:8819-8823; Prince, C. W., T. Oosawa, W. T. Butler, M. Tomana, A. S. Brown, and R. E. Schrohenloer. 1987. J. Biol. Chem. 262:2900-3907.). In this paper we use Northern blot analysis and in situ hybridization to localize expression of 2ar during mouse embryogenesis. 2ar RNA is first detected in developing limb bones and calvaria at 14.5 d p.c., in a population of cells distinct from those expressing SPARC (osteonectin). High levels of 2ar expression are also seen in the bone marrow-derived granulated metrial gland cells of the deciduum and placenta, and in a number of epithelial tissues, including embryonic and postnatal kidney tubules, uterine epithelium and sensory epithelium of the embryonic ear. The temporal and spatial pattern of 2ar expression seen in vivo suggests that the protein plays a wider role than previously realized, in processes which are not confined to bone development.

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