Pattern of Cytogenetic Abnormality by Fluorescence in Situ Hybridization (FISH) in Multiple Myeloma Patients in a Tertiary Hospital

Background: Multiple myeloma is a plasma cell neoplasm with acquired genetic abnormalities of clinical and prognostic importance, with survival duration ranging from a few months to more than 10 years. Cytogenetic abnormalities (CA) detected by fluorescence in situ hybridization (FISH) are of major prognostic significance since e.g. patients with del(17p), t(4;14) or gain 1q21 show dismal outcome. Objective: To evaluate the cytogenetic patterns by fluorescence in situ hybridization (FISH) of clinically diagnosed cases of multiple myeloma.Methods:This cross-sectional study was conducted in Department of Haematology, Dhaka Medical College Hospital, Dhaka, from January 2018 to December 2018. A total number of 30 patients with multiple myeloma were analyzed cytogenetically by interphase fluorescence in situ hybridization (iFISH). The collected data were analyzed by using the Statistical Package for Social Science (SPSS-24) for windows version 10.0.Results:Out of 30 diagnosed Multiple Myeloma cases the mean age was 56.37±10.38 years and male to female ratio was almost 3:1. Sixteen (56.7%) of 30 patients. Among 30 cases of 8 cases were thyrogenicity positive of 7(23.3%) patients was detected del 13q positive. Isolated del 13q was found in 4 cases. 2 cases were found coexistence of del 13q and del 17p positive ;1 case was found coexistence of del 13q and t(4;14) positive and rest of 1 case had del 17 p positive. There was no detectable t (11; 14) and t(14;16) in any of 30 cases.Conclusion:FISH panel for Multiple Myeloma including del (13q); t(11;14); t(4;14), del(17p), t(14;16) is very important molecular test for the prognosis , risk stratification, treatment modality of the patient. On the basis of cytogenetic abnormality Multiple Myeloma risk stratification is modified now a day. This Revised International Staging system R-ISS is a simple and powerful prognostic staging system.

CircFISH: A Novel Method for the Simultaneous Imaging of Linear and Circular RNAs

Circular RNAs (circRNAs) are regulatory RNAs which have recently been shown to have clinical significance in several diseases, including, but not limited to, various cancers, neurological diseases and cardiovascular diseases. The function of such regulatory RNAs is largely dependent on their subcellular localization. Several circRNAs have been shown to conduct antagonistic roles compared to the products of the linear isoforms, and thus need to be characterized distinctly from the linear RNAs. However, conventional fluorescent in situ hybridization (FISH) techniques cannot be employed directly to distinguish the signals from linear and circular isoforms because most circRNAs share the same sequence with the linear RNAs. In order to address this unmet need, we adapted the well-established method of single-molecule FISH by designing two sets of probes to differentiate the linear and circular RNA isoforms by virtue of signal colocalization. We call this method ‘circular fluorescent in situ hybridization’ (circFISH). Linear and circular RNAs were successfully visualized and quantified at a single-molecule resolution in fixed cells. RNase R treatment during the circFISH reduced the levels of linear RNAs while the circRNA levels remain unaltered. Furthermore, cells with shRNAs specific to circRNA showed the loss of circRNA levels, whereas the linear RNA levels were unaffected. The optimization of the in-situ RNase R treatment allowed the multiplexing of circFISH to combine it with organelle staining. CircFISH was found to be compatible with multiple sample types, including cultured cells and fresh-frozen and formalin-fixed tissue sections. Thus, we present circFISH as a versatile method for the simultaneous visualization and quantification of the distribution and localization of linear and circular RNA in fixed cells and tissue samples.

hsa_circ_0000231 Promotes colorectal cancer cell growth through upregulation of CCND2 by IGF2BP3/miR-375 dual pathway

Background Circular RNAs (circRNAs) have emerged as vital regulators of the initiation and progression of diverse kinds of human cancers. In this study, we explored the role of hsa_circ_0000231 and its downstream pathway in CRC. Methods The expression profile of circRNAs in 5 pairs of CRC tissues and adjacent normal tissues were analyzed by Microarray. Quantitative real-time PCR and in situ hybridization and Base Scope Assay were used to determine the level and prognostic values of hsa_circ_0000231. Then, functional experiments in vitro and in vivo were performed to investigate the effects of hsa_circ_0000231 on cell proliferation. Mechanistically, fluorescent in situ hybridization, dual luciferase reporter assay, RNA pull-down and RNA immunoprecipitation experiments were performed to confirm the interaction between hsa_circ_0000231 and IGF2BP3 or has_miR-375. Results We acquired data through circRNA microarray profiles, showing that the expression of hsa_circ_0000231 was upregulated in CRC primary tissues compared to adjacent normal tissues, which was indicated poor prognosis of patients with CRC. Functional analysis indicated that inhibition of hsa_circ_0000231 in CRC cell lines could suppress CRC cell proliferation as well as tumorigenesis in vitro and in vivo. The mechanistic analysis showed that hsa_circ_0000231 might, on the one hand, act as a competing endogenous RNA of miR-375 to promote cyclin D2 (CCND2) and, on the other hand, bind to the IGF2BP3 protein to prevent CCND2 degradation. Conclusions The findings suggested that hsa_circ_0000231 facilitated CRC progression by sponging miR-375 or binding to IGF2BP3 to modulate CCND2, implying that hsa_circ_0000231 might be a potential new diagnostic and therapeutic biomarker of CRC.

The Prognostic Impact of MYC Gene-Related Abnormalities on Multiple Myeloma Outcome through Fluorescence in situ Hybridization Analysis

Introduction: Chromosomal abnormalities (CAs) have been identified as important factors in determining the biological features and prognostic value of multiple myeloma (MM). MYC gene-related abnormalities (MYC GAs) are one of the CAs, but its unfavorable impact has not been fully investigated in daily clinical practice. Methods: This study retrospectively analyzed the prognostic impact of MYC GAs on 81 patients through fluorescence in situ hybridization analysis in our institute. Results: MYC GAs were associated with poor overall survival (hazard ratio [HR], 3.08; 95% confidence interval [CI], 1.23–7.73; p = 0.017), progression-free survival (PFS) (HR, 2.96; 95% CI, 1.58–5.53; p < 0.001), and time to next treatment (TNT) (HR, 2.11; 95% CI, 1.13–3.93; p = 0.018) in the median follow-up of 34.7 months. Furthermore, MYC GAs with an additional chromosome 8 (MYC-Ch8(+)) were associated with shorter PFS (HR, 3.15; 95% CI, 1.38–7.2; p = 0.0064), whereas MYC GAs without an additional chromosome 8 (MYC-Ch8(−)) were associated with shorter PFS (HR, 3.62; 95% CI, 1.51–8.68; p = 0.004) and shorter TNT (HR, 3.72; 95% CI, 1.41–9.81; p = 0.0078). Conclusion: These findings could help identify high-risk patients with MM. Further prospective studies are needed to confirm the significance of MYC GAs for the MM prognostic effect.

Avian haemosporidian parasites of accipitriform raptors

Background The order Accipitriformes comprises the largest group of birds of prey with 260 species in four families. So far, 21 haemosporidian parasite species have been described from or reported to occur in accipitriform birds. Only five of these parasite species have been characterized molecular genetically. The first part of this study involved molecular genetic screening of accipitriform raptors from Austria and Bosnia-Herzegovina and the first chromogenic in situ hybridization approach targeting parasites in this host group. The aim of the second part of this study was to summarize the CytB sequence data of haemosporidian parasites from accipitriform raptors and to visualize the geographic and host distribution of the lineages. Methods Blood and tissue samples of 183 accipitriform raptors from Austria and Bosnia-Herzegovina were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR, and tissue samples of 23 PCR-positive birds were subjected to chromogenic in situ hybridization using genus-specific probes targeting the parasites’ 18S rRNAs. All published CytB sequence data from accipitriform raptors were analysed, phylogenetic trees were calculated, and DNA haplotype network analyses were performed with sequences from clades featuring multiple lineages detected in this host group. Results Of the 183 raptors from Austria and Bosnia-Herzegovina screened by PCR and sequencing, 80 individuals (44%) were infected with haemosporidian parasites. Among the 39 CytB lineages detected, 18 were found for the first time in the present study. The chromogenic in situ hybridization revealed exo-erythrocytic tissue stages of Leucocytozoon parasites belonging to the Leucocytozoon toddi species group in the kidneys of 14 infected birds. The total number of CytB lineages recorded in accipitriform birds worldwide was 57 for Leucocytozoon, 25 for Plasmodium, and 21 for Haemoproteus. Conclusion The analysis of the DNA haplotype networks allowed identifying numerous distinct groups of lineages, which have not yet been linked to morphospecies, and many of them likely belong to yet undescribed parasite species. Tissue stages of Leucocytozoon parasites developing in accipitriform raptors were discovered and described. The majority of Leucocytozoon and Haemoproteus lineages are specific to this host group, but most Plasmodium lineages were found in birds of other orders. This might indicate local transmission from birds kept at the same facilities (raptor rescue centres and zoos), likely resulting in abortive infections. To clarify the taxonomic and systematic problems, combined morphological and molecular genetic analyses on a wider range of accipitriform host species are needed.

DNA-Methylation-Mediated lncRNA HOXB-AS4 Promotes Gastric Cancer Progression Through Regulating miR-130a-5p/PKP4

Background：Gastric cancer (GC) is one of the most common cancer in the world, possessing the second leading cause of cancer-related mortality. Long noncoding RNAs (lncRNAs) have been shown to play important roles in tumorigenesis. However, the effect of lncRNA HOXB-AS4 in GC progression and the underlying mechanisms remain unknown. Methods：Firstly, the expression of lncRNA HOXB-AS4 in gastric cancer tissues and cancer cells was investigated according to GEPIA database and Real time fluorescence quantitative PCR(qRT-PCR). Then, MTT, clone formation, Transwell and Western blot were used to study the effects of overexpression or down-regulation of HOXB-AS4 on the proliferation, invasion and epithelial mesenchymal transformation of cancer cells. We further studied the molecular mechanism of HOXB-AS4 by fluorescence in situ hybridization, bioinformatics analysis, luciferase reporting, methylation specific PCR (MSP) and chromatin immunoprecipitation (chip).Results:In the study, the GEPIA database and quantitative Real-Time PCR (qRT-PCR) assay showed that HOXB-AS4 was upregulated in GC tissues and cells. Then, MTT, clone formation, transwell, and western blot assays suggested that overexpression of HOXB-AS4 increased cell proliferation, migration, and invasion, and regulated epithelial-mesenchymal transition (EMT) markers expression, while knockdown of HOXB-AS4 showed the opposite effect. Fluorescence in situ hybridization (FISH) assay found that HOXB-AS4 localized in the cytoplasm of the GSE-1 and AGS cells. Further mechanism experiments, including bioinformatics, luciferase reporter, qRT-PCR, and western blot assays showed that HOXB-AS4 sponged to miR-130a-5p to regulate the PKP4 expression. Knockdown of miR-130a-5p obliterated the effect of HOXB-AS4, which was further abolished by knockdown of PKP4 in vitro and in vivo. Methylation-specific PCR (MSP) and chromatin immunoprecipitation (CHIP) assay showed that overexpression of HOXB-AS4 in GC was mediated by SP1-dependent DNA methylation. Abnormal upregulation of lncRNA HOXB-AS4 contributed to GC progression, which was mediated by DNA methylation. The study clarified that DNA-methylation-mediated HOXB-AS4 played its role through miR-130a-5p/PKP4 axis.Conclusions: Our study provides new insights for the understanding of epigenetic regulation on lncRNA expression in GC, and indicates that HOXB-AS4 could be a biomarker of GC prognosis. Moreover, targeting HOXB-AS4 /miR-130a-5p/PKP4 axis might be a promising strategy to treat GC.