Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Expression and coding potential analysis of Hmrhl. a. Quantitative real time PCR analysis of Hmrhl expression showed that it is expressed in all human tissues (Brain, Heart, Kidney, lung, liver, pancreas, spleen, thymus, small intestine, colon, skeletal muscle, testes, prostate, ovary, placenta, leukocyte, from left to right) examined. Lowest expression was found in skeletal muscle (SM) which was taken as control, the level of which was considered as 1 and all others were plotted in comparison to it. Highest expression was seen in spleen (spln) followed by pancreas (Pnc), testis (Tst) and other tissues. b. Northern blot detection of Hmrhl. Total RNA from HEK 293T and K562 cell lines were separated on agarose gel and subsequently hybridized with DIG labelled Hmrhl specific riboprobe to detect the transcript (i). In parallel, methylene blue staining was used to determine the size of HMRHL, using 28 S rRNA (5 kb) and 18s rRNA (1.9 kb) as reference (ii). Note that the size of Hmrhl is similar to that of 28s rRNA, revealing that Hmrhl is about 5 kb in size. c. Protein-coding potential as determined by Broad Institute's PhyloCSF data and visualized in UCSC Genome Browser, showing that Hmrhl has no coding potential. d. Circular phylogenetic tree built in iTOL (Interactive Tree of Life).

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Northern Blot, Agarose Gel Electrophoresis, Staining

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl locus exhibits hallmarks of enhancer. a. ENCODE data visualized through Integrated Genome Viewer (IGV) for DNase hypersensitive sites, p300 binding, enhancer specific histone marks, H3K27Ac and H3K4Me1 and the promoter specific histone mark, H3K4Me3 at the 5′ end of Hmrhl, only in K562 but not in GM12878 cells. Note the two prominent peaks (red) for the enhancer mark H3K27Ac in K562. b-c. Chromatin immunoprecipitation with Ab8895 (anti-H3K4Me1 antibody) and Ab4729 (anti-H3K27Ac antibody) followed by qPCR in K562 cells. Note the enrichment of both the enhancer marks at the 5′ end of Hmrhl in the IP fraction as compared to input/PIS/gene desert region (GD), that serves as a negative control.

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Negative Control

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl locus exhibits hallmarks of enhancer contd. a. Encode data shows the binding of various transcription and PolII at the 5′ end of Hmrhl. We have retained the H3K27Ac peaks in this figure also for a reference. b. Schematic for chromatin interaction analysis (ChiaPET data) for Hmrhl. The large purple-black peak representing histone marks on the extreme left denotes the promoter of phkb gene while the small purple peak at the far right represents the 5'end of Hmrhl. ChiaPET data shows the interaction of Hmrhl locus with phkb promoter, as represented by two black boxes (blue arrows) connected by a black line in b. The Hmrhl locus is expanded below in c , showing that this locus has enhancer properties only in K562 cell line (orange-yellow color), but not in other cell lines like GM12878, HepG2 or hESC. Genomic segments are colour coded by ENCODE as denoted in d , with red colour signifying active promoter ( phkb promoter at far left, black arrow in b ) while orange colour represents active enhancer at Hmrhl locus at far right (red arrow in b ).

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Binding Assay

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl is differentially expressed in various cancers. a. Expression of Hmrhl in various normal and cancer samples as observed by qPCR. Note that Hmrhl is highly upregulated in several lymphoma samples (bracket) in comparison to normal range (arrow). In fact, of all cancers, the highest levels of Hmrhl are seen in some of the lymphoma samples. b-c. qPCR analysis of Hmrhl and PHKB expression showing that both are over expressed in K562 leukemia condition as compared to GM12878 normal lymphocytes.

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Expressing

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl functions as enhancer RNA for phkb gene. a. Lucifaerase assay showing the intense signal of reporter activity in K562 cells with insert 3 cloned in enhancer vector. Note the low level of luciferase signal obtained with insert 2 both with promoter and enhancer vectors. b. siRNA (Sigma) mediated down-regulation of Hmrhl causes down-regulation of PHKB in K562 cells treated with Hmrhl specific siRNA pool as compared to control cells without transfection and cells treated with scrambled siRNA as negative control. c-d. Smart pool siRNA (Dharmacon) were used against the Hmrhl region to downregulate Hmrhl and subsequently expression level of PHKB gene were checked by qPCR in both K562 and GM12878 cell lines. Scrambled siRNA was used as a negative control. Note the down regulation of PHKB only in K562.

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Activity Assay, Clone Assay, Plasmid Preparation, Luciferase, Transfection, Negative Control, Expressing

Journal: BMC Cancer

Article Title: Jacarelhyperol A induced apoptosis in leukaemia cancer cell through inhibition the activity of Bcl-2 proteins

doi: 10.1186/1471-2407-14-689

Figure Lengend Snippet: The inhibitory activity of Jac-A on tumour cells via MTT Assay (IC 50 , μ M, n = 4, mean ± SD)

Article Snippet: 150 μg of K562 cell lysates in 500 μL of CHAPS lysis buffer were precleared for 60 min at 4°C with 20 μL of a 1:1 slurry of protein A/G Plus-Agarose (Santa Cruz Biotechnology, Cat.# sc 2003) and 1 μg of rabbit IgG.

Techniques: Activity Assay, MTT Assay

Journal: BMC Cancer

Article Title: Jacarelhyperol A induced apoptosis in leukaemia cancer cell through inhibition the activity of Bcl-2 proteins

doi: 10.1186/1471-2407-14-689

Figure Lengend Snippet: Jac-A caused K562 cell apoptosis. 10 6 of K562 cells were treated with different concentrations of Jac-A for 48 h. Cells were then stained with Annexin V-FITC and propidium iodide, and analysed using flow cytometry. Four different cell populations marked as the following: live cell population (PI - AV-), early apoptosis (PI - AV+), late apoptosis (PI + AV+) and dead cells (PI + AV-). Cells were treated with no Jac-A (A) , 0.5% DMSO (B) , or 0.1, 1, 5, 10 μM/L Jac-A (C, D, E and F) . The results are one representative of three independent experiments.

Article Snippet: 150 μg of K562 cell lysates in 500 μL of CHAPS lysis buffer were precleared for 60 min at 4°C with 20 μL of a 1:1 slurry of protein A/G Plus-Agarose (Santa Cruz Biotechnology, Cat.# sc 2003) and 1 μg of rabbit IgG.

Techniques: Staining, Flow Cytometry

Journal: BMC Cancer

Article Title: Jacarelhyperol A induced apoptosis in leukaemia cancer cell through inhibition the activity of Bcl-2 proteins

doi: 10.1186/1471-2407-14-689

Figure Lengend Snippet: Jac-A induced caspase-dependent apoptosis in K562 cells. (A) Cyt c release in Jac-A-induced apoptosis of K562 cells. 10 6 of K562 cells were treated with different concentration of Jac-A for 48 h. Cytosol and mitochondrial heavy membrane samples were prepared and subjected to immunoblot with anti-Cyt c specific antibodie as described in Materials and methods. The results are one representative of three independent experiments. (B) Western blot showing conspicuous cleavage of caspase-3, caspase-9, and PARP in K562 cells treated with Jac-A. K562 cells subjected to protein extract preparation after treated with 0 (control), 3, 6, 12 μM/L Jac-A for 48 h. Then, fifty micrograms extracted protein subjected to Western blot using anti-PARP, PARP, cleaved caspase-9, caspase-9, cleaved caspase-3, caspase-3, and β-actin. The results are one representative of three independent experiments. (C) Jac-A-induced apoptosis was inhibited in a concentration dependent manner by the Z-VAD-fmk. K562 cells were first treated with or without different concentrations of Z-VAD-fmk for 4 h, followed by the treatment of Jac-A (10 μM) for 48 h.

Article Snippet: 150 μg of K562 cell lysates in 500 μL of CHAPS lysis buffer were precleared for 60 min at 4°C with 20 μL of a 1:1 slurry of protein A/G Plus-Agarose (Santa Cruz Biotechnology, Cat.# sc 2003) and 1 μg of rabbit IgG.

Techniques: Concentration Assay, Membrane, Western Blot, Control

Journal: BMC Cancer

Article Title: Jacarelhyperol A induced apoptosis in leukaemia cancer cell through inhibition the activity of Bcl-2 proteins

doi: 10.1186/1471-2407-14-689

Figure Lengend Snippet: Jac-A inhibited the heterodimerization of antiapoptotic proteins with pro-apoptotic proteins. Jac-A inhibited the binding between Bax with Bcl-x L or Bcl-2 (A) and Bak with Mcl-1 (B) . K562 cells were treated with indicated concentrations of Jac-A for 48 h. 150 μg of K562 cell lysates were subjected to co-immunoprecipitation using anti-Bax and anti-Bak antibodies, respectively, further immunoblot with anti- Bcl-x L , Bcl-2, Mcl-1, Bax, or Bak antibody, as described in Materials and methods. The results are one representative of three independent experiments.

Article Snippet: 150 μg of K562 cell lysates in 500 μL of CHAPS lysis buffer were precleared for 60 min at 4°C with 20 μL of a 1:1 slurry of protein A/G Plus-Agarose (Santa Cruz Biotechnology, Cat.# sc 2003) and 1 μg of rabbit IgG.

Techniques: Binding Assay, Immunoprecipitation, Western Blot

Journal: BMC Cancer

Article Title: Jacarelhyperol A induced apoptosis in leukaemia cancer cell through inhibition the activity of Bcl-2 proteins

doi: 10.1186/1471-2407-14-689

Figure Lengend Snippet: Therapeutic study of Jac-A in the K562-bearing mice (n = 10/group). (A) Tumour volume plot of K562-bearing mice treated with vehicle or Jac-A at 2, 10, or 50 mg/kg by oral gavage for 21 days. The tumours were measured twice per week. The data are represented as the mean ± SEM. Tumour growth was inhibited significantly after treatment with Jac-A compared with the control group. *, P < 0.05; †, P < 0.01; ‡, P < 0.001 compared with the control group. (B) Selected nude mice models of different groups treated with Jac-A or vehicle at day 14 after therapy. (C) Sizes of selected tumours harvested from dead nude mice bearing K562 cells from different groups treated with the vehicle or Jac-A. (D) Kaplan-Meier survival plot of the K562-bearing nude mice. The survival of the K562-bearing nude mice was prolonged in the Jac-A treated groups compared with control group. (E) Body weight plot of the K562-bearing nude mice. The data are represented as the mean ± SEM. *, P < 0.05; †, P < 0.01; ‡, P < 0.001 compared with the control group.

Article Snippet: 150 μg of K562 cell lysates in 500 μL of CHAPS lysis buffer were precleared for 60 min at 4°C with 20 μL of a 1:1 slurry of protein A/G Plus-Agarose (Santa Cruz Biotechnology, Cat.# sc 2003) and 1 μg of rabbit IgG.

Techniques: Control