Journal: OncoTargets and therapy

Article Title: miR-486-5p attenuates tumor growth and lymphangiogenesis by targeting neuropilin-2 in colorectal carcinoma

doi: 10.2147/OTT.S103460

Figure Lengend Snippet: NRP2 is a target of miR-486-5p. Notes: ( A ) Effect of miR-486-5p on NRP2 expression as determined by a luciferase reporter assay. The data were normalized by determining the firefly and Renilla luciferase activities measured at 72 hours posttransfection; SW620 and HT-29 cells were transfected with miR-486-5p mimic (miR-486-5p group), mimic control (NC group), and blank control (blank group). ( B ) Transfection of miR-486-5p mimics to SW620 and HT-29 cells increases the expression of miR-486-5p detected by qRT-PCR. ( C ) NRP2 protein expression level was significantly decreased in miR-486-5p-transfected SW620 and HT-29 cells. ( D ) Relative NRP2 protein levels in figure ( C ) were quantified using the gray scale quantification analysis. ** P <0.01. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NRP2, neuropilin-2; qRT-PCR, quantitative real-time polymerase chain reaction; NC, nontarget control.

Article Snippet: Human CRC cell lines SW620 and HT-29 were purchased from Boster Company (Boster, Wuhan, People’s Republic of China).

Techniques: Expressing, Luciferase, Reporter Assay, Transfection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: OncoTargets and therapy

Article Title: miR-486-5p attenuates tumor growth and lymphangiogenesis by targeting neuropilin-2 in colorectal carcinoma

doi: 10.2147/OTT.S103460

Figure Lengend Snippet: miR-486-5p inhibited xenograft tumor growth by targeting NRP2 in vivo. Notes: ( A ) Groups of nude mice were implanted subcutaneously with SW620/PBS cells (Blank group), SW620/mimic-control cells (NC group), or SW620/miR-486-5p cells (miR-486-5p group), Tumor volumes were recorded at the indicated times. ( B ) The tumor mass was determined when the mice were sacrificed. ( C ) NRP2 expression in tumors was examined by Western blot. ( D ) Quantitative analysis of the NRP2 protein level using a gray scan analysis. One representative tumor mass group from three independent experiments with similar results is shown. * P <0.05, ** P <0.01. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NRP2, neuropilin-2; PBS, phosphate-buffered saline; NC, nontarget control.

Article Snippet: Human CRC cell lines SW620 and HT-29 were purchased from Boster Company (Boster, Wuhan, People’s Republic of China).

Techniques: In Vivo, Expressing, Western Blot

Journal: Oncotarget

Article Title: MiR-675-5p supports hypoxia induced epithelial to mesenchymal transition in colon cancer cells

doi: 10.18632/oncotarget.14464

Figure Lengend Snippet: ( A ) Real time-PCR for miR-675-5p in SW480 and SW620 cells. All data were normalized for U6 and ΔΔct was expressed as relative amount of miRNAs. Values are presented as the mean ± SD (of three independent experiments). SW620 vs SW480 ** p < 0.001. ( B ) Real time-PCR for miR-675-5p and EMT master genes: Snail, Slug and E-cadherin in SW480 and SW620 cells. Data were normalized for β-actin, while U6 was used for miRNA normalization. ΔΔct is expressed as Fold of induction (FOI) with respect to expression in control samples. Data are the mean ± SD of three independent experiments. SW480 miRNA inhibitor vs SW480 scramble control: * p < 0.05; *** p < 0.0001; SW620 miRNA inhibitor vs SW620 scramble control * p < 0.05; ** p < 0.001; *** p < 0.0001. ( C ) Immunofluorescences and median focal plane in confocal analysis for E-CADHERIN, ZO-1and SNAIL in SW480 and SW620 cells treated with miR-675-5p inhibitor or scramble control, in blue the nuclear staining with DAPI. ( D ) Migration assay: Phase contrast micrographs (10×) showing the migration of SW480 and SW620 cells pre-treated with miR-675-5p inhibitor or scramble control. Right panel: Quantification of migration by counting the number of migrated cells (violet) per field ( n = 6); * p < 0.05.

Article Snippet: SW620 cells were grown at a density of 100.000 cells/well in a 6 wells plate, and transfected by using Attractene Transfection Reagent (cat. number.1051531, Quiagen) for 6 hours with 0.2 mg/ml H19 siRNA (SR319206B Origene Technologies) or scramble negative control (SR30004 Origene Technologies) at the same dose following manufacturer's indications.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining, Migration

Journal: Oncotarget

Article Title: MiR-675-5p supports hypoxia induced epithelial to mesenchymal transition in colon cancer cells

doi: 10.18632/oncotarget.14464

Figure Lengend Snippet: Real time-PCR for HIF1α ( A ) and VEGF-A ( B ) in SW480 and SW620 cells treated with miR-675-5p inhibitor or scramble control. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to expression in control samples. SW480 miRNA inhibitor vs SW480 control * p < 0.05; SW620 miRNA inhibitor vs SW620 control * p < 0.05 ( C ) Real time-PCR for HIF1α in SW480 and SW620 cells treated with miR-675-5p inhibitor or scramble control after 6 hours of hypoxia. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to expression in normoxia. Values are presented as the mean ± SD. SW620 inhibitor vs SW620 ctr ** p < 0.001. ELISA assay for HIF-1α ( D ) and VEGFa ( E ) performed in SW480 and SW620 cells, transfected with miRNA inhibitor or scramble control, after 6 hours of hypoxia. Data are expressed as Absorbance (ABS) values at 450 nm. SW620 miRNA inhibitor vs SW620 scramble control ** p < 0.001. Real time-PCR for hypoxia targets genes and EMT regulator genes (VEGF, SNAIL, SLUG) in SW480 cells ( F ) and SW620 cells ( G ) treated with miR-675-5p inhibitor or scramble control, after 6 hours of hypoxia. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to expression in control samples. SW620 inhibitor vs SW620 control ** p < 0.001. ( H ) Real time-PCR for lncH19 and HIF1α in SW620 cells treated with siRNA H19 or scramble control and exposed to hypoxia for 6 hours. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to expression in control samples. SW620 siH19 vs SW620 control * p < 0.05;. ( I ) Real time-PCR for Snail and Slug in SW620 cells transfected with siRNA H19 or scrambled control and exposed to hypoxia for 6 hours. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to expression in control samples. ** p < 0.001. Data are the mean ± SD of three independent experiments.

Article Snippet: SW620 cells were grown at a density of 100.000 cells/well in a 6 wells plate, and transfected by using Attractene Transfection Reagent (cat. number.1051531, Quiagen) for 6 hours with 0.2 mg/ml H19 siRNA (SR319206B Origene Technologies) or scramble negative control (SR30004 Origene Technologies) at the same dose following manufacturer's indications.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Oncotarget

Article Title: MiR-675-5p supports hypoxia induced epithelial to mesenchymal transition in colon cancer cells

doi: 10.18632/oncotarget.14464

Figure Lengend Snippet: ( A ) ELISA assay for HIF-1α performed in SW620 cells nuclear extracts after mimic or scramble-control transfection. Data are expressed as Absorbance (ABS) values at 450nm. SW620 mimic vs SW620 scramble control ** p < 0.001. Data are the mean ± SD of three independent experiments. ( B ) Immunofluorescences and median focal plane of confocal analysis for HIF1α (red) in SW620 cells treated with miR-675-5p mimic or scrambled control, in blue the nuclear staining with DAPI. ( C ) Real-time PCR for HIF1α targets gene (Snail, Slug, VEGF, VEGFR-2) from SW620 cells after miR-675-5p mimic or scramble control transfection. Data were normalized for β-actin and ΔΔct is expressed as FOI of indicated genes after mimic transfection with respect to scramble control. ( D ) ELISA assay for VEGF levels in supernatants from SW620 cell lines 18 hours after mimic and scramble control transfection. Data are expressed as pg/ml of soluble VEGF. SW620 mimic vs SW620 scramble control *** p < 0,0001. Data are the mean ± SD of three independent experiments. ( E ) Migration assay: Phase contrast micrographs (10×) showing the migration of SW620 cells pre-treated with mimic miR-675-5p and scramble control. Right panel: Quantification of motility established by counting the number of migrated cells (violet) per field; SW620 mimic vs SW620 scramble control * p < 0.05.

Article Snippet: SW620 cells were grown at a density of 100.000 cells/well in a 6 wells plate, and transfected by using Attractene Transfection Reagent (cat. number.1051531, Quiagen) for 6 hours with 0.2 mg/ml H19 siRNA (SR319206B Origene Technologies) or scramble negative control (SR30004 Origene Technologies) at the same dose following manufacturer's indications.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Staining, Real-time Polymerase Chain Reaction, Migration

Journal: Oncotarget

Article Title: MiR-675-5p supports hypoxia induced epithelial to mesenchymal transition in colon cancer cells

doi: 10.18632/oncotarget.14464

Figure Lengend Snippet: ( A ) Left: predicted seed sequence for miR-675-5p on DDB2 gene. Right: Real-time PCR for DDB-2 gene from SW620 cells after 18 hours of miR-675-5p mimic or miR-675-5p inhibitor or scramble control transfection. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to scramble control. SW620 mimic and inhibitor vs SW620 ** p < 0,001. Data are the mean ± SD of three independent experiments. ( B ) Western blot for DDB2, HIF1α and β-actin in SW620 cells after 18 hours of miR-675-5p scramble control or inhibitor transfection.

Article Snippet: SW620 cells were grown at a density of 100.000 cells/well in a 6 wells plate, and transfected by using Attractene Transfection Reagent (cat. number.1051531, Quiagen) for 6 hours with 0.2 mg/ml H19 siRNA (SR319206B Origene Technologies) or scramble negative control (SR30004 Origene Technologies) at the same dose following manufacturer's indications.

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Transfection, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: miR‐210 expression and inhibition profiles in colorectal tumour cells. (A) The metastatic colorectal cancer (CRC) cell line SW620 has the highest level of miR‐210 transcripts compared to primary CRC SW480 cells (both cell lines originated from the same patient at different stages of cancer progression) and normal colon epithelia (CCD841). (B) PMIS‐miR‐210 inhibits miR‐210 expression in SW620 cells, but does not inhibit other miRs. (C and D) RNA sequencing from PMIS‐miR‐210 SW620 cells revealed different sets of genes regulated by miR‐210 compared to PMIS‐miR‐210 ‐excised murine xenograft tumours. Only 120 regulated genes were similar in the two sets of RNA‐seq data. (E) These genes were further analyzed for their gene ontology pathways and histone modifying genes including H3K27ac and H3K4me3, which were upregulated. * p < .05; ** p < .01

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Expressing, Inhibition, RNA Sequencing

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: Small colorectal tumours are inhibited or ablated by injection of PMIS‐miR‐210 . (A) Transduced PMIS‐miR‐210 or PMIS‐EV SW620 cells (5 × 10 5 ) were transplanted into the flanks of Foxn1 nu/j nude mice and allowed to grow for 4 weeks, excised and analyzed. Five of the eight PMIS‐miR‐210 SW620 implanted cells failed to grow tumours compared to controls, which all grew large tumours. (B and C) SW620 cells (5 × 10 5 ) were transplanted into nude mice and allowed to grow for 7–10 days and form solid tumours. After 7–10 days, different doses (2.5, 5 or 10 μg) of PMIS‐miR‐210 or PMIS‐EV plasmid DNA were directly injected into the tumour. After 35–40 days, the tumours were removed and analyzed; interestingly, in three of the five mice (at different DNA concentrations) no tumours were found in the PMIS‐miR‐210 ‐injected tumours. (D and E) SW620 cells were transduced with a luciferase construct (5 × 10 5 ) to allow for live imaging of tumour formation and after 7–10 days of growth, tumours were injected with 10 μg of PMIS‐miR‐210 or PMIS‐EV plasmid DNA. After 35–40 days, the tumours were imaged, removed and quantitated. In two PMIS‐miR‐210 ‐injected mice, the tumours were not detectable by BLI or visible by dissection.

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Injection, Plasmid Preparation, Transduction, Luciferase, Construct, Imaging, Dissection

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: Large colorectal tumours are significantly reduced in NSG xenograft mice with PMIS‐miR‐210 injections. (A) SW620 cells were transduced with a luciferase construct and (1 × 10 6 ) cells (2× more than Figure ) were transplanted into each flank of NSG mice and allowed to grow and imaged after 7–10 days. (B) After 7–10 days, SW620 tumours were injected with 5 μg of PMIS‐miR‐210 or PMIS‐EV plasmid DNA and 5 days later another dose of 5 μg plasmid DNA was administered to the tumours. Capecitabine was given to the mice as a control treatment to compare the efficacy of PMIS‐miR‐210 treatments. After 35–40 days, the mice were imaged for tumour growth. (C and D) Tumours 35–40 days after treatments were removed and analyzed and tumour weights were recorded, each mouse had two tumours, one on each flank. N = 8 per group, except for capecitabine treatment ( N = 4); Student's t ‐tests conducted between groups

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Transduction, Luciferase, Construct, Injection, Plasmid Preparation, Control

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: Large colorectal tumours are significantly reduced in Foxn1 nu/j xenograft mice with PMIS‐miR‐210 injections. (A) SW620 cells were transduced with a luciferase construct and (1 × 10 6 ) cells were transplanted into each flank of Foxn1 nu/j mice and allowed to grow and imaged after 7–10 days. (B) After 7–10 days, SW620 tumours were injected with 5 μg of PMIS‐miR‐210 or PMIS‐EV plasmid DNA and 5 days later another dose of 5 μg plasmid DNA was administered to the tumours. Capecitabine was given to the mice as a control treatment to compare the efficacy of PMIS‐miR‐210 treatments. After 35–40 days, the mice were imaged for tumour growth. (C and D) Tumours 35–40 days after treatments were removed and analyzed and tumour weights were recorded, each mouse had two tumours, one on each flank. N = 8 per group; N = 4 for capecitabine treatment; Student's t ‐tests conducted between groups

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Transduction, Luciferase, Construct, Injection, Plasmid Preparation, Control

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: PMIS‐miR‐210 is widely expressed in the injected tumours. (A) PMIS‐EV and PMIS‐miR‐210 injected SW620 tumours from Foxn1 nu/j (from Figure ) mice were sectioned and stained for GFP, and Ki67 expression. GFP is expressed from both PMIS‐EV and PMIS‐miR‐210 expression constructs. (B and C) The relative intensities of GFP and Ki67 immunofluorescence were calculated using ImageJ and values shown in the graphs. Four independent sections for each of the two tumours per group were used, N = 4 each. (D) Serial sections from the tumours were probed for cleaved caspase 3 (CC3) expression to demonstrate active apoptosis. (E) CC3 expression measured by immunofluorescence was significantly increased in SW620 tumours expressing PMIS‐miR‐210 . N = 3; * p < .05

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Injection, Staining, Expressing, Construct, Immunofluorescence

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: The tumour suppressor NME1 is regulated by miR‐210 . (A) RNA‐seq from SW620 cells and tumours were specifically analyzed for XIST , NME1 , BRCA1 , FGFRL1 , BRK1 and MYC expression with and without PMIS‐miR‐210 expression. (B) Transcript levels of the genes in panel A were validated by qPCR in SW620 cells with and without PMIS‐miR‐210 expression. N = 3. (C) NME1 protein levels were increased in PMIS‐miR‐210 ‐transduced SW620 cells compared to PMIS‐EV and untreated cell controls. (D and E) NME1 expression measured by immunofluorescence was significantly increased in SW620 tumours expressing PMIS‐miR‐210 . N = 6; * p < .05

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: RNA Sequencing, Expressing, Immunofluorescence

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: miR‐210 regulates XIST transcript levels. (A) RNA‐seq and qPCR showing that miR‐210 levels were inhibited over 80% in SW620 cells and XIST transcript were increased 143‐fold in PMIS‐miR‐210 ‐transduced SW620 cells. (B) miR‐210 expression increases in metastatic colorectal SW620 cells. (C) Demonstration of an inverse relationship between miR‐210 and XIST expression. Increasing amounts of transfected miR‐210 expression vector resulted in decreasing amounts of endogenous XIST transcripts. Increasing amounts of transfected PMIS‐miR‐210 correlate with decreased miR‐210 and increased XIST transcripts. The mouse (Mu) and Human (Hu) XIST transcripts contain miR‐210 binding sites. (D) Dual luciferase reporter assays in 293 cells with a Luc‐XIST reporter, 2.5 μg (contains miR‐210 binding sequence) and Luc‐XIST mut reporter 2.5 μg ( miR‐210 binding sequences mutated). miR‐210 , miR‐200c , scrambled and empty vector (EV) expression constructs (5 μg) were transfected in 293 cells. Luciferase was measured after 48 h post transfection. N = 3, * p < .05; ** p < .01

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: RNA Sequencing, Expressing, Transfection, Plasmid Preparation, Binding Assay, Luciferase, Sequencing, Construct

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: Epigenetic regulation of NME1 by miR‐210 and XIST . (A) Analyses of epigenetic regulators activating NME1 expression revealed that H3K27Ac and H3K4me3 both marked the NME1 transcription start site (TSS) and identified NME1 as an actively transcribed gene. (B) ChIP‐qPCR experiments demonstrate in SW620 cells transduced with PMIS‐miR‐210 that both H3K27Ac and H3K4me3 marks were increased at the proximal promoter. As a control, Brk1 was not affected, N = 4. (C and D) ChIP‐qPCR experiments in SW620 cells demonstrate that XIST overexpression increased H3K27ac and H3K4me3 chromatin marks at the NME1 TSS but not at the NME1 3′UTR, used as a control, N = 5 (D). SW620 cells transfected with PMIS‐miR‐210 increased both epigenetic marks at the NME1 TSS (C) but not at the NME1 3′UTR (D). SW620 cells co‐transfected with PMIS‐miR‐210 and shXIST , decreased the epigenetic marks compared to PMIS‐miR‐210 alone, indicating that miR‐210 inhibition of XIST plays a role in NME1 epigenetic regulation. N = 5, * p < .05; ** p < .01

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Expressing, ChIP-qPCR, Transduction, Control, Over Expression, Transfection, Inhibition

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: Cell‐to‐cell transfer of PMIS‐miR‐210 by extracellular vesicles (ECVs). (A) Transmission electron microscopy (TEM) photo of ECVs secreted from SW620 and SW620 PMIS‐miR‐210 cells. (B) Isolated ECVs were probed for CD63 and CD9 to prove their identity. (C) ECVs isolated from PMIS‐miR‐210 ‐transduced SW620 and 293 cells were probed by qPCR for PMIS‐miR‐210 transcripts. SW620 PMIS‐miR‐210 cells secreted high levels of PMIS‐miR‐210 in ECVs compared to controls and higher levels than 293 PMIS‐miR‐210 cells. (D) Transwell experiments demonstrate that SW620 cells transduced with PMIS‐miR‐210 (donor cells) could transfect and express PMIS‐miR‐210 in SW620 recipient cells. A ratio of 1/1 (2 × 10 5 donor cells to 2 × 10 5 recipient cells) showed that the recipient cells expressed the PMIS‐miR‐210 transcript after 72 h. Furthermore, miR‐210 expression was decreased in the recipient cells at a 1/1 ratio. The concentration of donor cells was decreased by half at 1 × 10 5 (0.5/1), while the recipient cell concentration was maintained at 2 × 10 5 and the recipient cells showed less expression of PMIS‐miR‐210 compared to the 1/1 ratio. miR‐210 expression was decreased but less than the 1/1 ratio. A ratio of 0.25 donor/1 recipient cell did not show a significant effect. As controls, the donor cells express high levels of PMIS‐miR‐210 and decreased levels of miR‐210 , while the recipient cells alone did not express PMIS‐miR‐210 . (E) Identical experiments show that the long non‐coding RNA (LncRNA X‐inactive specific transcript) XIST levels were increased in the recipient cells at 1/1 and 0.5/1 ratios. XIST is highly expressed in the SW620 PMIS‐miR‐210 ‐transduced cells but not in the SW620 cells. (F) The tumour suppressor NME1 is also increased in the recipient cells at 1/1 and 0.5/1 ratios. As a control, we show that P21 was not affected as it is not a target of miR‐210 . NME1 transcripts are increased in the SW620 PMIS‐miR‐210 ‐transduced donor cells. N = 3 for transwell experiments; * p < .05; ** p < .01

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Transmission Assay, Electron Microscopy, Isolation, Transduction, Expressing, Concentration Assay, Control

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: miR‐210 controls cytoplasmic and nuclear accumulation of XIST . (A) Immunofluorescence of XIST transcripts in SW620 cells and PMIS‐miR‐210 ‐transduced SW620 cells by in situ hybridization. The nuclei are stained with DAPI (blue). (B) miR‐210 and XIST transcripts are decreased in SW620 cells after treatment with actinomycin D, measured by qPCR and C t values plotted. However, XIST transcripts are relatively stable in PMIS‐miR‐210 ‐transduced SW620 cells, while miR‐210 transcripts are rapidly degraded in the presence of actinomycin D. β‐Tubulin was used as a control and rapidly degraded after 3 days of actinomycin treatment. N = 3; representative experiment shown

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Immunofluorescence, In Situ Hybridization, Staining, Control

Journal: International journal of cancer

Article Title: G protein-coupled receptor GPR55 promotes colorectal cancer and has opposing effects to cannabinoid receptor 1

doi: 10.1002/ijc.31030

Figure Lengend Snippet: (A, B) mRNA expression levels were determined by qRT-PCR in the colon of healthy control mice (healthy) and in colon tissue collected from mice subjected to the colitis-associated CRC model (non-tumor and tumor), reference gene: Hprt. (A) CB1 mRNA was found to be up-regulated in non-tumor tissue and down-regulated in tumor tissue compared to healthy colon tissue. (B) GPR55 mRNA, in contrast, was down-regulated in non-tumor tissue compared to healthy control colon whereas it was up-regulated in tumors. Statistical analysis for graphs A and B was performed using one-way ANOVA and Tukey’s posthoc test. * P < .05, *** P < .001 (C-F) Data of CRC patients were obtained from OncoTrack. (C, D) mRNA expression levels were obtained by RNAseq. Tumors (n=86) were TNM staged according to the guidelines of the Union internationale contre le cancer (UICC). Control samples (n=21) were obtained from tumor-adjacent non-neoplastic colon of CRC patients. Statistical analysis was done by one-way ANOVA followed by Tukey’s posthoc test. * P < .05, *** P < .001 (C) CB1 mRNA was down-regulated in tumors but increased with disease severity. (D) In contrast, GPR55 mRNA decreased with stage. (E, F) DNA methylation status was assessed in tumor samples (n=81, white bars) and in tumor-free control tissue (n=67, black bars). Each set of bars shows the mean + SEM degree of methylation in one CpG. The hg19 coordinate of each CpG is indicated by the x-axis label. Statistical analysis was performed with a regularized t-test that was conducted using the limma package in R as described in detail elsewhere27; P values were adjusted for multiple-testing using the false-discovery method. * P < .01, ** P < .001, *** P < .0001 (E) In tumor samples, CNR1 methylation was increased at CpG islands surrounding the promoter region whereas it was decreased in the body of the gene. (F) GPR55 lacks CpG islands and rather exhibits a global decrease in methylation in tumor samples. (G) GPR55 mRNA levels were increased in DLD-1, HCT116, SW620, and HT29 cells after treatment with 5-Aza-dC. Data shown are means + SEM of three independent experiments, reference gene: HPRT1. (H) Stable over-expression of GPR55 led to a growth advantage of SW480 cells. Data shown are means + SD of six independent experiments. Statistical analysis was done by one-way ANOVA and Tukey’s posthoc test. * P < .05, ** P < .01

Article Snippet: Cell culture Colon cancer cell lines HCT116, SW480, SW620, HT29 and DLD-1 were obtained from Interlab Cell Line Collection (Genova, Italy), CaCo-2 from ATCC (Manassas, VA, USA).

Techniques: Expressing, Quantitative RT-PCR, DNA Methylation Assay, Methylation, Over Expression