Journal: Journal of Translational Medicine

Article Title: Reduction of miR-744 delivered by NSCLC cell-derived extracellular vesicles upregulates SUV39H1 to promote NSCLC progression via activation of the Smad9/BMP9 axis

doi: 10.1186/s12967-020-02654-9

Figure Lengend Snippet: miR-744 transferred by cancer cell-derived EVs represses the development of NSCLC by regulating the SUV39H1/Smad9/BMP4 axis in A549 cells. a Overexpression efficiency of Smad9 determined by RT-qPCR in A549 cells. b Western blot analysis validating the overexpression efficiency of Smad9 in A549 cells (* p < 0.05, vs. OE-NC-treated A549 cells). c Expression of miR-744, SUV39H1, Smad9, and BMP4 determined by RT-qPCR in OE-Smad9-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells. d Representative Western blots of SUV39H1, Smad9, and BMP4 and p-Smad9 and their quantitation in OE-Smad9-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells. e Viability of OE-Smad9-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells measured using CCK-8 assay. f Colony formation of OE-Smad9-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells measured using colony formation assay (* p < 0.05, vs. si-NC-transfected A549 cells treated with EVs from BEAS-2B cells; # p < 0.05, vs. si-NC-transfected A549 cells treated with EVs from H1299 cells). g – j Results of in vivo tumor growth . A549 cells with or without Smad9 overexpression were injected into mice to establish the xenograft model and the EVs were injected into the tumors 7 days after transplantation. Tumor volume ( g ) was measured every 3 days; and final tumor volume ( h ) and weight ( i ) were measured, averaged, and compared after 4 weeks. The expression of proliferation marker, Ki-67 ( j ), was measured by IHC staining images were taken (* p < 0.05, vs. mice injected with A549 cells treated with BEAS-2B-EVs; # p < 0.05, vs. mice injected with A549 cells treated with H1299-EVs). k Knockdown efficiencies of 3 siRNAs targeting BMP4 determined by RT-qPCR in A549 cells. l Western blot analysis validating the knockdown efficiencies of 3 siRNAs targeting BMP4 in A549 cells (* p < 0.05, vs. A549 cells transfected with si-NC). m Expression of SUV39H1, Smad9, and BMP4 in si-BMP4-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells. n Western blots of SUV39H1, Smad9, BMP4 and p-Smad9 and their quantitation in si-BMP4-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells. o Viability of si-BMP4-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells measured using CCK-8 assay. p Colony formation of si-BMP4-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells measured using colony formation assay (* p < 0.05, vs. si-NC-transfected A549 cells treated with EVs from BEAS-2B cells; # p < 0.05, vs. si-NC-transfected A549 cells treated with EVs from H1299 cells). Data are summarized as mean ± standard deviation. Data from two groups were compared using unpaired t- test and data from multiple groups were assessed by one-way ANOVA with Tukey's tests. Data obtained at different time points were assessed by Bonferroni-corrected repeated measures ANOVA. Mice: n = 8/group. Each experiment was repeated three times

Article Snippet: A549 cells were transfected with miR-744 mimic or inhibitor (Ambion, Carlsbad, CA, USA), overexpression (OE) plasmids (OE-SUV39H1, OE-Smad9) (GeneChem, Shanghai, China), or small interfering RNAs (siRNAs) (si-SUV39H1, si-Smad9, or si-BMP4) (RiboBio, Guangdong, China), respectively.

Techniques: Derivative Assay, Over Expression, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Quantitation Assay, CCK-8 Assay, Colony Assay, In Vivo, Injection, Transplantation Assay, Marker, Immunohistochemistry, Knockdown, Standard Deviation

Journal: Scientific Reports

Article Title: Up-regulation of chemokine receptor CCR4 is associated with Human Hepatocellular Carcinoma malignant behavior

doi: 10.1038/s41598-017-10267-4

Figure Lengend Snippet: Clinicopathological data.

Article Snippet: Green fluorescent protein (GFP)-labeled lentiviral vector and four short hairpin RNA (shRNA) targeting CCR4 were purchased from Genepharma Shanghai.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Up-regulation of chemokine receptor CCR4 is associated with Human Hepatocellular Carcinoma malignant behavior

doi: 10.1038/s41598-017-10267-4

Figure Lengend Snippet: Immunohistochemistry to determine expression of CCR4 and its clinical significance in HCC patients. ( A ) CCR4 expression level in HCC tumor tissues and the paired normal tissues evaluated by immunohistochemical staining with tissue microarray. (Upper) 20×; (lower) 200×. ( B and C ) CCR4 scores based on the cytoplasmic or nuclear levels of expression in 75 HCC patients, compared with matched normal tissues. ( D and E ) HCC patients with high expression of CCR4 presented with worse overall survival, and disease free survival compared with that of low expression of CCR4. *p < 0.05, **p < 0.03, ***p < 0.01. Data represent the mean ± SD and are representive of three independent experiments.

Article Snippet: Green fluorescent protein (GFP)-labeled lentiviral vector and four short hairpin RNA (shRNA) targeting CCR4 were purchased from Genepharma Shanghai.

Techniques: Immunohistochemistry, Expressing, Immunohistochemical staining, Staining, Microarray

Journal: Scientific Reports

Article Title: Up-regulation of chemokine receptor CCR4 is associated with Human Hepatocellular Carcinoma malignant behavior

doi: 10.1038/s41598-017-10267-4

Figure Lengend Snippet: Relationship between CCR4 expression, HCC Differention, and Nuclear Grade.

Article Snippet: Green fluorescent protein (GFP)-labeled lentiviral vector and four short hairpin RNA (shRNA) targeting CCR4 were purchased from Genepharma Shanghai.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Up-regulation of chemokine receptor CCR4 is associated with Human Hepatocellular Carcinoma malignant behavior

doi: 10.1038/s41598-017-10267-4

Figure Lengend Snippet: CCR4 expression does not affect the proliferation of HCC cells in vitro . ( A and B ) CCR4 expression level in eight HCC cell lines detected by qRT-PCR or western blot. ( C ) Left panel : effect of shCCR4 on CCR4 expression in BEL-7405 cells compared with shNC detected by western blot; Right panel : ectopic expression of CCR4 in HepG2 transfected with lentivirus-CCR4 and lentivirus-Vector detected by western blot. ( D ) Proliferation curve doesn’t show significantly effect on HCC cell growth for CCR4 down-regulate group or CCR4 up-regulate group. Data represent the mean ± SD and are representive of three independent experiments.

Article Snippet: Green fluorescent protein (GFP)-labeled lentiviral vector and four short hairpin RNA (shRNA) targeting CCR4 were purchased from Genepharma Shanghai.

Techniques: Expressing, In Vitro, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Up-regulation of chemokine receptor CCR4 is associated with Human Hepatocellular Carcinoma malignant behavior

doi: 10.1038/s41598-017-10267-4

Figure Lengend Snippet: CCR4 facilitates HCC cells angiogenesis in vitro and in vivo . ( A ) Silencing CCR4 in BEL-7405 cells could significantly decrease the tubular formation ability of HUVEC while CCR4 overexpression could increase tubular formation ability significantly. ( B ) Volumes of CCR4 knockdown nude mice tumors were significantly smaller than those in control nude mice tumors. ( C ) Volumes of CCR4 overexpress nude mice tumors were significantly larger than those in control nude mice tumors. ( D , E and F ) Expression of tumor vasculogenic mimicry marker VEGF and CD31 were significantly decreased in CCR4 knockdown nude mice tumors. *p < 0.05, **p < 0.03, ***p < 0.01. Data represent the mean ± SD and are representive of three independent experiments.

Article Snippet: Green fluorescent protein (GFP)-labeled lentiviral vector and four short hairpin RNA (shRNA) targeting CCR4 were purchased from Genepharma Shanghai.

Techniques: In Vitro, In Vivo, Over Expression, Knockdown, Control, Expressing, Marker

Journal: Scientific Reports

Article Title: Up-regulation of chemokine receptor CCR4 is associated with Human Hepatocellular Carcinoma malignant behavior

doi: 10.1038/s41598-017-10267-4

Figure Lengend Snippet: CCR4 promotes HCC cells metastasis in vitro and in vivo . ( A and B ) Wound-healing assay shows a significant decrease or increase in healing rate of the scramble wound in BEL-7405/shCCR4 and HepG2/CCR4 respectively. ( C ) Silencing CCR4 in BEL-7405 cells could reduce the migrated cells through transwell assay. ( D ) Overexpress CCR4 in HepG2 cells could significantly increase the migrated cells through transwell assay. ( E ) Typical image of the effect on lung metastases of HCC cells via tail vein injection. The arrows in the upper panel indicate lung metastasis tumors. Representative images of 18 F-FLT micro-PET/CT images of mice are shown at the middle panel , arrow indicates 18 F-FLT uptake positivity in thoracic metastatic lesions. While the pathological image showed in the lower panel , arrow indicate metastatic tumors. *p < 0.05, **p < 0.03, ***p < 0.01. Data represent the mean ± SD and are representive of three independent experiments.

Article Snippet: Green fluorescent protein (GFP)-labeled lentiviral vector and four short hairpin RNA (shRNA) targeting CCR4 were purchased from Genepharma Shanghai.

Techniques: In Vitro, In Vivo, Wound Healing Assay, Transwell Assay, Injection, Micro-PET

Journal: Scientific Reports

Article Title: Up-regulation of chemokine receptor CCR4 is associated with Human Hepatocellular Carcinoma malignant behavior

doi: 10.1038/s41598-017-10267-4

Figure Lengend Snippet: CCR4 induces Epithelial-mesenchymal transition (EMT) in HCC cells. ( A and B ) Western blot analyses showed that knockdown CCR4 in BEL-7405 cells could significantly decrease the expression of E-cadherin and increase the expression of N-cadherin and Vimentin. ( C ) Immunofluorescent staining showed that changes in EMT marker expression: E-cadherin expression is increased while Vimentin expression is decreased in BEL-7405/shCCR4 cells. ( D and E ) Western blot analyses showed that overexpress CCR4 in HepG2 cells could significantly decrease the expression of E-cadherin and increase the expression of N-cadherin and Vimentin. ( F ) Immunofluorescent staining showed that changes in EMT marker expression: Vimentin expression is increased while E-cadherin expression is decreased in HepG2/CCR4 cells. Data represent the mean ± SD and are representive of three independent experiments.

Article Snippet: Green fluorescent protein (GFP)-labeled lentiviral vector and four short hairpin RNA (shRNA) targeting CCR4 were purchased from Genepharma Shanghai.

Techniques: Western Blot, Knockdown, Expressing, Staining, Marker

Journal: Scientific Reports

Article Title: Up-regulation of chemokine receptor CCR4 is associated with Human Hepatocellular Carcinoma malignant behavior

doi: 10.1038/s41598-017-10267-4

Figure Lengend Snippet: MMP2 plays a crucial role in HCC cells invasion mediated by CCR4. ( A ) Eight metastasis-related genes showed a more than 2-fold mRNA differential expression in Tumor Metastasis PCR array. ( B ) Effect of shMMP2 on MMP2 expression in BEL-7405 and HepG2/CCR4 cells detected by western blot. ( C ) Wound-healing assay shows a significant inhibitory role of siMMP2 in healing rate of the scramble wound in BEL-7405 and HepG2/CCR4 cells respectively. ( D ) MMP2 expression level in 75 cases of HCC tumor tissues and the paired normal tissues evaluated by IHC staining. (Upper) 20×; (lower) 200×. ( E ) Expression correlation of CCR4 and MMP2 was analyzed in 75HCC patients using IHC. *p < 0.05, **p < 0.03, ***p < 0.01. Data represent the mean ± SD and are representive of three independent experiments.

Article Snippet: Green fluorescent protein (GFP)-labeled lentiviral vector and four short hairpin RNA (shRNA) targeting CCR4 were purchased from Genepharma Shanghai.

Techniques: Quantitative Proteomics, Expressing, Western Blot, Wound Healing Assay, Immunohistochemistry, Paraffin-embedded Immunohistochemistry

Journal: Scientific Reports

Article Title: Up-regulation of chemokine receptor CCR4 is associated with Human Hepatocellular Carcinoma malignant behavior

doi: 10.1038/s41598-017-10267-4

Figure Lengend Snippet: CCR4 up-regulates MMP2 expression through ERK/MAPK/AKT signaling pathway. ( A ) Effect of shCCR4 on p-ERK, p-P38, p-AKT, JNK and MMP2 expressions in BEL-7405 cells were detected by western blot. ( B ) Densitometry represents the expression of the proteins relative to GAPDH. ( C ) Effect of overexpress CCR4 on p-ERK, p-P38, p-AKT, JNK and MMP2 expressions in HepG2 cells were detected by western blot. ( D ) Densitometry represents the expression of the proteins relative to GAPDH. ( E and F ) Schematic representation of the effect of CCR4 facilitates HCC tumor biological behavior. Data represent the mean ± SD and are representive of three independent experiments.

Article Snippet: Green fluorescent protein (GFP)-labeled lentiviral vector and four short hairpin RNA (shRNA) targeting CCR4 were purchased from Genepharma Shanghai.

Techniques: Expressing, Western Blot