Journal: Cancer Cell International

Article Title: Overexpression of microRNA-367 inhibits angiogenesis in ovarian cancer by downregulating the expression of LPA1

doi: 10.1186/s12935-020-01551-x

Figure Lengend Snippet: Primer sequences for RT-qPCR

Article Snippet: The membrane was then incubated overnight at 4 °C with diluted primary antibody, i.e. one of the rabbit antibodies LPA1 (R&D system, Minneapolis, MN, USA, AF9963, 1:2000), MCM2 (R&D system, AF5778, 1:2000), MMP2 (R&D system, AF902, 1:2000), MMP9 (R&D system, AF909, 1:2000), VEGF (R&D system, AF-493-NA, 1:2000), and GAPDH (R&D system, AF5718, 1:2000).

Techniques:

Journal: Cancer Cell International

Article Title: Overexpression of microRNA-367 inhibits angiogenesis in ovarian cancer by downregulating the expression of LPA1

doi: 10.1186/s12935-020-01551-x

Figure Lengend Snippet: miR-367 is poorly expressed but LPA1 is highly expressed in ovarian cancer cells. a Differential expression of miR-367 in cancer tissue samples and adjacent normal tissue samples in ovarian cancer microarray dataset GSE48485. b Binding site map of miR-367 to the target gene LPA1 in the StarBase database. c Box diagram of differential expression of LPA1 gene in cancer tissue samples and adjacent normal tissue samples in ovarian cancer microarray dataset GSE66957

Article Snippet: The membrane was then incubated overnight at 4 °C with diluted primary antibody, i.e. one of the rabbit antibodies LPA1 (R&D system, Minneapolis, MN, USA, AF9963, 1:2000), MCM2 (R&D system, AF5778, 1:2000), MMP2 (R&D system, AF902, 1:2000), MMP9 (R&D system, AF909, 1:2000), VEGF (R&D system, AF-493-NA, 1:2000), and GAPDH (R&D system, AF5718, 1:2000).

Techniques: Quantitative Proteomics, Microarray, Binding Assay

Journal: Cancer Cell International

Article Title: Overexpression of microRNA-367 inhibits angiogenesis in ovarian cancer by downregulating the expression of LPA1

doi: 10.1186/s12935-020-01551-x

Figure Lengend Snippet: miR-367 downregulates LPA1 expression. a Bioinformatics prediction of binding sites for miR-367 and LPA1. b Luminescence activity of LPA1 by dual luciferase reporter gene assay. c RIP assay detection of the binding of miR-367 and LPA1 to Ago2. d Detection of relative expression of LPA1 in cancer tissues and adjacent normal tissues by RT-qPCR. e Analysis of protein level of LPA1 in cancer and adjacent normal tissues by western blot analysis. f Statistical expression of protein level of LPA1. g Correlation analysis between miR-367 and LPA1. h RT-qPCR results of the relative expression level of LPA1 in each group of cells. i Western blot analysis of LPA1 protein levels in each group of cells. j Statistical protein expression level of LPA1. Measurement data were expressed as mean ± standard deviation. Data in compliance with normal distribution and homogeneity between two groups were compared using t test. Comparisons among multiple groups were conducted by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05 vs. normal tissues or cells; # p < 0.05 vs. Inhibitor NC The experiment was repeated three times independently

Article Snippet: The membrane was then incubated overnight at 4 °C with diluted primary antibody, i.e. one of the rabbit antibodies LPA1 (R&D system, Minneapolis, MN, USA, AF9963, 1:2000), MCM2 (R&D system, AF5778, 1:2000), MMP2 (R&D system, AF902, 1:2000), MMP9 (R&D system, AF909, 1:2000), VEGF (R&D system, AF-493-NA, 1:2000), and GAPDH (R&D system, AF5718, 1:2000).

Techniques: Expressing, Binding Assay, Activity Assay, Luciferase, Reporter Gene Assay, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Cancer Cell International

Article Title: Overexpression of microRNA-367 inhibits angiogenesis in ovarian cancer by downregulating the expression of LPA1

doi: 10.1186/s12935-020-01551-x

Figure Lengend Snippet: Low expression of miR-367 upregulates the expression of LPA1 to promote proliferation, invasion, and angiogenesis of ovarian cancer A2780 cells. a RT-qPCR analysis results of the efficiency of LPA1 silencing by appropriate siRNAs. b miR-367 and LPA1 expression in cells determined by using RT-qPCR. c A2780 cell proliferation ability detected by Transwell assay (scale bar = 25 μm). d Invasion ability of A2780 cells measured by Transwell assay (scale bar = 50 μm). e Relative length of blood vessels. f Relative number of blood vessel branches. g – j Statistics of CAM blood vessel number, area, vessel area ratio and tissues area. k RT-qPCR detection of mRNA levels of related factors. l Western blot analysis of protein levels of related factors. m Statistics of protein expression levels of related factors. Measurement data were expressed as mean ± standard deviation. Data in compliance with normal distribution and homogeneity among multiple groups were conducted by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05 vs. NC. # p < 0.05 vs. cells treated with si-LPA1. The experiment was repeated three times independently

Article Snippet: The membrane was then incubated overnight at 4 °C with diluted primary antibody, i.e. one of the rabbit antibodies LPA1 (R&D system, Minneapolis, MN, USA, AF9963, 1:2000), MCM2 (R&D system, AF5778, 1:2000), MMP2 (R&D system, AF902, 1:2000), MMP9 (R&D system, AF909, 1:2000), VEGF (R&D system, AF-493-NA, 1:2000), and GAPDH (R&D system, AF5718, 1:2000).

Techniques: Expressing, Quantitative RT-PCR, Transwell Assay, Western Blot, Standard Deviation

Journal: Cancer Cell International

Article Title: Overexpression of microRNA-367 inhibits angiogenesis in ovarian cancer by downregulating the expression of LPA1

doi: 10.1186/s12935-020-01551-x

Figure Lengend Snippet: Low expression of LPA1 inhibits tumorigenic ability of ovarian cancer in nude mice. a Volume of mice tumor. b Weight of mice tumor. c RT-qPCR detection results of mRNA levels of related factors in tumor tissues. d Western blot analysis of the protein levels of related factors in tumor tissues. e Statistical graph of protein levels of related factors. f Immunohistochemistry for detecting CD34 expression (200×). Measurement data were expressed as mean ± standard deviation. Data in compliance with normal distribution and homogeneity between two groups were compared using t test. Comparisons among multiple groups were conducted by one-way ANOVA followed by Tukey’s post hoc test. Statistics of tumor volume at different time points was analyzed using repeated measures ANOVA. * p < 0.05 vs. normal tissues or cells. The experiment was repeated three times independently

Article Snippet: The membrane was then incubated overnight at 4 °C with diluted primary antibody, i.e. one of the rabbit antibodies LPA1 (R&D system, Minneapolis, MN, USA, AF9963, 1:2000), MCM2 (R&D system, AF5778, 1:2000), MMP2 (R&D system, AF902, 1:2000), MMP9 (R&D system, AF909, 1:2000), VEGF (R&D system, AF-493-NA, 1:2000), and GAPDH (R&D system, AF5718, 1:2000).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Standard Deviation

Journal: Cancer Cell International

Article Title: Overexpression of microRNA-367 inhibits angiogenesis in ovarian cancer by downregulating the expression of LPA1

doi: 10.1186/s12935-020-01551-x

Figure Lengend Snippet: The regulatory mechanism map. Overexpressed miR-367 could reduce the expression of LPA1 and inhibit the proliferation and invasion of cancer cells, and the process of tumor-induced angiogenesis, hereby repressing ovarian cancer development

Article Snippet: The membrane was then incubated overnight at 4 °C with diluted primary antibody, i.e. one of the rabbit antibodies LPA1 (R&D system, Minneapolis, MN, USA, AF9963, 1:2000), MCM2 (R&D system, AF5778, 1:2000), MMP2 (R&D system, AF902, 1:2000), MMP9 (R&D system, AF909, 1:2000), VEGF (R&D system, AF-493-NA, 1:2000), and GAPDH (R&D system, AF5718, 1:2000).

Techniques: Expressing

Journal: Cancer Cell International

Article Title: Overexpression of microRNA-367 inhibits angiogenesis in ovarian cancer by downregulating the expression of LPA1

doi: 10.1186/s12935-020-01551-x

Figure Lengend Snippet: Primer sequences for RT-qPCR

Article Snippet: Then tissue sections were washed with PBS, placed in 0.01 M citrate buffer, boiled at 95 °C for 20 min, cooled to room temperature, rinsed with PBS, and blocked with normal goat serum working solution with 37 °C for 10 min. Tissue sections were incubated with anti-rabbit CD34 (R&D system, AF4117, 1:100) and LPA1 (R&D system, AF9963, 1:100) at 4 °C for 12 h. After washing with PBS, the corresponding biotin-labeled secondary antibody IgG goat anti-rabbit (R&D system, AB-105-C, 1:20,000) was added and kept for 10 min. Next, horseradish peroxidase-labeled streptavidin working solution (S-A/HRP) was added and kept for 10 min. Tissue sections were developed with diaminobenzidine (DAB) and stored in the dark for 8 min. Then cells were washed with tap water, stained with hematoxylin, dehydrated, transparentized, blocked, and observed by light microscopy.

Techniques:

Journal: Cancer Cell International

Article Title: Overexpression of microRNA-367 inhibits angiogenesis in ovarian cancer by downregulating the expression of LPA1

doi: 10.1186/s12935-020-01551-x

Figure Lengend Snippet: miR-367 is poorly expressed but LPA1 is highly expressed in ovarian cancer cells. a Differential expression of miR-367 in cancer tissue samples and adjacent normal tissue samples in ovarian cancer microarray dataset GSE48485. b Binding site map of miR-367 to the target gene LPA1 in the StarBase database. c Box diagram of differential expression of LPA1 gene in cancer tissue samples and adjacent normal tissue samples in ovarian cancer microarray dataset GSE66957

Article Snippet: Then tissue sections were washed with PBS, placed in 0.01 M citrate buffer, boiled at 95 °C for 20 min, cooled to room temperature, rinsed with PBS, and blocked with normal goat serum working solution with 37 °C for 10 min. Tissue sections were incubated with anti-rabbit CD34 (R&D system, AF4117, 1:100) and LPA1 (R&D system, AF9963, 1:100) at 4 °C for 12 h. After washing with PBS, the corresponding biotin-labeled secondary antibody IgG goat anti-rabbit (R&D system, AB-105-C, 1:20,000) was added and kept for 10 min. Next, horseradish peroxidase-labeled streptavidin working solution (S-A/HRP) was added and kept for 10 min. Tissue sections were developed with diaminobenzidine (DAB) and stored in the dark for 8 min. Then cells were washed with tap water, stained with hematoxylin, dehydrated, transparentized, blocked, and observed by light microscopy.

Techniques: Quantitative Proteomics, Microarray, Binding Assay

Journal: Cancer Cell International

Article Title: Overexpression of microRNA-367 inhibits angiogenesis in ovarian cancer by downregulating the expression of LPA1

doi: 10.1186/s12935-020-01551-x

Figure Lengend Snippet: miR-367 downregulates LPA1 expression. a Bioinformatics prediction of binding sites for miR-367 and LPA1. b Luminescence activity of LPA1 by dual luciferase reporter gene assay. c RIP assay detection of the binding of miR-367 and LPA1 to Ago2. d Detection of relative expression of LPA1 in cancer tissues and adjacent normal tissues by RT-qPCR. e Analysis of protein level of LPA1 in cancer and adjacent normal tissues by western blot analysis. f Statistical expression of protein level of LPA1. g Correlation analysis between miR-367 and LPA1. h RT-qPCR results of the relative expression level of LPA1 in each group of cells. i Western blot analysis of LPA1 protein levels in each group of cells. j Statistical protein expression level of LPA1. Measurement data were expressed as mean ± standard deviation. Data in compliance with normal distribution and homogeneity between two groups were compared using t test. Comparisons among multiple groups were conducted by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05 vs. normal tissues or cells; # p < 0.05 vs. Inhibitor NC The experiment was repeated three times independently

Article Snippet: Then tissue sections were washed with PBS, placed in 0.01 M citrate buffer, boiled at 95 °C for 20 min, cooled to room temperature, rinsed with PBS, and blocked with normal goat serum working solution with 37 °C for 10 min. Tissue sections were incubated with anti-rabbit CD34 (R&D system, AF4117, 1:100) and LPA1 (R&D system, AF9963, 1:100) at 4 °C for 12 h. After washing with PBS, the corresponding biotin-labeled secondary antibody IgG goat anti-rabbit (R&D system, AB-105-C, 1:20,000) was added and kept for 10 min. Next, horseradish peroxidase-labeled streptavidin working solution (S-A/HRP) was added and kept for 10 min. Tissue sections were developed with diaminobenzidine (DAB) and stored in the dark for 8 min. Then cells were washed with tap water, stained with hematoxylin, dehydrated, transparentized, blocked, and observed by light microscopy.

Techniques: Expressing, Binding Assay, Activity Assay, Luciferase, Reporter Gene Assay, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Cancer Cell International

Article Title: Overexpression of microRNA-367 inhibits angiogenesis in ovarian cancer by downregulating the expression of LPA1

doi: 10.1186/s12935-020-01551-x

Figure Lengend Snippet: Low expression of miR-367 upregulates the expression of LPA1 to promote proliferation, invasion, and angiogenesis of ovarian cancer A2780 cells. a RT-qPCR analysis results of the efficiency of LPA1 silencing by appropriate siRNAs. b miR-367 and LPA1 expression in cells determined by using RT-qPCR. c A2780 cell proliferation ability detected by Transwell assay (scale bar = 25 μm). d Invasion ability of A2780 cells measured by Transwell assay (scale bar = 50 μm). e Relative length of blood vessels. f Relative number of blood vessel branches. g – j Statistics of CAM blood vessel number, area, vessel area ratio and tissues area. k RT-qPCR detection of mRNA levels of related factors. l Western blot analysis of protein levels of related factors. m Statistics of protein expression levels of related factors. Measurement data were expressed as mean ± standard deviation. Data in compliance with normal distribution and homogeneity among multiple groups were conducted by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05 vs. NC. # p < 0.05 vs. cells treated with si-LPA1. The experiment was repeated three times independently

Article Snippet: Then tissue sections were washed with PBS, placed in 0.01 M citrate buffer, boiled at 95 °C for 20 min, cooled to room temperature, rinsed with PBS, and blocked with normal goat serum working solution with 37 °C for 10 min. Tissue sections were incubated with anti-rabbit CD34 (R&D system, AF4117, 1:100) and LPA1 (R&D system, AF9963, 1:100) at 4 °C for 12 h. After washing with PBS, the corresponding biotin-labeled secondary antibody IgG goat anti-rabbit (R&D system, AB-105-C, 1:20,000) was added and kept for 10 min. Next, horseradish peroxidase-labeled streptavidin working solution (S-A/HRP) was added and kept for 10 min. Tissue sections were developed with diaminobenzidine (DAB) and stored in the dark for 8 min. Then cells were washed with tap water, stained with hematoxylin, dehydrated, transparentized, blocked, and observed by light microscopy.

Techniques: Expressing, Quantitative RT-PCR, Transwell Assay, Western Blot, Standard Deviation

Journal: Cancer Cell International

Article Title: Overexpression of microRNA-367 inhibits angiogenesis in ovarian cancer by downregulating the expression of LPA1

doi: 10.1186/s12935-020-01551-x

Figure Lengend Snippet: Low expression of LPA1 inhibits tumorigenic ability of ovarian cancer in nude mice. a Volume of mice tumor. b Weight of mice tumor. c RT-qPCR detection results of mRNA levels of related factors in tumor tissues. d Western blot analysis of the protein levels of related factors in tumor tissues. e Statistical graph of protein levels of related factors. f Immunohistochemistry for detecting CD34 expression (200×). Measurement data were expressed as mean ± standard deviation. Data in compliance with normal distribution and homogeneity between two groups were compared using t test. Comparisons among multiple groups were conducted by one-way ANOVA followed by Tukey’s post hoc test. Statistics of tumor volume at different time points was analyzed using repeated measures ANOVA. * p < 0.05 vs. normal tissues or cells. The experiment was repeated three times independently

Article Snippet: Then tissue sections were washed with PBS, placed in 0.01 M citrate buffer, boiled at 95 °C for 20 min, cooled to room temperature, rinsed with PBS, and blocked with normal goat serum working solution with 37 °C for 10 min. Tissue sections were incubated with anti-rabbit CD34 (R&D system, AF4117, 1:100) and LPA1 (R&D system, AF9963, 1:100) at 4 °C for 12 h. After washing with PBS, the corresponding biotin-labeled secondary antibody IgG goat anti-rabbit (R&D system, AB-105-C, 1:20,000) was added and kept for 10 min. Next, horseradish peroxidase-labeled streptavidin working solution (S-A/HRP) was added and kept for 10 min. Tissue sections were developed with diaminobenzidine (DAB) and stored in the dark for 8 min. Then cells were washed with tap water, stained with hematoxylin, dehydrated, transparentized, blocked, and observed by light microscopy.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Standard Deviation

Journal: Cancer Cell International

Article Title: Overexpression of microRNA-367 inhibits angiogenesis in ovarian cancer by downregulating the expression of LPA1

doi: 10.1186/s12935-020-01551-x

Figure Lengend Snippet: The regulatory mechanism map. Overexpressed miR-367 could reduce the expression of LPA1 and inhibit the proliferation and invasion of cancer cells, and the process of tumor-induced angiogenesis, hereby repressing ovarian cancer development

Article Snippet: Then tissue sections were washed with PBS, placed in 0.01 M citrate buffer, boiled at 95 °C for 20 min, cooled to room temperature, rinsed with PBS, and blocked with normal goat serum working solution with 37 °C for 10 min. Tissue sections were incubated with anti-rabbit CD34 (R&D system, AF4117, 1:100) and LPA1 (R&D system, AF9963, 1:100) at 4 °C for 12 h. After washing with PBS, the corresponding biotin-labeled secondary antibody IgG goat anti-rabbit (R&D system, AB-105-C, 1:20,000) was added and kept for 10 min. Next, horseradish peroxidase-labeled streptavidin working solution (S-A/HRP) was added and kept for 10 min. Tissue sections were developed with diaminobenzidine (DAB) and stored in the dark for 8 min. Then cells were washed with tap water, stained with hematoxylin, dehydrated, transparentized, blocked, and observed by light microscopy.

Techniques: Expressing