Journal: Cell Death & Disease

Article Title: Identification of microRNA signature in the progression of gestational trophoblastic disease

doi: 10.1038/s41419-017-0108-2

Figure Lengend Snippet: a Validation of the microarray profiling data. Expression levels of the most differentially expressed 16 miRNAs were examined by qRT-PCR in 34 cases of GTN and 60 cases of CHM trophoblastic tissues and then normalized to the housekeeping gene RNU6 with the 2 −ΔΔCt method. Differences were evaluated by using Mann–Whitney U -test. Fold changes were 0.26 for miR-370-3p, 217.56 for miR-371a-5p, 19.5 for miR-518a-3p, 9.63 for miR-519d-3p, 6.41 for miR-520a-3p, and 2.52 for miR-934. b miR-371a-5p and miR-518a-3p expression levels quantified by qRT-PCR in villous primary human trophoblasts (PHT), immortalized normal trophoblastic cell line HTR-8/SVneo, and choriocarcinoma cell lines BeWo, JAR, and JEG-3. Relative expressions in choriocarcinoma cells were normalized against corresponding miRNA levels in PHT cells. * P < 0.05, ** P < 0.01, *** P < 0.001. c Representative micrographs of miR-371a-5p and miR-518a-3p detected with fluorescence in situ hybridization. Positive signals for miR-371a-5p or miR-518a-3p are visualized in green, while blue depicts DAPI nuclear stain. Magnification 100×

Article Snippet: Two double-digoxigenin (DIG)-labeled oligonucleotide probes, complementary to mature miR-371a-5p (Exon Biotechnology, Guangzhou, China) and miR-518a-3p (Genscript, Nanjing, China), respectively, were synthesized.

Techniques: Biomarker Discovery, Microarray, Expressing, Quantitative RT-PCR, MANN-WHITNEY, Fluorescence, In Situ Hybridization, Staining

Journal: Cell Death & Disease

Article Title: Identification of microRNA signature in the progression of gestational trophoblastic disease

doi: 10.1038/s41419-017-0108-2

Figure Lengend Snippet: BeWo, JAR, and JEG-3 cells in 96-well plates were transfected with 30 nM miR-371a-5p or miR-518a-3p mimics ( a , left) or 40 nM inhibitors ( b , right). Cell viabilities were measured by CCK-8 assay at indicated time points post transfection. N = 12, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Two double-digoxigenin (DIG)-labeled oligonucleotide probes, complementary to mature miR-371a-5p (Exon Biotechnology, Guangzhou, China) and miR-518a-3p (Genscript, Nanjing, China), respectively, were synthesized.

Techniques: Transfection, CCK-8 Assay

Journal: Cell Death & Disease

Article Title: Identification of microRNA signature in the progression of gestational trophoblastic disease

doi: 10.1038/s41419-017-0108-2

Figure Lengend Snippet: BeWo, JAR, and JEG-3 cells in 24-well plates were transfected with 100 nM miR-371a-5p or miR-518a-3p mimics ( a , left) or 150 nM inhibitors ( b , right). Forty-eight hours post transfection, cells were stained with propidium iodide and quantified by flow cytometric analysis. N = 6, * P < 0.05, ** P < 0.01

Article Snippet: Two double-digoxigenin (DIG)-labeled oligonucleotide probes, complementary to mature miR-371a-5p (Exon Biotechnology, Guangzhou, China) and miR-518a-3p (Genscript, Nanjing, China), respectively, were synthesized.

Techniques: Transfection, Staining

Journal: Cell Death & Disease

Article Title: Identification of microRNA signature in the progression of gestational trophoblastic disease

doi: 10.1038/s41419-017-0108-2

Figure Lengend Snippet: BeWo, JAR, and JEG-3 cells were transfected with miR-371a-5p or miR-518a-3p mimics ( a , left) or inhibitors ( b , right). Twenty-four hours post transfection, cells were pretreated with mitomycin C and then harvested and seeded into transwell inserts with (for invasion) or without (for migration) Matrigel. After incubation, cells were fixed and stained, and then visualized under a microscope at 100× magnification. Five random selected fields were counted, and the average number was calculated for statistical analysis. N = 3, ** P < 0.01, *** P < 0.001

Article Snippet: Two double-digoxigenin (DIG)-labeled oligonucleotide probes, complementary to mature miR-371a-5p (Exon Biotechnology, Guangzhou, China) and miR-518a-3p (Genscript, Nanjing, China), respectively, were synthesized.

Techniques: Transfection, Migration, Incubation, Staining, Microscopy

Journal: Cell Death & Disease

Article Title: Identification of microRNA signature in the progression of gestational trophoblastic disease

doi: 10.1038/s41419-017-0108-2

Figure Lengend Snippet: a qRT-PCR quantification of the mRNA levels of BCCIP , BNIP3L , and SOX2 genes. BeWo, JAR, and JEG-3 cells were transfected with miR-371a-5p mimic/inhibitor or control mimic/inhibitor. Cells were harvested at 36 h post transfection, and α-tubulin was used as the endogenous reference gene. b Western blotting analysis of BCCIP, BNIP3L, and SOX2. Cells were harvested at 48 h post transfection, and α-tubulin was used as the endogenous control. c Direct interaction of miR-371a-5p and BCCIP . Upper: bioinformatic predicted miR-371a-5p targeting site in 3′-UTR of BCCIP . Lower: luciferase activity assay. At 48 h post co-transfection with BCCIP–WT or BCCIP–MUT and miR-371a-5p mimic/inhibitor or control mimic/inhibitor, luciferase activities were measured in HTR-8/SVneo cells. Renilla luciferase activities in corresponding controls were normalized to 1. Mck stands for control mimic and ick stands for control inhibitor. N = 9, * P < 0.05, *** P < 0.001

Article Snippet: Two double-digoxigenin (DIG)-labeled oligonucleotide probes, complementary to mature miR-371a-5p (Exon Biotechnology, Guangzhou, China) and miR-518a-3p (Genscript, Nanjing, China), respectively, were synthesized.

Techniques: Quantitative RT-PCR, Transfection, Control, Western Blot, Luciferase, Activity Assay, Cotransfection

Journal: Cell Death & Disease

Article Title: Identification of microRNA signature in the progression of gestational trophoblastic disease

doi: 10.1038/s41419-017-0108-2

Figure Lengend Snippet: Top 10 upregulated genes induced by miR-371a-5p or miR-518a-3p knockdown in BeWo, JAR, and JEG-3 cells

Article Snippet: Two double-digoxigenin (DIG)-labeled oligonucleotide probes, complementary to mature miR-371a-5p (Exon Biotechnology, Guangzhou, China) and miR-518a-3p (Genscript, Nanjing, China), respectively, were synthesized.

Techniques: Knockdown

Journal: Cell Death & Disease

Article Title: Identification of microRNA signature in the progression of gestational trophoblastic disease

doi: 10.1038/s41419-017-0108-2

Figure Lengend Snippet: BeWo, JAR, and JEG-3 cells were transfected with miR-371a-5p/miR-518a-3p inhibitors or control inhibitors. Transcriptomes were sequenced at 36 h post transfection, and the upregulated genes were identified with >2-fold changes and the adjusted P < 0.05. a Classification based on the upregulated genes after miR-371a-5p knockdown. Totally, 237, 132, and 277 genes were identified in BeWo, JAR, and JEG-3 cells, respectively. Left: BeWo cells; middle: JAR cells; right: JEG-3 cells. b Classification based on the upregulated genes after miR-518a-3p knockdown. Totally, 229, 269, and 191 genes were identified in BeWo, JAR, and JEG-3 cells, respectively. Left: BeWo cells; middle: JAR cells; right: JEG-3 cells

Article Snippet: Two double-digoxigenin (DIG)-labeled oligonucleotide probes, complementary to mature miR-371a-5p (Exon Biotechnology, Guangzhou, China) and miR-518a-3p (Genscript, Nanjing, China), respectively, were synthesized.

Techniques: Transfection, Control, Knockdown