Journal: Nature Communications

Article Title: Akt-p53-miR-365-cyclin D1/cdc25A axis contributes to gastric tumorigenesis induced by PTEN deficiency

doi: 10.1038/ncomms3544

Figure Lengend Snippet: ( a ) Profiling data of the upregulated (red) and downregulated (green) miRNAs in PTEN -mutant gastric epithelium at P20 and P60 (mean change fold >2; P <0.01, Student’s t -test). ( b ) The expression of miR-365 in PTEN -mutant and control epithelium at P20 and P60 was measured using northern blot analysis ( n= 4). ( c ) Cell viability was determined by cell count. Gastric cancer cell lines AGS, BGC-823 and SGC-7901 were transfected with miR-365 (365) or scrambled (Scr) mimic for 72 h ( n= 4–8). BGC-823 cells were transfected with miR-365 antisense oligonucleotides (anti-365) or scrambled antisense oligonucleotides (anti-Scr) for 72 h ( n= 4). ( d ) Tumour formation assay of stably miR-365-expressing (miR-365) and control (vector) BGC-823 cells in a mouse xenograft ( n= 8). Photographs illustrated representative feature of tumour growth ~4 weeks after injection. Tumour growth was determined by tumour volume. The lines between controls and miR-365-overexpressing xenograft tumours represented the connected data from the corresponding sides of the same nude mouse. ** P <0.01 was calculated using Pair-Sample t -test. ( e ) Luciferase reporter activities of reporter constructs. BGC-823 cells were cotransfected with the reporter constructs containing wild-type (WT) 3′-UTRs of cyclin D1 or cdc25A, as well as mutant (Mut) 3′-UTRs with the putative target sites mutated (As illustrated in ), together with miR-365 or scrambled mimic ( n= 6). ( f ) The protein levels of cyclin D1 and cdc25A were determined using western blot analysis. BGC-823 cells were transfected with miR-365 (365) or scrambled (Scr) mimic as well as miR-365 (anti-365) and scrambled (anti-Scr) antisense oligonucleotides ( n= 6). ( g ) Cell viability was determined by cell count. BGC-823 cells were cotransfected with miR-365 mimic and pCMV-HA-cyclin D1 or pCMV6-cdc25A-GFP construct for 24 h ( n= 3). The upper bands in western blot indicated the exogenous expression of cyclin D1 or cdc25A. The lower bands indicated the endogenous ones. Values represent the mean±s.d. * P <0.05 and ** P <0.01 were calculated using Student’s t -test.

Article Snippet: The Akt siRNA (CST; no. 6211), p53 siRNA (CST; no. 6512), cyclin D1 siRNA (Santa Cruz; SC-29286) and cdc25A siRNA (Santa Cruz; SC-29254) were transfected with a final concentration of 100 nM.

Techniques: Mutagenesis, Expressing, Northern Blot, Cell Counting, Transfection, Tube Formation Assay, Stable Transfection, Plasmid Preparation, Injection, Luciferase, Construct, Western Blot

Journal: Nature Communications

Article Title: Akt-p53-miR-365-cyclin D1/cdc25A axis contributes to gastric tumorigenesis induced by PTEN deficiency

doi: 10.1038/ncomms3544

Figure Lengend Snippet: ( a ) Schematic of orthotopic miRNA injection protocols. Beginning at P25, 50 μg (5 μg μl −1 ) miR-365 mimic was injected into the ventral submucosa of PTEN -mutant gastric corpus, and 50 μg control mimic was injected into the opposite dorsal submucosa, every 4 days for 16 days. ( b ) miR-365 expression was detected using qRT–PCR 3 days after injection ( n =3). ( c ) HE and immunohistochemical analysis of the expression of p-Akt, BrdU and cyclin D1 in miR-365 and control mimic-injected gastric epithelium ( n =5). Scale bar represents 100 μm. ( d ) The expression of cyclin D1 and cdc25A was detected using qRT–PCR in miR-365 and control mimic-injected gastric epithelium ( n =5). Values represent the mean±s.d. * P <0.05 and ** P <0.01 were calculated using Student’s t -test.

Article Snippet: The Akt siRNA (CST; no. 6211), p53 siRNA (CST; no. 6512), cyclin D1 siRNA (Santa Cruz; SC-29286) and cdc25A siRNA (Santa Cruz; SC-29254) were transfected with a final concentration of 100 nM.

Techniques: Injection, Mutagenesis, Expressing, Quantitative RT-PCR, Immunohistochemical staining

Journal: Nature Communications

Article Title: Akt-p53-miR-365-cyclin D1/cdc25A axis contributes to gastric tumorigenesis induced by PTEN deficiency

doi: 10.1038/ncomms3544

Figure Lengend Snippet: ( a ) The relative level of miR-365 in 127 human gastric tumour tissues (black) and matched normal tissues distal to tumour regions (grey) detected using qRT–PCR. The band represents the mean. P <0.001 was calculated using Pair-Sample t -test. ( b ) A correlation between miR-365 and PTEN expression was determined using Spearman coefficient analysis in human gastric tumour tissues ( n =24, r 2 =0.682, P <0.001). ( c , d ) Correlations between miR-365 and cyclin D1 or cdc25A expression were determined using Spearman coefficient in human gastric tumour tissues (cyclin D1: n =24, r 2 =0.651, P <0.001; cdc25A: n =25, r 2 =0.596, P <0.01).

Article Snippet: The Akt siRNA (CST; no. 6211), p53 siRNA (CST; no. 6512), cyclin D1 siRNA (Santa Cruz; SC-29286) and cdc25A siRNA (Santa Cruz; SC-29254) were transfected with a final concentration of 100 nM.

Techniques: Quantitative RT-PCR, Expressing