With propidium iodide (PI, 50 mg/ml) for 30 min in the dark. The stained cells were analyzed with fluorescence-activated cell sorting (FACS) by flow cytometry (FACSCalibur, Becton Dickinson,Bedford, MA). The cell debris and Iloprost biological activity fixation artifacts were gated out and cell populations that were at the G0/G1, S, and G2/M phases were quantified using the ModFit software (Becton Dickinson). At least 10,000 cells in each sample were analyzed to obtain a measurable signal. For apoptosis analysis, an Annexin-V-FLUOS Staining kit (Roche, Mannheim, Germany) was used according to the manufacturer’s instructions 48 h after CASIN web transfection. Apoptosis was analyzed with FACS using the CellQuest software (Becton Dickinson). Annexin-V-FLUOS-positive cells were regarded as apoptotic cells.Immunohistochemistry and in situ HybridizationImmunohistochemical staining was performed with a two-step detection kit 23727046 (Zhongshan Goldenbridge, Beijing, China) as described previously [24]. The primary antibodies were Cyclin D1 (Santa Cruz, CA, 1:100 dilution) and Bcl-2 (Invitrogen, Carlsbad, CA, 1:200 dilution). A four-grade scoring system was used to evaluate the degree of Cyclin D1 immunostaining: score 0,Vector Construction and Luciferase Reporter AssayThe human Bcl-2 and Cyclin D1 39-UTRs, which contained predicted targets of miR-195, were amplified by PCR and cloned into a modified version of pcDNA3.1(+) that contained a fireflyMiR-195 Is a Prognostic Factor for TSCC PatientsTable 1. Relationship between expression of miR-195, Cyclin D1, and Bcl-2 and clinicopathologic factors in 81 TSCC patients.miR-195 (T/N) Characteristics Sex Male Female Age ,60 y 60 y Tumor size T1 2 T3 4 Differentiation Well Moderate Poor Clinical stage I I III V Node metastasis Yes No Status Survival Death 48 33 0.81060.755 0.43360.418 42 39 0.60360.592 0.70560.728 0.010 48 33 0.78060.770 0.46660.394 0.493 35 38 8 0.70960.753 0.59260.590 0.68760.582 0.019 53 28 0.77260.762 0.42560.297 0.747 45 36 0.70860.762 0.57060.502 0.005 45 36 0.62560.623 0.67460.693 0.321 No. Mean ?SDCyclin D1( )Bcl-2 ( ) No. of high expressionP0.No. of low expressionP0.No. of low expressionNo. of high expressionP0.2223 15 0.2718 10 0.2223 15 0.2619 9 0.3320 18 0.3716 12 0.18 2117 17 4 0.23 2612 12 4 0.2721 17 0.3216 12 0.1824 14 0.2616 12 0.28203513Abbreviations: T, tumor; N, nonmalignant tissue; T1 4: T stage of TNM classification system. doi:10.1371/journal.pone.0056634.tluciferase reporter gene (gift from Brigid L.M. Hogan, Duke University, Durham, NC, USA) [26], at a position downstream of the luciferase reporter, between the EcoRI and XhoI cloning sites. The vectors were named wild type 39UTRs and the primers for cloning the 39-UTRs of Cyclin D1 and Bcl-2 were as follows: Cyclin D1, sense, 59-GAT GAA TTC TTA TCC CCT GCC CCT TCC-39 and antisense, 59-TAT CTC GAG TGG GTC CAC CAT GGC TAA GTG A-39; Bcl-2, sense, 59-GAC GAA TTC AAT GCA GTG GTG CTT AC-39 and antisense, 59-CTT CTC GAG GAG GAG GTT CTC AGA TGT T-39. Site-directed mutagenesis of the miR-195 binding sites in Cyclin D1 and Bcl-2 39UTRs were performed using Site-Directed Mutagenesis Kit (SBS Genetech, Beijing, China) and named as mutant 39UTRs. The primers for cloning the mutant 39-UTRs of Cyclin D1 and Bcl-2 were as follows: Cyclin D1 binding site 1, sense, 59- TTT CTT ATT GCG CAC GTA CCG TTG ACT TCC AG-39 and antisense, 59- CTG GAA GTC AAC GGT ACG TGC GCA ATA AGA AA -39; Cyclin D1 binding site 2,sense, 59- CTT TCA CAT TGT TTG GAC CTA TTG GAG GAT CAG -39 and anti.With propidium iodide (PI, 50 mg/ml) for 30 min in the dark. The stained cells were analyzed with fluorescence-activated cell sorting (FACS) by flow cytometry (FACSCalibur, Becton Dickinson,Bedford, MA). The cell debris and fixation artifacts were gated out and cell populations that were at the G0/G1, S, and G2/M phases were quantified using the ModFit software (Becton Dickinson). At least 10,000 cells in each sample were analyzed to obtain a measurable signal. For apoptosis analysis, an Annexin-V-FLUOS Staining kit (Roche, Mannheim, Germany) was used according to the manufacturer’s instructions 48 h after transfection. Apoptosis was analyzed with FACS using the CellQuest software (Becton Dickinson). Annexin-V-FLUOS-positive cells were regarded as apoptotic cells.Immunohistochemistry and in situ HybridizationImmunohistochemical staining was performed with a two-step detection kit 23727046 (Zhongshan Goldenbridge, Beijing, China) as described previously [24]. The primary antibodies were Cyclin D1 (Santa Cruz, CA, 1:100 dilution) and Bcl-2 (Invitrogen, Carlsbad, CA, 1:200 dilution). A four-grade scoring system was used to evaluate the degree of Cyclin D1 immunostaining: score 0,Vector Construction and Luciferase Reporter AssayThe human Bcl-2 and Cyclin D1 39-UTRs, which contained predicted targets of miR-195, were amplified by PCR and cloned into a modified version of pcDNA3.1(+) that contained a fireflyMiR-195 Is a Prognostic Factor for TSCC PatientsTable 1. Relationship between expression of miR-195, Cyclin D1, and Bcl-2 and clinicopathologic factors in 81 TSCC patients.miR-195 (T/N) Characteristics Sex Male Female Age ,60 y 60 y Tumor size T1 2 T3 4 Differentiation Well Moderate Poor Clinical stage I I III V Node metastasis Yes No Status Survival Death 48 33 0.81060.755 0.43360.418 42 39 0.60360.592 0.70560.728 0.010 48 33 0.78060.770 0.46660.394 0.493 35 38 8 0.70960.753 0.59260.590 0.68760.582 0.019 53 28 0.77260.762 0.42560.297 0.747 45 36 0.70860.762 0.57060.502 0.005 45 36 0.62560.623 0.67460.693 0.321 No. Mean ?SDCyclin D1( )Bcl-2 ( ) No. of high expressionP0.No. of low expressionP0.No. of low expressionNo. of high expressionP0.2223 15 0.2718 10 0.2223 15 0.2619 9 0.3320 18 0.3716 12 0.18 2117 17 4 0.23 2612 12 4 0.2721 17 0.3216 12 0.1824 14 0.2616 12 0.28203513Abbreviations: T, tumor; N, nonmalignant tissue; T1 4: T stage of TNM classification system. doi:10.1371/journal.pone.0056634.tluciferase reporter gene (gift from Brigid L.M. Hogan, Duke University, Durham, NC, USA) [26], at a position downstream of the luciferase reporter, between the EcoRI and XhoI cloning sites. The vectors were named wild type 39UTRs and the primers for cloning the 39-UTRs of Cyclin D1 and Bcl-2 were as follows: Cyclin D1, sense, 59-GAT GAA TTC TTA TCC CCT GCC CCT TCC-39 and antisense, 59-TAT CTC GAG TGG GTC CAC CAT GGC TAA GTG A-39; Bcl-2, sense, 59-GAC GAA TTC AAT GCA GTG GTG CTT AC-39 and antisense, 59-CTT CTC GAG GAG GAG GTT CTC AGA TGT T-39. Site-directed mutagenesis of the miR-195 binding sites in Cyclin D1 and Bcl-2 39UTRs were performed using Site-Directed Mutagenesis Kit (SBS Genetech, Beijing, China) and named as mutant 39UTRs. The primers for cloning the mutant 39-UTRs of Cyclin D1 and Bcl-2 were as follows: Cyclin D1 binding site 1, sense, 59- TTT CTT ATT GCG CAC GTA CCG TTG ACT TCC AG-39 and antisense, 59- CTG GAA GTC AAC GGT ACG TGC GCA ATA AGA AA -39; Cyclin D1 binding site 2,sense, 59- CTT TCA CAT TGT TTG GAC CTA TTG GAG GAT CAG -39 and anti.
