Of 39-UTRs of Cyclin D1 or Bcl-2 by miR-195. SCC-15 and CAL27 cells were co-transfected with firefly luciferase reporter plasmids containing wildtype (Wt) and mutant wildtype and mutant (Mut) 39-UTRs of Cyclin D1 or Bcl-2, and pRL-TK plasmid (a plasmid expressing rellina luciferase) and pcDNA3.0-miR-195 (miR-195) or pcDNA3.0 as indicated. After 48 h, firefly luciferase activities were measured and normalized by renilla luciferase activities. Data were presented as mean 6 SD (n = 3) (**P,0.01). (C), Inhibition of Gracillin protein expression of Cyclin D1 and Bcl-2. SCC-15 and CAL27 cells were transfected with pcDNA3.0 as a negative control (NC) or with pcDNA3.0-miR-195 (miR-195) as indicated. After 48 h, Cyclin D1, Bcl-2 and internal control b-actin were detected by Western blotting. doi:10.1371/journal.pone.0056634.gMiR-195 Is a Prognostic Factor for TSCC PatientsMiR-195 Is a Prognostic Factor for TSCC PatientsFigure 6. Inhibition of Cyclin D1 and Bcl-2 was responsible for the tumor suppressive effects of miR-195. (A), Inhibition of cell cycle progression by knockdown of Cyclin D1. SCC-15 and CAL27 cells were transfected with control RNA (Ctrl RNA) or Cyclin D1 siRNA as indicated. Cells were stained with propidium iodide (PI) at 48 h post-transfection and analyzed with FACS (*P,0.05, **P,0.01). (B), Promotion of apoptosis by knockdown of Bcl-2. SCC-15 and CAL27 cells were transfected with control RNA (Ctrl RNA) or Bcl-2 siRNA as indicated. Apoptotic cells were monitored with FACS after Annexin V and PI staining (**P,0.01). doi:10.1371/journal.pone.0056634.gstable levels in the patient samples in question. In this 
respect, miRNAs are relatively stable as compared with other biological macromolecules. They can be well preserved in tissue samples even after formalin fixation and paraffin-embedding, and can be efficiently extracted and evaluated [28,29]. Therefore, based on our observations that decreased miR-195 expression was associated with poor overall survival in TSCC patients, we anticipate that miR-195 could be a Anlotinib web useful prognostic factor for TSCC and that its stability should allow the development of practical, economical methods for TSCC detection. Moreover, the development of cancers involves the altered expression of multiple genes, so the protein product of a single oncogene may not accurately reflect the status of the disease. However, just as other single miRNAs are known to target multiple messenger RNAs to regulate gene expression [30], miR-195 can also regulate multiple coding genes that are related to tumor growth [31]. Thus, expression of miR-195 is likely to reflect altered physiology of TSCC more precisely and effectively than any of the target genes alone. Although our data failed to establish evidence for Cyclin D1 and Bcl-2 expression as prognostic markers in TSCC, we did demonstrate for the first time that immunostaining of Cyclin D1 and 1326631 Bcl-2 is inversely correlated with miR-195 levels in TSCC tissues. Because Cyclin D1 and Bcl-2 have been shown to be direct targets of miR-195 [16,17] and their expression in TSCC may account for the effect of miR-195, we examined the expression of these two proteins in paraffin sections of TSCC samples using immunohistochemistry. In this study, the expression of the Cyclin D1 was only statistically significantly associated with the tumor size of TSCC but not with other clinicopathological factors analyzed, whereas the expression of Bcl-2 in TSCC was not statistically significantly associated.Of 39-UTRs of Cyclin D1 or Bcl-2 by miR-195. SCC-15 and CAL27 cells were co-transfected with firefly luciferase reporter plasmids containing wildtype (Wt) and mutant wildtype and mutant (Mut) 39-UTRs of Cyclin D1 or Bcl-2, and pRL-TK plasmid (a plasmid expressing rellina luciferase) and pcDNA3.0-miR-195 (miR-195) or pcDNA3.0 as indicated. After 48 h, firefly luciferase activities were measured and normalized by renilla luciferase activities. Data were presented as mean 6 SD (n = 3) (**P,0.01). (C), Inhibition of protein expression of Cyclin D1 and Bcl-2. SCC-15 and CAL27 cells were transfected with pcDNA3.0 as a negative control (NC) or with pcDNA3.0-miR-195 (miR-195) as indicated. After 48 h, Cyclin D1, Bcl-2 and internal control b-actin were detected by Western blotting. doi:10.1371/journal.pone.0056634.gMiR-195 Is a Prognostic Factor for TSCC PatientsMiR-195 Is a Prognostic Factor for TSCC PatientsFigure 6. Inhibition of Cyclin D1 and Bcl-2 was responsible for the tumor suppressive effects of miR-195. (A), Inhibition of cell cycle progression by knockdown of Cyclin D1. SCC-15 and CAL27 cells were transfected with control RNA (Ctrl RNA) or Cyclin D1 siRNA as indicated. Cells were stained with propidium iodide (PI) at 48 h post-transfection and analyzed with FACS (*P,0.05, **P,0.01). (B), Promotion of apoptosis by knockdown of Bcl-2. SCC-15 and CAL27 cells were transfected with control RNA (Ctrl RNA) or Bcl-2 siRNA as indicated. Apoptotic cells were monitored with FACS after Annexin V and PI staining (**P,0.01). doi:10.1371/journal.pone.0056634.gstable levels in the patient samples in question. In this respect, miRNAs are relatively stable as compared with other biological macromolecules. They can be well preserved in tissue samples even after formalin fixation and paraffin-embedding, and can be efficiently extracted and evaluated [28,29]. Therefore, based on our observations that decreased miR-195 expression was associated with poor overall survival in TSCC patients, we anticipate that miR-195 could be a useful prognostic factor for TSCC and that its stability should allow the development of practical, economical methods for TSCC detection. Moreover, the development of cancers involves the altered expression of multiple genes, so the protein product of a single oncogene may not accurately reflect the status of the disease. However, just as other single miRNAs are known to target multiple messenger RNAs to regulate gene expression [30], miR-195 can also regulate multiple coding genes that are related to tumor growth [31]. Thus, expression of miR-195 is likely to reflect altered physiology of TSCC more precisely and effectively than any of the target genes alone. Although our data failed to establish evidence for Cyclin D1 and Bcl-2 expression as prognostic markers in TSCC, we did demonstrate for the first time that immunostaining of Cyclin D1 and 1326631 Bcl-2 is inversely correlated with miR-195 levels in TSCC tissues. Because Cyclin D1 and Bcl-2 have been shown to be direct targets of miR-195 [16,17] and their expression in TSCC may account for the effect of miR-195, we examined the expression of these two proteins in paraffin sections of TSCC samples using immunohistochemistry. In this study, the expression of the Cyclin D1 was only statistically significantly associated with the tumor size of TSCC but not with other clinicopathological factors analyzed, whereas the expression of Bcl-2 in TSCC was not statistically significantly associated.