Mor tissues compared with matched nonmalignant tissues (Fig. 1B; P,0.01). Moreover, miR-195 expression was also statistically significantly decreased in the TSCC cell lines SCC-15 and CAL27, compared with TSCC adjacent nonmalignant tissues (Fig. 1C). To further determine whether there was an association between miR-195 expression and tumor prognosis, we looked for a correlation between miR-195 expression and the clinicopathological features of TSCC. By normalizing miR-195 expression 1655472 levels in the tumor tissues with those in adjacent nonmalignant tissues (Tumor/Nonmalignant, T/N), we found that there were statistically significant relationships between miR-195 expression (T/N) and some clinicopathologic features of TSCC (Table 1), including tumor size (P = 0.005), clinical stage (P = 0.019), and patient mortality (P = 0.010).Decreased miR-195 Expression was Associated with Poor Overall Survival in TSCC PatientsWe further analyzed the correlation of miR-195 expression and postoperative overall survival of TSCC patients. With the mean fold change (T/N = 0.652) in miR-195 expression chosen as the cut-off point, we divided the patients into a high miR-195 expression group (T/N fold change .0.652) and a low miR-195 expression group (T/N fold change ,0.652). Patients with high miR-195 expression survived statistically significantly longer than those with low miR-195 expression (Fig. 2; P = 0.006). To identify whether miR-195 is an independent prognostic covariate for TSCC, we did a multivariable Cox proportional hazards analysis. In the final multivariable Cox regression model, low levels of miR195 expression in TSCC were associated with a poor prognosis in terms of overall survival (P = 0.025, relative risk = 0.322), independent of other clinical covariates (Table 2), suggesting that miR195 might be used as an independent prognostic factor for TSCC.Figure 1. miR-195 expression was reduced in human TSCC and cell lines. (A), Relative levels of miR-195 in 81 surgical specimens of TSCC and matched adjacent nonmalignant tissues was quantified by qRT-PCR. Data were presented as log2 fold change (DDCT values, TSCC/ 15857111 Nonmalignant, T/N). (B), Means of miR-195 relative levels for 81 surgical specimens of TSCC and the matched adjacent nonmalignant tissues. Data were presented as 22DCt (miR-195-U6) values (**P,0.01). (C), miR-195 expression was examined by qRT CR for 81 surgical specimens of nonmalignant tissues and cell lines as indicated. Data were presented as 22DCt (miR-195-U6) values (***P,0.001). doi:10.1371/journal.pone.0056634.gMiR-195 Is a Prognostic Factor for TSCC Patientsin human TSCC development, we analyzed the association of the expression of Cyclin D1 and Bcl-2 with clinicopathological Solvent Yellow 14 parameters. As shown in Table 1, the expression of Cyclin D1 was associated with tumor size of TSCC (P = 0.023), whereas the expression of Bcl-2 was not statistically significantly associated with any of the clinicopathologic parameters.MedChemExpress TA02 Overexpression of miR-195 Inhibited Cell Cycle Progression and Promoted Apoptosis in TSCC Cell LinesTo understand the biological function of miR-195 in TSCC, miR-195 was overexpressed in TSCC cell lines. In the first of these experiments, we transfected TSCC cells with miR-195 and performed cell counting assays to evaluate the effects of miR195 expression on cell proliferation and viability. Overexpression of miR-195 inhibited the viability of SCC-15 and CAL27 cells (Fig. 4A), leading to substantial accumulation of the cell p.Mor tissues compared with matched nonmalignant tissues (Fig. 1B; P,0.01). Moreover, miR-195 expression was also statistically significantly decreased in the TSCC cell lines SCC-15 and CAL27, compared with TSCC adjacent nonmalignant tissues (Fig. 1C). To further determine whether there was an association between miR-195 expression and tumor prognosis, we looked for a correlation between miR-195 expression and the clinicopathological features of TSCC. By normalizing miR-195 expression 1655472 levels in the tumor tissues with those in adjacent nonmalignant tissues (Tumor/Nonmalignant, T/N), we found that there were statistically significant relationships between miR-195 expression (T/N) and some clinicopathologic features of TSCC (Table 1), including tumor size (P = 0.005), clinical stage (P = 0.019), and patient mortality (P = 0.010).Decreased miR-195 Expression was Associated with Poor Overall Survival in TSCC PatientsWe further analyzed the correlation of miR-195 expression and postoperative overall survival of TSCC patients. With the mean fold change (T/N = 0.652) in miR-195 expression chosen as the cut-off point, we divided the patients into a high miR-195 expression group (T/N fold change .0.652) and a low miR-195 expression group (T/N fold change ,0.652). Patients with high miR-195 expression survived statistically significantly longer than those with low miR-195 expression (Fig. 2; P = 0.006). To identify whether miR-195 is an independent prognostic covariate for TSCC, we did a multivariable Cox proportional hazards analysis. In the final multivariable Cox regression model, low levels of miR195 expression in TSCC were associated with a poor prognosis in terms of overall survival (P = 0.025, relative risk = 0.322), independent of other clinical covariates (Table 2), suggesting that miR195 might be used as an independent prognostic factor for TSCC.Figure 1. miR-195 expression was reduced in human TSCC and cell lines. (A), Relative levels of miR-195 in 81 surgical specimens of TSCC and matched adjacent nonmalignant tissues was quantified by qRT-PCR. Data were presented as log2 fold change (DDCT values, TSCC/ 15857111 Nonmalignant, T/N). (B), Means of miR-195 relative levels for 81 surgical specimens of TSCC and the matched adjacent nonmalignant tissues. Data were presented as 22DCt (miR-195-U6) values (**P,0.01). (C), miR-195 expression was examined by qRT CR for 81 surgical specimens of nonmalignant tissues and cell lines as indicated. Data were presented as 22DCt (miR-195-U6) values (***P,0.001). doi:10.1371/journal.pone.0056634.gMiR-195 Is a Prognostic Factor for TSCC Patientsin human TSCC development, we analyzed the association of the expression of Cyclin D1 and Bcl-2 with clinicopathological parameters. As shown in Table 1, the expression of Cyclin D1 was associated with tumor size of TSCC (P = 0.023), whereas the expression of Bcl-2 was not statistically significantly associated with any of the clinicopathologic parameters.Overexpression of miR-195 Inhibited Cell Cycle Progression and Promoted Apoptosis in TSCC Cell LinesTo understand the biological function of miR-195 in TSCC, miR-195 was overexpressed in TSCC cell lines. In the first of these experiments, we transfected TSCC cells with miR-195 and performed cell counting assays to evaluate the effects of miR195 expression on cell proliferation and viability. Overexpression of miR-195 inhibited the viability of SCC-15 and CAL27 cells (Fig. 4A), leading to substantial accumulation of the cell p.
