SIRT1 expression and BRAF V600E mutation status in PTC tissues and their correlations with clinicopathological features
We examined the mRNA and protein expression of SIRT1 in primary PTC tissues and matched adjacent normal tissues (ANT). Significantly higher expression of SIRT1 was found in 23 of 42 (54.76%) cancerous tissues compared with adjacent normal tissues (Fig. 1A and 1B). In addition, the correlation between SIRT1 levels and patients’ clinicopathological characteristics was assessed to determine its clinical significance (Table 1). We found that the expression of SIRT1 was significantly correlated with lymph node metastasis (p < 0.05) and a BRAF V600E mutation (p < 0.01). SIRT1 expression was higher in 65.63% (21/32) of the lymph node metastatic patients and in 67.74% (21/31) of the BRAF V600E-mutated patients. Furthermore, we also confirmed that the BRAF V600E mutation was significantly correlated with lymph node metastasis in PTC patients (Table 2).
BRAF V600E-mutated PTC cells possessed the highest level of SIRT1 expression
SIRT1 expression was also detected in two human PTC cell lines, TPC-1 (with wild-type BRAF) and BCPAP (with the BRAF V600E mutation) and one normal thyroid follicular epithelial cell line (Nthy-ori 3 − 1), using western blot analysis (Fig. 2). We found that TPC-1 and BCPAP cells exhibited a notable upregulation of SIRT1 compared with Nthy-ori 3 − 1 cells (p < 0.05). In addition, BCPAP cells had significantly higher SIRT1 levels compared to TPC-1 cells (p < 0.05). Therefore, to assess the relationship between SIRT1 and the BRAF V600E mutation in PTC, we used the TPC-1 and BCPAP cell lines for further examination.
SIRT1 promoted cell proliferation of BRAF V600E-mutated cells
The siRNAs were used to downregulate endogenous SIRT1 expression in TPC-1 and BCPAP cells. For SIRT1 overexpression, we synthetized and transfected CRISPR RNA-guided activation of SIRT1 (CRISPR SIRT1) and their negative controls into the two cell lines. The knockdown or activation of SIRT1 in PTC cells resulted in efficient changes in SIRT1 levels, as confirmed by western blot analyses (Fig. 3A and 3B).
To assess the role of SIRT1 on the proliferation of PTC cells, we performed a CCK-8 assay on TPC-1 and BCPAP cells. After incubating for four days, we found that knocking down SIRT1 significantly inhibited the cell viability (p < 0.01) and that the CRISPR SIRT1-transfected BCPAP cells had a higher cell proliferation than that in the control group (p < 0.05) at 48 h (Fig. 3C). However, we failed to observe any significant variations in TPC-1 cells (Fig. 3D).
SIRT1 facilitated the cell cycle progression in BRAF V600E-mutated cells
To determine whether the effect of SIRT1 on cell proliferation was mediated by influencing the cell cycle progression, flow cytometry was performed (Fig. 4A and 4B). Downregulation of SIRT1 led to a significant reduction in the number of BCPAP cells in the G1 phase and a significant accumulation of cells in S phase compared with the control (both p < 0.05); moreover, when the upregulated SIRT1 expression was induced, fewer cells were hindered in S phase (p < 0.05). Consistent with these observations, SIRT1 overexpression facilitated cyclin D1 expression in BCPAP cells (Fig. 4C). No significant variations were observed in TPC-1 cells (Fig. 4B and 4D).
SIRT1 reduced apoptosis in BRAF V600E-mutated cells
Next, we performed a TUNEL assay to determine whether SIRT1 played a significant role in the apoptosis of TPC-1 and BCPAP cells. As shown in Fig. 5A, the percentage of BCPAP cells undergoing apoptosis was significantly increased in the SIRT1 siRNA group compared with that in the control cells at 48 h (p < 0.01). In addition, the number of TUNEL-positive apoptotic cells in the SIRT1 overexpression group in BCPAP cells was significantly lower than that in the control group. However, we failed to observe any significant variations in TPC-1 cells (Fig. 5B). Consistently, the levels of apoptotic proteins, including BAX and BCL-2, in SIRT1-regulated TPC-1 and BCPAP cells were in agreement with the above results (p < 0.05; Fig. 5C and 5D). Therefore, SIRT1 is an important participant in the regulation of cell apoptosis in BCPAP cells.
SIRT1 enhanced the migration and invasion potential of BRAF V600E-mutated cells
Then, we investigated the functional role of SIRT1 in the migration and invasion of PTC cells. In wound healing assays, the distance of the scratch wound in the BCPAP SIRT1 siRNA group was significantly larger than that in the control group, whereas SIRT1 overexpression promoted the migration of BCPAP cells (both p < 0.01; Fig. 6A). Nevertheless, no significant variations were observed in TPC-1 cells (Fig. 6B). Similar results were also found in the Matrigel invasion assay (Fig. 6C and 6D), in which the number of cells migrating through the chamber in the si-SIRT1 BCPAP group was significantly less than that in the control group (p < 0.01). In addition, the stimulatory effect of SIRT1 on BCPAP cell invasion was also confirmed (p < 0.01). However, we observed no significant variations in TPC-1 cells (Fig. 6D). These results indicated that SIRT1 enhanced the migration and invasion potential of BCPAP cells.
SIRT1 facilitated phosphorylation of MAPK pathway proteins in BRAF V600E-mutated cells
The above results showed that SIRT1 has different biological effects on TPC-1 and BCPAP cells. Since the distinct difference between these two cells is the BRAF V600E mutation status, we examined the activity of the MAPK signaling pathway. When SIRT1 was knocked down in BCPAP cells, the expression of p-ERK1/2 and p-JNK (active forms) were significantly inhibited (both p < 0.01), whereas those of their inactive forms (ERK1/2 and JNK) were unchanged (Fig. 7A). Thus, the loss of SIRT1 resulted in an overall reduced MAPK activity. In contrast, SIRT1 overexpression promoted their active forms in BCPAP cells, as confirmed by western blot analyses (Fig. 7A). However, no significant variations were found in TPC-1 cells (Fig. 7B). These data indicate that SIRT1 promoted the activity of ERK1/2 and JNK in the MAPK pathway and that SIRT1 is an important modulator of the MAPK pathway activity in BRAF V600E-mutated cells.