Glioma tissues and the adjacent normal tissues from 50 patients were collected after surgery in Zhejiang Cancer Hospital. The tissues were embedded in paraffin or immediately frozen and stored at -80 ℃ for the next experiments. Approval for this study was obtained from Zhejiang Cancer Hospital Ethics Committee. Written informed consents were acquired from all participants. Patient characteristics are shown in Table 1.

Human embryonic brain cells (HEB), human glioma cell lines A-172 (glioblastoma grade II), U-138 MG (glioblastoma grade III) and U-251 MG (glioblastoma grade IV) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in DMEM/F-12 media containing 10% fetal bovine serum (GIBCO, Life Technologies, Carlsbad, CA, USA) at an incubator with 37 ℃ and 5% CO₂.

For knocking down of MST1 or SIRT6, siRNA (siMST1-1: 5'-GCAGGUCAACUUACAGAUATT-3’; siMST1-2: 5’-GACUGAUGGAGCCAAUACUTT-3’; siSIRT6-1: 5’-AAGAATGTGCCAAGTGTAAGA-3’; siSIRT6-2: 5’-CCGGCTCTGCACCGTGGCTAA-3’; siFOXO3a-1: 5’-ATGGCAAGCACAGAGTTGGATGAAGTCC A-3’; siFOXO3a-2: 5’-ATGGCTAGTACCGAACTGGACGAGG TGCA-3’) and the negative control (siNC: 5’-UUCUCCGAACG UGUCACGUTT-3’) were synthesized by Vigenebio (Jinan, China). U-251 MG cells (4 × 10⁵ per well) were seeded in 12-well plates overnight. The culture medium was then removed, and washed with Phosphate-buffered saline (PBS). The cells were transfected with 40 nM siRNAs by Lipofectamine 2000 (Invitrogen). For the over-expression of MST1 and SIRT6, the primary precursor sequences were PCR and subcloned into plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA, USA). Cells were then transfected with 40 nM pcDNA3.1-MST1 or pcDNA3.1-SIRT6 by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were harvested for further analysis 48 hours after transfection.

RNAs from tissues or cells were extracted by Trizol reagent (Invitrogen). The RNAs were then reverse transcribed into cDNAs using miScript Reverse Transcription kit (Qiagen, Hilden, Germany). cDNAs (10 ng) were amplified by using SYBR1 Premix Ex Taq^TM II (Takara, Shiga, Japan) under the following conditions: Denaturation at 95 ℃ for 15 s, annealing at 60 ℃ for 30 s and extension at 72 ℃ for 30 s in a total 40 cycles. U6 was used as the internal reference. Three technological replicates were used to ensure the reliability of the analysis. For relative quantification, the fold change was determined with 2^-△△Ct method, The primer sequences were shown: MST1, 5’-ACAAATCCTCCTCCCACATTCCG-3’ (forward) and 5’-CACTCCTGACAAATGGGTGCTG-3’ (reverse); SIRT6, 5’-CCAAGTTCGACACCACCTTT-3’ (forward) and 5’CGGACGTACTGCGTCTTACA-3’ (reverse); FOXO3a, 5’-AGTGGATGGTGCGCTGTGT-3’ (forward) and 5’-CTGTGCAGGGACAGGTTGT -3’ (reverse); U6, 5’-CTCGCTTCGGCAGCACA-3’ (forward) and 5’-AACGCTTCACGAATTTGCGT-3’ (reverse); GAPDH, 5’-TGTTCGTCATGGGTGTGAAC-3’ (forward) and 5’-ATGGCATGGACTGTGGTCAT -3’ (reverse).

The extracted glioma tumor specimens and the adjacent normal tissues were cut into slices of 5 µm. The slices were embedded in paraffin, and then dewaxed in xylene and rehydrated in ethanol. The sections were then blocked for endogenous peroxidase by incubating in 3% H₂O₂ and then washed in PBS containing 0.05 M EDTA followed by 4% paraformaldehyde. Tissues were incubated in 4% dry milk and 0.3% goat serum in PBS solution for 20 minutes to block non-specific binding. Then the sections were in cubated overnight at room temperature with anti-MST1 or SIRT6 (6 µg/mL; Santa Cruz, CA, USA) antibody in the presence of 10% rabbit serum. After washing, sections were incubated for another 2 h with horseradish peroxidase (HRP) goat anti-rabbit IgG secondary antibody. Slides were counterstained with hematoxylin to stain cell nuclei in order to identify cells. Slides were then dehydrated and examined under a light microscope (Olympus, Tokyo, Japan).

Sample proteins were extracted from tissues or cells using RIPA buffer (KeyGen, Nanjin, China). Protein concentration was determined via BCA protein assay kit (Boster, China). Protein lysates (30 µg) were loaded onto 10% SDS-PAGE, and then transferred to PVDF membrane. The membrane was blocked in PBS-T with 5% BSA for 2 hours. PVDF membranes were then probed with rabbit anti-MST1 antibody (80 kDa, 1 : 1500, Abcam, Cambridge, MA, USA), Bcl-2 (26 kDa) or Bax (21 kDa) (1 : 1000, Abcam), Cleaved caspase-3 (17 kDa) or Cleaved Poly(ADP-Ribose)Polymerase (PARP; 25 kDa) (1 : 2000, Abcam), SIRT6 (37 kDa) or FOXO3a (72 kDa) (1:2500, Abcam), β-actin (1 : 3000, Abcam) overnight at 4 ℃. The PVDF membrane was washed with Tris Buffered Saline with Tween 20 (TBST, 10 min × 3 times) and labeled with horseradish peroxidase (HRP)-conjugated secondary antibodies (1 : 5000; Abcam) for 1 hour. Immunoreactivities were detected by enhanced chemiluminescence (KeyGen, Nanjin, China), and the band intensity was quantified by ImageJ software. The relative protein levels were calculated based on βactin protein as a loading control.

U-251 MG cells were cultured in 96-well plates at concentration of 1 × 10⁴ per well for 24 hours and then were treated with Anlotinib for another 24 hours. The 3-(4, 5-dimethylthiazal-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay (Solarbio, Beijing, China) was utilized to estimate cell viability. The results were calculated by the means of spectrophotometric plate reader (BioTek, VT, USA) at 490 nm. All the results were tested at least by three independent experiments.

Following heat to 56 ℃, the DMEM (4.1 mL) and agar (4%, 0.9 mL) were then cooled for precipitation. The 1 × 104 cells/mL single-cell suspension was made via dilution of 1 × 106 U-251 MG cells per mL in DMEM with 0.36% agar. The colonies were stained with 0.04% crystal violet-2% ethanol in PBS and then in- cubated at 37 ℃ for 3 weeks. The stained colonies were pho- tographed under a light microscope (Olympus).

U-251 MG cells were harvested at concentration of 1 × 10⁶ per well, and then fixed with 4% paraformaldehyde at 27 ℃ for 1 hour. Permeated with 0.1% Triton-X 100 and 0.1% sodium citrate for 2 minutes, the cells were then added by terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) solution (In Situ Cell Death Detection Kit; Roche, IN, USA) and incubated at dark in 37 ℃ for 1 hour. 0.5 µg/mL 4’, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) was utilized to stain the nuclei. The fluorescence was also analyzed by fluorescent microscopy (Olympus, Tokyo, Japan).

The promoter sequence of SIRT6 mRNA was cloned into pGLluciferase plasmid (Promega, Madison, Wisconsin, USA) to construct luciferase reporter plasmid. U-251 MG cells were seeded at concentration of 3 × 10⁴ per well for triplicates in 24-well plates 24 hours before transfection, and co-transfected with siSIRT6, siFOXO3a or siNC. Forty-eight hours later, the cells were then harvested and lysed, and the luciferase activities were performed with the Lucifer Reporter Assay System (Promega).

All data are expressed as mean ± SEM of at least 3 independent experiments. All statistical analyses were using GraphPad Prism software (GraphPad Prism Software Inc., San Diego, USA). Statistical analyses were performed with one-way analysis of variance (ANOVA). P < 0.05 was considered as significant.

We firstly determine the expression level of MST1 in glioma tissues. As shown in Fig. 1A, the mRNA expression level of MST1 in 50 glioma tissues was lower than that in the adjacent normal tissues. The mRNA expression level of SIRT6 in glioma tissues was also lower than that in the adjacent normal tissues (Fig. 1A). Consistently, the downregulation of MST1 and SIRT6 in glioma tissues were also confirmed by immunohistochemical staining, demonstrating lower protein expression of MST1 and SIRT6 in glioma tissues than those in adjacent normal tissues (Fig. 1B). The correlation analysis showed a positive relationship between MST1 level and SIRT6 expression (Fig. 1C), suggesting the potential relationship with glioma.

Figure 1.

Down-regulation of MST1 in glioma. (A) The mRNA expression of MST1 (P = 0.00018) and SIRT6 (P = 0.00029) in human glioma tissues (n = 50, Tumor) were lower than that in adjacent normal tissues (n = 50, Normal). ^*** represents Tumor vs. Normal. (B) Immunohistochemistry (IHC) staining showed lower expression of MST1 and SIRT6 in glioma tissues than that in Normal tissues. (C) Positive correlation between MST1 level and SIRT6 expression (P = 0.00033).

MST1 expression profiles were also detected in three independent glioma cell lines (A-172, U-138 MG and U-251 MG) and human embryonic brain cell line (HEB). The results showed that the mRNA (Fig. 2A) and protein (Fig. 2B) expression of MST1 in glioma cell lines were significantly down-regulated as compared with that in HEB cells. Of which, U-138 MG cell line demonstrated a 50% reduction in both mRNA and protein expression of MST1, and A-172 demonstrated a 20% reduction in both mRNA and protein expression of MST1 (Fig. 2A,B). Particularly, U-251 MG cell line performing about 50% reduction in mRNA expression and more than 50% reduction in protein expression than that in HEB cells, respectively (Fig. 2A,B), which was selected for the following functional analysis. U-251 MG cell line with stable over-expression of MST1 was established, and the expression of MST1 was higher about ninefold in U-251 MG cells transfected with MST1 over-expression than that in the control cells (Fig. 2C). MTT assay revealed that MST1 over-expression significantly decreased viability of U-251 MG cells (Fig. 2D). MST1 over-expression also suppressed colony formation of U-251 MG cells (Fig. 2E), as shown by the decreased number of colonies in cells with MST1 over-expression compared to control cells. Moreover, TUNEL staining analysis showed that MST1 over-expression also induced apoptosis in cells with MST1 over-expression as compared to the control cells (Fig. 2F). Besides that, the expression of anti-apoptotic protein Bcl-2 was decreased and the expression of pro-apoptotic proteins Bax, cleaved-caspase 3 and cleaved PARP were increased in cells with MST1 over-expression as compared to the control cells (Fig. 2G). These results revealed that MST1 over-expression inhibited cell viability and colony formation and induced cell apoptosis of glioma cells.

Figure 2.

Over-expression of MST1 inhibited cell viability and colony formation and induced cell apoptosis of glioma cells. (A) The mRNA expression of MST1 in glioma cell lines (A-172 (P = 0.0085), U-138 MG (P = 0.0004) and U-251 MG (P = 0.0002)) was lower than that in human embryonic brain cells (HEB). All results are expressed as mean ± SEM of at least 3 independent experiments. *, * * * represents glioma cell lines vs. HEB. (B) The protein expression of MST1 in glioma cell lines (A-172 (P = 0.031), U-138 MG (P = 0.0076) and U-251 MG (P = 0.00083)) was lower than human embryonic brain cells (HEB). All results are expressed as mean ± SEM of at least 3 independent experiments. *, **, * * * represents glioma cell lines vs. HEB. (C) MST1 was increased in U-251 MG cell line after transfecting with pcDNA-MST1 (MST1) (P = 0.0001). All results are expressed as mean ± SEM of at least 3 independent experiments. * * * represents MST1 vs. Control. (D) MST1 decreased viability of U-251 MG cells by MTT assay (P = 0.00067). All results are expressed as mean ± SEM of at least 3 independent experiments. * * * represents MST1 vs. Control. (E) MST1 suppressed colony formation of U-251 MG cells, which was detected by colony formation assay (P = 0.0063). All results are expressed as mean ± SEM of at least 3 independent experiments. * * * represents MST1 vs. Control. (F) MST1 induced apoptosis of U-251 MG cells by TUNEL assay (P = 0.0006). All results are expressed as mean ± SEM of at least 3 independent experiments. * * * represents MST1 vs. Control. (G) MST1 decreased protein expression of Bcl-2 (P = 0.0030) and increased protein expression of MST1 (P = 0.0014), Bax (P = 0.0090), cleaved-caspase 3 (P = 0.0082) and cleaved PARP (P = 0.03) in U-251 MG cells by western blot. All results are expressed as mean ± SEM of at least 3 independent experiments. *, ** represents MST1 vs. Control.

The underlying mechanism was then explored. MST1 was knocked down in U-251 MG cell line by siRNAs (siMST1-1# and siMST1-2#), and the transfection efficiency was confirmed by the down-regulated MST1 mRNA (Fig. 3A) and protein expression (Fig. 3B). Of which, siMST1-2# performing about 60% reduction in mRNA expression and 50% reduction in protein expression (Fig. 3A,B), which was selected for the subsequent analysis. The luciferase reporter assay demonstrated that siMST1 decreased the luciferase activity of SIRT6 promoter while MST1 over-expression increased the activity as compared to the corresponding controls (Fig. 3C). Besides, siMST1 performed about 60% reduction, and MST1 over-expression performed 50% induction of SIRT6 protein expression as compared to the corresponding controls (Fig. 3D). These investigations suggested that MST1 positively regulated SIRT6 expression.

Figure 3.

MST1 positively regulated SIRT6 expression via FOXO3a (A) MST1 mRNA expression was decreased in U-251 MG cells that transfected with siMST1-1# (P = 0.0030) or siMST1-2# (P = 0.00054) as compared to the control group. All results are expressed as mean ± SEM of at least 3 independent experiments. **, *** represents siMST1 vs. siNC. (B) MST1 protein expression was decreased in U-251 MG cells that transfected with siMST1-1# (P = 0.0378) or siMST1-2# (P = 0.0065) as compared to the control group. All results are expressed as mean ± SEM of at least 3 independent experiments. *, ** represents siMST1 vs. siNC. (C) siMST1 decreased the luciferase activity of SIRT6 (P = 0.00036) promoter while MST1 over-expression increased the activity (P = 0.00019) by luciferase reporter assay. All results are expressed as mean ± SEM of at least 3 independent experiments. * * * represents siMST1 vs. siNC or MST1 vs. Control. (D) siMST1 decreased the protein expression of SIRT6 (P = 0.0003) while MST1 over-expression increased the protein expression of SIRT6 (P = 0.004) in U-251 MG cells by western blot. All results are expressed as mean ± SEM of at least 3 independent experiments. **, *** represents siMST1 vs. siNC or MST1 vs. Control. (E) FOXO3a mRNA expression was decreased in U-251 MG cells that transfected with siFOXO3a-1# (P = 0.0035) or siFOXO3a-2 (P = 0.00078). All results are expressed as mean ± SEM of at least 3 independent experiments. **, * * * represents siFOXO3a vs. siNC. (F) FOXO3a protein expression was decreased in U-251 MG cells that transfected with siFOXO3a-1# (P = 0.01) or siFOXO3a-2as compared to the control group(P = 0.0017). All results are expressed as mean ± SEM of at least 3 independent experiments. *, ** represents siFOXO3a vs. siNC. (G) The increased luciferase activity induced by MST1 over-expression (P = 0.0015) was decreased after transfecting an addition of siFOXO3a (P = 0.0018) by luciferase reporter assay. All results are expressed as mean ± SEM of at least 3 independent experiments. ** represents MST1 vs. Control or MST1 + siFOXO3a. (H) The increased SIRT6 protein expression in U-251 MG cells induced by MST1 over-expression (P = 0.0046) was decreased after transfecting an addition of siFOXO3a (P = 0.0040) by western blot. All results are expressed as mean ± SEM of at least 3 independent experiments. ** represents MST1 vs. Control or MST1 + siFOXO3a.

FOXO3a transcriptional factor was shown to positively regulate the expression of SIRT6 (Kim et al., 2010). In our study, U-251 MG cell was transfected with siRNAs (siFOXO3a-1# and siFOXO3a-2#) , showing 40% and 60% reduction in both mRNA (Fig. 3E) and protein (Fig. 3F) expression as compared to the corresponding controls, respectively. Of which, siFOXO3a-2# performing less mRNA and protein expression of FOXO3a (Fig. 3E,F), and was selected for the following assays. Indeed, luciferase reporter assay revealed the increased luciferase activity induced by MST1 over-expression was decreased after co-transfection of siFOXO3a and MST1 over-expression (Fig. 3G). Moreover, the increased SIRT6 protein expression induced by MST1 overexpression was also decreased by co-transfection of siFOXO3a and MST1 over-expression (Fig. 3H). Overall our results indicate that MST1 positively regulated SIRT6 expression via FOXO3a.

Based on the results, it raises the possibility that SIRT6 may be involved in the effect of MST1 on glioma progression. We thus used SIRT6 specific siRNAs (siSIRT6-1# and siSIRT6-2#) to knock down SIRT6 expression, as shown in Fig. 4A. Of which, siSIRT6-1# demonstrated 70% reduction and siSIRT6-2# demonstrated 50% reduction of SIRT6 mRNA expression as compared to the control (Fig. 4A), and siSIRT6-1# was selected for the following experiments. Firstly, MTT assay showed that the cell viability inhibited by MST1 was promoted by addition of siSIRT6 (Fig. 4B). Secondly, the decreased expression level Bcl-2 or the increased expression levels of Bax, cleaved-caspase 3 and cleaved PARP by MST1 over-expression were increased or decreased by addition of siSIRT6, respectively (Fig. 4C). The colony formation assay showed that the inhibitory activity of MST1 on colony formation of glioma cells was induced by addition of siSIRT6 (Fig. 4D). These results demonstrated that the regulatory effects of MST1 on glioma cell viability and colony formation was at least partially through suppression of SIRT6. As shown in Fig.5, MST1 could promote the phosphorylation of FOXO3a, and destruct the binding of FOXO3a to 14-3-3 protein in the cytoplasm, thus dissociating FOXO3a and promoting translocation into nucleus. Moreover, FOXO3a binds with promoter of SIRT6, and positively regulates the expression of SIRT6, thus leading to the inhibition of cell proliferation and promotion of cell apoptosis.

Figure 4.

MST1 inhibited cell viability and induced cell apoptosis of glioma cells through regulating SIRT6 expression (A)SIRT6 mRNA expression was decreased in U-251 MG cells transfected with siSIRT6-1# (P = 0.0002) or siSIRT6-2# (P = 0.0017). All results are expressed as mean ± SEM of at least 3 independent experiments. **, *** represents siSIRT6 vs. siNC. (B) The decreased viability of U-251 MG cells induced by MST1 over-expression (P = 0.00059) was increased after transfecting with siSIRT6 (P = 0.00047) by MTT assay. All results are expressed as mean ± SEM of at least 3 independent experiments. * represents MST1 vs. Control or MST1 + siSIRT6. (C) MST1 decreased expression of Bcl-2 (P = 0.028) and increased expression of SIRT6 (P = 0.046), Bax (P = 0.00063), cleaved-caspase 3 (P = 0.0013) and cleaved PARP (P = 0.00057) protein expression of U-251 MG cells by western blot. Co-transfection of MST1 over-expression and siSIRT6 increased expression of Bcl-2 (P = 0.046) and decreased expression of SIRT6 (P = 0.040), Bax (P = 0.00038), cleaved-caspase 3 (P = 0.0059) and cleaved PARP (P = 0.00046) protein expression. All results are expressed as mean ± SEM of at least 3 independent experiments. *, **, *** represents MST1 vs. Control or MST1 + siSIRT6. (D) The decreased number of colonies of U-251 MG cells induced by MST1 over-expression (P = 0.0075) was increased after transfecting with siSIRT6 (P = 0.0048) (P = 0.0082) by colony formation assay. All results are expressed as mean ± SEM of at least 3 independent experiments. ** represents MST1 vs. Control or MST1 + siSIRT6.

Figure 5.

Summary diagram to illustrate the hypothesized pathway and effects of MST1 on glioma progression. MST1 could promote the phosphorylation of FOXO3a, and destruct the binding of FOXO3a to 14-3-3 protein in the cytoplasm, thus dissociating FOXO3a and promoting translocation into nucleus. Moreover, FOXO3a binds with promoter of SIRT6, and positively regulates the expression of SIRT6, thus leading to the inhibition of cell proliferation and promotion of cell apoptosis.