Sirt3 enhances glioma cell viability by stabilizing Ku70–BAX interaction

Figures & data

Table 1 The correlations between clinical parameters and Sirt3 expression in glioma patients

Variables	Patients	Sirt3 expression		P-value
	(n=114)	Low (n=60)	High (n=54)
Gender				0.713
Female	48	27	21 (43.8%)
Male	66	33	33 (50.0%)
Age (years)				0.238
≤50	55	27	28 (50.9%)
>50	59	33	26 (44.1%)
Tumor size				0.879
≤5 cm	69	47	22 (31.9%)
>5 cm	45	13	32 (71.1%)
WHO grade				0.004*
II	27	21	6 (22.2%)
III	45	21	24 (53.3%)
IV	42	18	24 (57.1%)
Karnofsky score				0.379
≤90	69	39	30 (43.5%)
>90	45	21	24 (53.3%)
Surgery				0.122
GTR	48	29	19 (39.6%)
STR	35	18	17 (48.6%)
PR/biopsy	31	13	18 (58.1%)

Note:

* Statistically significant.

Abbreviations: GTR, gross total resection; PR, partial resection; Sirt3, sirtuin 3; STR, subtotal resection.

Figure 1 Sirt3 is upregulated in glioma tissues.

Notes: Representative low (A) and high (B) Sirt3 protein expression in clinical glioma tissues by IHC staining, showing the location of Sirt3 in cytoplasm. Magnification: 400×. (C) RT-qPCR results showed a significantly higher RNA level of Sirt3 in glioma tissues than that in normal brain tissues (P=0.038). *P<0.05 by Student’s t-test. (D) Higher Sirt3 RNA transcription indicated a poorer overall survival of glioma patients. *P<0.05 by log-rank test.
Abbreviations: IHC, immunohistochemistry; RT-qPCR, quantitative real-time PCR; Sirt3, sirtuin 3.

Table 2 Univariate analysis for overall survival of glioma patients

Variables	Patients	Overall survival time (months)		P-value
	(n=114)	Mean ± SD	Median
Gender				0.433
Female	48	36.7±3.2	38.0
Male	66	30.0±3.1	17.0
Age (years)				0.808
≤50	55	30.8±3.5	17.0
>50	59	34.6±2.9	32.0
Tumor size				0.027*
≤5 cm	69	36.9±2.8	35.0
>5 cm	45	25.9±3.4	17.0
WHO grade				0.012*
II	27	44.1±3.4	49.0
III	45	34.6±3.8	33.0
IV	42	23.7±3.5	11.0
Karnofsky score				0.768
≤90	69	33.7±2.9	32.0
>90	45	31.7±3.6	27.0
Surgery				<0.001*
GTR	48	39.7±3.5	46.0
STR	35	32.8±3.7	33.0
PR/biopsy	31	21.6±3.7	10.0
Sirt3 expression				0.003*
Low	60	39.3±2.9	39.0
High	54	25.0±3.1	11.0

Note:

* Statistically significant.

Abbreviations: GTR, gross total resection; PR, partial resection; Sirt3, sirtuin 3; STR, subtotal resection.

Figure 2 Analyses of the overall survival of glioma patients.

Notes: The overall survival curve for our enrolled cohort was showed by Kaplan–Meier plotting (A). Additionally, log-rank test was used to identify prognostic factors based on patients’ gender (B), age (C), tumor size (D), WHO grade (E), Karnofsky score (F), surgical treatment (G), and Sirt3 protein level (H), respectively. *P<0.05.
Abbreviations: GTR, gross total resection; PR, partial resection; Sirt3, sirtuin 3; STR, subtotal resection.

Table 3 Multivariate analysis for the prognostic factors of glioma patients

Variables	HR	95% CI	P-value
Tumor size	1.105	0.702–1.739	0.666
WHO grade	1.927	1.320–2.814	0.001*
Surgery	1.799	1.164–2.781	0.008*
Sirt3 expression	1.602	1.089–2.446	0.017*

Note:

* Statistically significant.

Abbreviation: Sirt3, sirtuin 3.

Figure 3 Sirt3 enhances glioma cell viability.

Notes: (A) The protein expression level of Sirt3 was significantly higher in U87 and U251 glioma cell lines than that in normal human astrocytes (NHA cells). Overexpressing Sirt3 enhanced growth viability of U87 (B) and U251 cells (C), while silencing Sirt3 showed opposite effects. (D) Immunoprecipitation experiments demonstrated the interaction between Sirt3 and Ku70 proteins. (E) Sirt3 negatively regulated the acetylation of Ku70 without affecting its total protein level. (F) Silencing Sirt3 upregulated the BAX protein translocation into mitochondria and promoted the release of cytochrome c and cleavage of caspase 3. On the other hand, overexpressing Sirt3 showed opposite effects on the apoptotic proteins. All experiments were performed in triplicate and repeated for three times. *P<0.05 compared to control (CTL) cells by Student’s t-test.
Abbreviations: BAX, Bcl-2-associated X protein; IB, immunoblotting; IP, immunoprecipitation; NHA, normal human astrocytes; ov, overexpression; si, siRNA; Sirt3, sirtuin 3.

Figure 4 Silencing Ku70 attenuates the pro-proliferative effects of Sirt3 in glioma cells.

Notes: (A) CCK-8 assay showed that Ku70 siRNA significantly inhibited cell proliferation of U87 cells that were stably expressing Sirt3. (B) Treatment by Ku70 siRNA enhanced BAX protein translocation into mitochondria and promoted the release of cytochrome c.
Abbreviations: BAX, Bcl-2-associated X protein; CTL, control; si, siRNA.