Targeting EZH2 for glioma therapy with a novel nanoparticle–siRNA complex

Figures & data

Table S1 Blood biochemical test

	ALB (g/L)	SEM	ALP (U/L)	SEM	ALT (U/L)	SEM
NS	23.7	0.3	81.4	3.7	27.0	5.7
EM-DMC	24.3	0.2	76.2	6.7	28.7	5.7
Consi	23.1	0.3	82.3	10.4	27.3	4.8
EZH2si	25.6	0.3	81.6	7.9	27.7	3.2
	AST (U/L)	SEM	BUN (μmol/L)	SEM	CHOL (mmol/L)	SEM
NS	277.3	0.4	8.2	1.2	2.7	0.1
EM-DMC	261	0.6	8.3	0.8	2.4	0.2
Consi	281.3	0.4	8.7	1.4	2.4	0.1
EZH2si	263.2	0.3	9.3	1.0	2.2	0.1
	CK (U/L)	SEM	CREA (μmol/L)	SEM	GLU (U/L)	SEM
NS	497.0	73.6	5.0	1.2	3.3	0.2
EM-DMC	516.7	67.2	4.8	0.6	3.2	0.3
Consi	501.0	135.4	4.3	1.3	3.7	0.1
EZH2si	497.3	6.3	4.1	0.7	4.1	0.3
	HDL (U/L)	SEM	LDH (U/L)	SEM	LDL (mmol/L)	SEM
NS	1.1	0.1	1,679.67	277.8	0.2	0.01
EM-DMC	0.9	0.1	1,789.3	291.1	0.2	0.01
Consi	1.0	0.1	1,596.3	222.1	0.2	0.01
EZH2si	1.1	0.1	1,492.3	199.5	0.2	0.01
	TBIL (mg/dL)	SEM	TG (mmol/L)	SEM	TP (g/L)	SEM
NS	7.2	1.5	1.6	0.3	48.7	1.3
EM-DMC	7.1	1.3	1.5	0.3	50.1	1.2
Consi	7.2	1.1	1.6	0.2	51.8	2.0
EZH2si	8.0	1.2	1.7	0.2	51.7	1.8
	UA (μmol/L)	SEM	XAMY (U/L)	SEM
NS	121.7	12.1	1,797.3	43.5
EM-DMC	119.3	12.5	1,753.3	65.7
Consi	114.7	10.7	1,877.0	102.7
EZH2si	116.7	11.3	1,776.7	83.6

Table S2 Blood routine test

Group	PLT (10^9/L)	SEM	RBC (10^12/L)	SEM
NS	319.7	96.8	9.5	0.1
EG	291.7	82.7	9.1	0.2
DM	307.7	61.7	9.3	0.2
DMG	311.3	33.0	9.5	0.2
	HGB (g/L)	SEM	WBC (10^9/L)	SEM
NS	170.7	9.2	7.6	1.2
EG	171.1	7.3	7.2	2.1
DM	163.0	8.6	7.4	0.9
DMG	170.0	16.5	7.6	1.2

Figure 1 Preparation of DMC.

Notes: Preparation of DMC and EZH2si complex: first, a novel gene carrier was prepared by a self-assembly method. MPEG-PCL and DOTAP were assembled into a new gene carrier, DOTAP/MPEG-PCL micelles (DMC). Then, EZH2 siRNA was carried into cancer cells by DMC.

Abbreviation: DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane.

Figure 2 Physicochemical properties of DMC.

Notes: (A) Particle size of DMC; (B) zeta potential of DMC; (C) morphological characteristics of DMC by TEM observation; and (D) gel retardation assay of siRNA and complexes. Lane 1, DNA marker; lane 2, naked siRNA; lanes 3–6, different weight ratios of siRNA with DMC. siRNA was completely incorporated into DMC at a weight ratio of 1:50, and complexes were prepared without free siRNA.

Abbreviation: TEM, transmission electron microscope.

Figure 3 Transfection efficiency measurement of DMC.

Notes: DMC, containing 4 µg siRNA, was used to transfect U87 cells for 4 hours. The transfection efficiencies at both weight ratios (siRNA vs DMC as 1:100 and 1:150) were determined using (A) fluorescence microscope and (B) flow cytometry.

Abbreviation: FITC, fluorescein isothiocyanate.

Figure 4 RT-PCR, Western blot and MTT test of cell activity.

Note: When U87 cells were transfected with DMC, Consi-DMC or EZH2si-DMC for 72 hours, EZH2 expression was tested by RT-PCR (A) and Western blot (B), and cell activity was tested by MTT test (C). *P<0.01, EZH2si-DMC versus NS, DMC, Consi-DMC.

Abbreviations: RT-PCR, reverse transcription PCR; NS, normal saline.

Figure 5 Cell apoptosis test.

Notes: When U87 cells were transfected with DMC (A), Consi-DMC (B) or EZH2si-DMC (C) for 72 hours, U87 cells with different treatments were stained with Annexin V-FITC/PI and tested by flow cytometry. (D) Statistics of tumor cell apoptosis. *P<0.05.

Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 6 Molecular mechanism of siEZH2-DMC antiglioma.

Notes: (A) The effect of siEZH2-DMC on apoptosis-related proteins by Western blotting analysis. (B) The phosphorylation of Akt and p44/42 MAPK in U87 was inhibited by siEZH2-DMC.

Figure 7 Antiglioma effect of EZH2si-DMC in vivo.

Notes: (A) Tumor growth curves; (B) tumor weight; (C) tumor photos of NS, DMC, DMC-Consi and DMC-EZH2si treatment groups; and (D) body weight of different groups. (Mean ± SEM, n=5) (*P<0.01, DMC-EZH2si vs NS, DMC, DMC-Consi (A); *P<0.01, DMC-EZH2 vs NS, DMC, and DMC-Consi (B).

Figure 8 Bioimaging analysis in the GL261 orthotopic mice model.

Notes: (A) Intracerebral glioma was seen by using fluorescence markers. Tumor was inhibited more effectively by α-MM, compared with other groups. (B) Life survival time assessment. Both α-M and α-MM extended life spans of mice, while mice in the α-MM group reached a longer survival time.

Figure 9 EZH2 expression detection in vivo.

Notes: (A) Intracerebral glioma was seen by using fluorescence markers. Tumor was inhibited more effectively by DMC-EZH2si complex, compared with other groups. (B) Life survival time assessment. DMC-EZH2si complex extended life spans of mice. The EZH2 expression of NS (A), DMC (B) and DMC-Consi (C) were higher than DMC-EZH2si (D). Results indicated that the EZH2 was successfully knocked down by the DMC-EZH2si complex.

Figure 10 Cell proliferation detection.

Notes: (A) Cell proliferation was assessed by counting the number of Ki67-positive cells in the field (five high power fields per slide). (B) Mean cell proliferation every five fields was added. EZH2si-DMC was superior to other controls in inhibiting cell proliferation. EZH2si-DMC significantly inhibited cell proliferation. *P<0.01, EZH2si-DMC versus NS, DMC, Consi-DMC.

Figure 11 Cell apoptosis detection.

Notes: (A) Cell apoptosis was assessed by counting the number of TUNEL-positive cells in the field (five high power fields per slide), and EZH2si-DMC was superior to other controls in increasing cell apoptosis. (B) Mean apoptosis rate every five fields. EZH2si-DMC significantly increased apoptosis (*P<0.01, EZH2si-DMC vs NS, DMC and Consi-DMC).

Figure S1 RT-PCR and MTT test of cell activity.

Notes: When GL261 cells were transfected with DMC, Consi-DMC or EZH2si-DMC for 72 hours, EZH2 expression was tested by RT-PCR (A) and cell activity was tested by MTT test (B). (siEZH2-1:GGATACAGCCTGTGCACAT; siEZH2-2:GCTTTGGACAACAAGCCTT; siEZH2-3:GCAAATTCTCGGTGTCAAA).

Abbreviation: RT-PCR, reverse transcription PCR.

Figure S2 Toxicity assessment in vivo with pathological section.

Notes: Histological examinations of HE-stained (A) heart, (B) liver, (C) lung, (D) kidney, and (E) spleen. No significant pathological changes were detected. Scale bar is 50 µm.