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Original Research

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

, , , , &
Pages 2637-2653 | Published online: 15 Apr 2019

Figures & data

Table S1 Blood biochemical test

Table S2 Blood routine test

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 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 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 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 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 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 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 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 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 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 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 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 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.

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.