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Cholesterol-conjugated let-7a mimics: antitumor efficacy on hepatocellular carcinoma in vitro and in a preclinical orthotopic xenograft model of systemic therapy.

Liu YM, Xia Y, Dai W, Han HY, Dong YX, Cai J, Zeng X, Luo FY, Yang T, Li YZ, Chen J, Guan J - BMC Cancer (2014)

Bottom Line: Furthermore, let-7a abundance in orthotopic xenografts was coincident with a reduction in the expression of 3 human ras mRNAs and RAS proteins.Chol-let-7a exerted significant antitumor effects by down-regulating all human ras genes at the transcriptional and translational levels.Chol-let-7a represents a potential useful modified molecule for systemic HCC therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences (CAMS), Beijing, China. xhblk@163.com.

ABSTRACT

Background: A major challenge to the clinical utility of let-7 for hepatocellular carcinoma (HCC) therapy is the lack of an effective carrier to target tumours. We confirmed the high transfection efficiency of cholesterol-conjugated let-7a miRNA mimics (Chol-let-7a) in human HCC cells, as well as their high affinity for liver tissue in nude mice. However, their antitumor efficacy via systemic delivery remains unknown.

Methods: We explored the effects of Chol-let-7a on HCC in vitro and in vivo. Cell viability and mobility, let-7a abundance and the target ras genes was measured. Live-cell image and cell ultrastructure was observed. Antitumor efficacy in vivo was analyzed by ultrasonography, hispatholgogy and transmission electronic microscopy in a preclinical model of HCC orthotopic xenografts with systemic therapy.

Results: Chol-let-7a inhibited the viability and mobility of HCC cells. Chol-let-7a was primarily observed in the cytoplasm and induced organelle changes, including autophagy. Mild changes were observed in the cells treated with negative control miRNA. Chol-let-7a reached HCC orthotopic tumours, significantly inhibited tumour growth, and prevented local invasion and metastasis. Compared to control tumours, Chol-let-7a-treated tumours showed more necrosis. Tumour cells showed no significant atypia, and mitoses were very rare after systemic Chol-let-7a therapy. Furthermore, let-7a abundance in orthotopic xenografts was coincident with a reduction in the expression of 3 human ras mRNAs and RAS proteins.

Conclusions: Chol-let-7a exerted significant antitumor effects by down-regulating all human ras genes at the transcriptional and translational levels. Chol-let-7a inhibited cell proliferation, growth, and metastasis, and mainly functioned in the cytoplasm. Chol-let-7a represents a potential useful modified molecule for systemic HCC therapy.

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Live-cell images. Live-cell images were taken from 24 h to 39 h post-transfection. The parental cells with no treatment were observed as blank controls. Laser confocal images of GFP-labelled live cells (green) were recorded in the 3 groups. Cy5-labelled Chol-let-7a or Chol-miRCtrl appears as distinct red bodies. GFP-labelled living HepG2 or SMMC7721 cells appear green. Dead cells lose their green fluorescence. The images show more dead or apoptotic cells in Chol-let-7a-treated cells (yellow arrows). A: HepG2 cells. B: SMMC7721 cells. Magnification: 160×.
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Fig2: Live-cell images. Live-cell images were taken from 24 h to 39 h post-transfection. The parental cells with no treatment were observed as blank controls. Laser confocal images of GFP-labelled live cells (green) were recorded in the 3 groups. Cy5-labelled Chol-let-7a or Chol-miRCtrl appears as distinct red bodies. GFP-labelled living HepG2 or SMMC7721 cells appear green. Dead cells lose their green fluorescence. The images show more dead or apoptotic cells in Chol-let-7a-treated cells (yellow arrows). A: HepG2 cells. B: SMMC7721 cells. Magnification: 160×.

Mentions: Images of live HCC cells were taken after treatment with Chol-let-7a or the negative control miRNA (Chol-miRCtrl). The parental cells served as blank controls that were visually inspected to evaluate potential off-target interactions of Chol-miRCtrl. Living HepG2 and SMMC7721 cells labelled by GFP were identified by green fluorescence. Images taken at the various observation time points are shown in Figure 2. The red fluorescence that indicated Chol-let-7a and Chol-miRCtrl was primarily focused in the cytoplasm (Figure 2). Through analysis of live images, we found that most of the Chol-let-7a-treated cells lost GFP fluorescence earlier than the 2 control groups (Figure 2). Some Chol-let-7a-treated cells showed typical features of apoptosis (Figure 2, Additional file 2). The numbers of GFP positive cells in the Chol-let-7a, Chol-miRCtrl, and parental cell groups were 40/50, 49/50, and 49/50, respectively, at 24 h after transfection, and 5/50, 40/50, and 49/50, respectively, at 39 h after transfection. Approximately 10/50 cells had lost GFP fluorescence in the Chol-miRCtrl group at 39 h after transfection. In addition, a few cells in which cytoplasmic Chol-let-7a was observed did not undergo cell death, and these cells subsequently lost their red fluorescence. This observation shows that some Chol-let-7a-treated cells did not die.Figure 2


Cholesterol-conjugated let-7a mimics: antitumor efficacy on hepatocellular carcinoma in vitro and in a preclinical orthotopic xenograft model of systemic therapy.

Liu YM, Xia Y, Dai W, Han HY, Dong YX, Cai J, Zeng X, Luo FY, Yang T, Li YZ, Chen J, Guan J - BMC Cancer (2014)

Live-cell images. Live-cell images were taken from 24 h to 39 h post-transfection. The parental cells with no treatment were observed as blank controls. Laser confocal images of GFP-labelled live cells (green) were recorded in the 3 groups. Cy5-labelled Chol-let-7a or Chol-miRCtrl appears as distinct red bodies. GFP-labelled living HepG2 or SMMC7721 cells appear green. Dead cells lose their green fluorescence. The images show more dead or apoptotic cells in Chol-let-7a-treated cells (yellow arrows). A: HepG2 cells. B: SMMC7721 cells. Magnification: 160×.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4289300&req=5

Fig2: Live-cell images. Live-cell images were taken from 24 h to 39 h post-transfection. The parental cells with no treatment were observed as blank controls. Laser confocal images of GFP-labelled live cells (green) were recorded in the 3 groups. Cy5-labelled Chol-let-7a or Chol-miRCtrl appears as distinct red bodies. GFP-labelled living HepG2 or SMMC7721 cells appear green. Dead cells lose their green fluorescence. The images show more dead or apoptotic cells in Chol-let-7a-treated cells (yellow arrows). A: HepG2 cells. B: SMMC7721 cells. Magnification: 160×.
Mentions: Images of live HCC cells were taken after treatment with Chol-let-7a or the negative control miRNA (Chol-miRCtrl). The parental cells served as blank controls that were visually inspected to evaluate potential off-target interactions of Chol-miRCtrl. Living HepG2 and SMMC7721 cells labelled by GFP were identified by green fluorescence. Images taken at the various observation time points are shown in Figure 2. The red fluorescence that indicated Chol-let-7a and Chol-miRCtrl was primarily focused in the cytoplasm (Figure 2). Through analysis of live images, we found that most of the Chol-let-7a-treated cells lost GFP fluorescence earlier than the 2 control groups (Figure 2). Some Chol-let-7a-treated cells showed typical features of apoptosis (Figure 2, Additional file 2). The numbers of GFP positive cells in the Chol-let-7a, Chol-miRCtrl, and parental cell groups were 40/50, 49/50, and 49/50, respectively, at 24 h after transfection, and 5/50, 40/50, and 49/50, respectively, at 39 h after transfection. Approximately 10/50 cells had lost GFP fluorescence in the Chol-miRCtrl group at 39 h after transfection. In addition, a few cells in which cytoplasmic Chol-let-7a was observed did not undergo cell death, and these cells subsequently lost their red fluorescence. This observation shows that some Chol-let-7a-treated cells did not die.Figure 2

Bottom Line: Furthermore, let-7a abundance in orthotopic xenografts was coincident with a reduction in the expression of 3 human ras mRNAs and RAS proteins.Chol-let-7a exerted significant antitumor effects by down-regulating all human ras genes at the transcriptional and translational levels.Chol-let-7a represents a potential useful modified molecule for systemic HCC therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences (CAMS), Beijing, China. xhblk@163.com.

ABSTRACT

Background: A major challenge to the clinical utility of let-7 for hepatocellular carcinoma (HCC) therapy is the lack of an effective carrier to target tumours. We confirmed the high transfection efficiency of cholesterol-conjugated let-7a miRNA mimics (Chol-let-7a) in human HCC cells, as well as their high affinity for liver tissue in nude mice. However, their antitumor efficacy via systemic delivery remains unknown.

Methods: We explored the effects of Chol-let-7a on HCC in vitro and in vivo. Cell viability and mobility, let-7a abundance and the target ras genes was measured. Live-cell image and cell ultrastructure was observed. Antitumor efficacy in vivo was analyzed by ultrasonography, hispatholgogy and transmission electronic microscopy in a preclinical model of HCC orthotopic xenografts with systemic therapy.

Results: Chol-let-7a inhibited the viability and mobility of HCC cells. Chol-let-7a was primarily observed in the cytoplasm and induced organelle changes, including autophagy. Mild changes were observed in the cells treated with negative control miRNA. Chol-let-7a reached HCC orthotopic tumours, significantly inhibited tumour growth, and prevented local invasion and metastasis. Compared to control tumours, Chol-let-7a-treated tumours showed more necrosis. Tumour cells showed no significant atypia, and mitoses were very rare after systemic Chol-let-7a therapy. Furthermore, let-7a abundance in orthotopic xenografts was coincident with a reduction in the expression of 3 human ras mRNAs and RAS proteins.

Conclusions: Chol-let-7a exerted significant antitumor effects by down-regulating all human ras genes at the transcriptional and translational levels. Chol-let-7a inhibited cell proliferation, growth, and metastasis, and mainly functioned in the cytoplasm. Chol-let-7a represents a potential useful modified molecule for systemic HCC therapy.

Show MeSH
Related in: MedlinePlus