Limits...
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.

Show MeSH

Related in: MedlinePlus

Organelle changes afterChol-let-7atherapy under transmission electron microscopy. HepG2 and SMMC7721 cells were transfected with Chol-let-7a or the negative control miRNA mimic (Chol-miRCtrl). Cells were collected at 48 h and 60 h and observed under TEM. The figure shows the cytoplasmic ultrastructure of Chol-let-7a-treated cells at 48 h post-transfection. Also shown are heterolysosomes as phagophores (white arrows), multivesicular bodies (MVBs, yellow arrows), and multilamellar bodies (MLBs, orange arrows). Mitophagosomes (double-membrane-enclosed damaged mitochondria) and enlarged irregular mitochondria (mt) are shown with blue arrows. Red arrows indicate the dilated RER with degranulation. Scale bars are 0.5 μm or 0.2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Organelle changes afterChol-let-7atherapy under transmission electron microscopy. HepG2 and SMMC7721 cells were transfected with Chol-let-7a or the negative control miRNA mimic (Chol-miRCtrl). Cells were collected at 48 h and 60 h and observed under TEM. The figure shows the cytoplasmic ultrastructure of Chol-let-7a-treated cells at 48 h post-transfection. Also shown are heterolysosomes as phagophores (white arrows), multivesicular bodies (MVBs, yellow arrows), and multilamellar bodies (MLBs, orange arrows). Mitophagosomes (double-membrane-enclosed damaged mitochondria) and enlarged irregular mitochondria (mt) are shown with blue arrows. Red arrows indicate the dilated RER with degranulation. Scale bars are 0.5 μm or 0.2 μm.

Mentions: We observed Chol-let-7a- and Chol-miRCtrl-treated cells under transmission electronic microscopy (TEM) at 48 h and 60 h post-transfection. Abnormal organelles were observed in the cytoplasm of Chol-let-7a-treated cells (Figure 3). Increased autophagocytic activity in HepG2 and SMMC7721 cells was observed 48 h after Chol-let-7a treatment, as revealed by the presence of abundant lysosomes and phagolysosomes exhibiting heterolysosomes such as phagophores, multivesicular bodies (MVBs), and multilamellar bodies (MLBs) in the cytoplasm (Figure 3), but only slight changes in nuclear morphology were observed. Enlarged irregular mitochondria with disorganized mitochondrial crests and dilated rough endoplasmic reticulum (RER), which are often accompanied by degranulation, were also clearly observed in the Chol-let-7a-treated cells. Furthermore, vacuolated organelles were found in individual cells. Some changes observed in the Chol-let-7a-treated cells were also found in Chol-miRCtrl-treated HCC cells (see Additional file 3A); however, these effects were relatively mild in the negative control cells.Figure 3


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)

Organelle changes afterChol-let-7atherapy under transmission electron microscopy. HepG2 and SMMC7721 cells were transfected with Chol-let-7a or the negative control miRNA mimic (Chol-miRCtrl). Cells were collected at 48 h and 60 h and observed under TEM. The figure shows the cytoplasmic ultrastructure of Chol-let-7a-treated cells at 48 h post-transfection. Also shown are heterolysosomes as phagophores (white arrows), multivesicular bodies (MVBs, yellow arrows), and multilamellar bodies (MLBs, orange arrows). Mitophagosomes (double-membrane-enclosed damaged mitochondria) and enlarged irregular mitochondria (mt) are shown with blue arrows. Red arrows indicate the dilated RER with degranulation. Scale bars are 0.5 μm or 0.2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Organelle changes afterChol-let-7atherapy under transmission electron microscopy. HepG2 and SMMC7721 cells were transfected with Chol-let-7a or the negative control miRNA mimic (Chol-miRCtrl). Cells were collected at 48 h and 60 h and observed under TEM. The figure shows the cytoplasmic ultrastructure of Chol-let-7a-treated cells at 48 h post-transfection. Also shown are heterolysosomes as phagophores (white arrows), multivesicular bodies (MVBs, yellow arrows), and multilamellar bodies (MLBs, orange arrows). Mitophagosomes (double-membrane-enclosed damaged mitochondria) and enlarged irregular mitochondria (mt) are shown with blue arrows. Red arrows indicate the dilated RER with degranulation. Scale bars are 0.5 μm or 0.2 μm.
Mentions: We observed Chol-let-7a- and Chol-miRCtrl-treated cells under transmission electronic microscopy (TEM) at 48 h and 60 h post-transfection. Abnormal organelles were observed in the cytoplasm of Chol-let-7a-treated cells (Figure 3). Increased autophagocytic activity in HepG2 and SMMC7721 cells was observed 48 h after Chol-let-7a treatment, as revealed by the presence of abundant lysosomes and phagolysosomes exhibiting heterolysosomes such as phagophores, multivesicular bodies (MVBs), and multilamellar bodies (MLBs) in the cytoplasm (Figure 3), but only slight changes in nuclear morphology were observed. Enlarged irregular mitochondria with disorganized mitochondrial crests and dilated rough endoplasmic reticulum (RER), which are often accompanied by degranulation, were also clearly observed in the Chol-let-7a-treated cells. Furthermore, vacuolated organelles were found in individual cells. Some changes observed in the Chol-let-7a-treated cells were also found in Chol-miRCtrl-treated HCC cells (see Additional file 3A); however, these effects were relatively mild in the negative control cells.Figure 3

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