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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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SystemicChol-let-7atherapy modulatedras/RAS abundance in HCC orthotopic xenografts. A: Let-7a expression in HepG2 orthotopic xenografts was examined by quantitative real-time PCR. Relative quantification of let-7a was calculated using the comparative cycle threshold (CT) method (2 ΔΔct) with let-7a normalized to U6. The results shown represent the mean and standard error from 3 independent experiments, *p <0.05, **p <0.01 in comparison with controls. T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.008; Chol-let-7a vs. blank, p = 0.013; Chol-miRCtrl vs. blank, p = 0.128. B: RAS protein expression in xenografts examined by western blotting. KRAS, HRAS, and NRAS protein expression was measured in xenografts by western blotting. Representative data are shown from 2 experiments. C: qRT-PCR analysis of ras mRNA in xenografts. Relative quantification of target genes was calculated using the comparative cycle threshold (CT) method (2 ΔΔct) with genes normalized to GAPDH. The results shown represent the mean and standard error from 3 independent experiments.*p <0.05, ** p <0.01 in comparison with controls. Analysis revealed deregulated expression of k-ras, h-ras, and n-ras mRNA. T-test: n-ras (Chol-let-7a vs. Chol-miRCtrl, p = 0.002; Chol-let-7a vs. blank, p = 0.002; Chol-miRCtrl vs. blank, p = 0.837); h-ras (Chol-let-7a vs. Chol-miRCtrl, p = 0.016; Chol-let-7a vs. blank, p = 0.033; Chol-miRCtrl vs. blank, p = 0.801); and k-ras (Chol-let-7a vs. Chol-miRCtrl, p = 0.041; Chol-let-7a vs. blank, p = 0.005; Chol-miRCtrl vs. blank, p = 0.84).
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Fig4: SystemicChol-let-7atherapy modulatedras/RAS abundance in HCC orthotopic xenografts. A: Let-7a expression in HepG2 orthotopic xenografts was examined by quantitative real-time PCR. Relative quantification of let-7a was calculated using the comparative cycle threshold (CT) method (2 ΔΔct) with let-7a normalized to U6. The results shown represent the mean and standard error from 3 independent experiments, *p <0.05, **p <0.01 in comparison with controls. T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.008; Chol-let-7a vs. blank, p = 0.013; Chol-miRCtrl vs. blank, p = 0.128. B: RAS protein expression in xenografts examined by western blotting. KRAS, HRAS, and NRAS protein expression was measured in xenografts by western blotting. Representative data are shown from 2 experiments. C: qRT-PCR analysis of ras mRNA in xenografts. Relative quantification of target genes was calculated using the comparative cycle threshold (CT) method (2 ΔΔct) with genes normalized to GAPDH. The results shown represent the mean and standard error from 3 independent experiments.*p <0.05, ** p <0.01 in comparison with controls. Analysis revealed deregulated expression of k-ras, h-ras, and n-ras mRNA. T-test: n-ras (Chol-let-7a vs. Chol-miRCtrl, p = 0.002; Chol-let-7a vs. blank, p = 0.002; Chol-miRCtrl vs. blank, p = 0.837); h-ras (Chol-let-7a vs. Chol-miRCtrl, p = 0.016; Chol-let-7a vs. blank, p = 0.033; Chol-miRCtrl vs. blank, p = 0.801); and k-ras (Chol-let-7a vs. Chol-miRCtrl, p = 0.041; Chol-let-7a vs. blank, p = 0.005; Chol-miRCtrl vs. blank, p = 0.84).

Mentions: To confirm that Chol-let-7a effectively carried let-7a to target tumours in vivo, we measured let-7a abundance in HepG2 orthotopic xenografts by qRT-PCR after systemic therapy. Using miRNA-specific primers, we found significant increases in let-7a miRNA abundance in treated xenografts (Figure 4A) (Chol-let-7a vs. Chol-miRCtrl, p = 0.008; Chol-let-7a vs. blank, p = 0.013; Chol-miRCtrl vs. blank, p =0.128). These results verified that Chol-let-7a successfully reached tumour tissues.Figure 4


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)

SystemicChol-let-7atherapy modulatedras/RAS abundance in HCC orthotopic xenografts. A: Let-7a expression in HepG2 orthotopic xenografts was examined by quantitative real-time PCR. Relative quantification of let-7a was calculated using the comparative cycle threshold (CT) method (2 ΔΔct) with let-7a normalized to U6. The results shown represent the mean and standard error from 3 independent experiments, *p <0.05, **p <0.01 in comparison with controls. T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.008; Chol-let-7a vs. blank, p = 0.013; Chol-miRCtrl vs. blank, p = 0.128. B: RAS protein expression in xenografts examined by western blotting. KRAS, HRAS, and NRAS protein expression was measured in xenografts by western blotting. Representative data are shown from 2 experiments. C: qRT-PCR analysis of ras mRNA in xenografts. Relative quantification of target genes was calculated using the comparative cycle threshold (CT) method (2 ΔΔct) with genes normalized to GAPDH. The results shown represent the mean and standard error from 3 independent experiments.*p <0.05, ** p <0.01 in comparison with controls. Analysis revealed deregulated expression of k-ras, h-ras, and n-ras mRNA. T-test: n-ras (Chol-let-7a vs. Chol-miRCtrl, p = 0.002; Chol-let-7a vs. blank, p = 0.002; Chol-miRCtrl vs. blank, p = 0.837); h-ras (Chol-let-7a vs. Chol-miRCtrl, p = 0.016; Chol-let-7a vs. blank, p = 0.033; Chol-miRCtrl vs. blank, p = 0.801); and k-ras (Chol-let-7a vs. Chol-miRCtrl, p = 0.041; Chol-let-7a vs. blank, p = 0.005; Chol-miRCtrl vs. blank, p = 0.84).
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Related In: Results  -  Collection

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Fig4: SystemicChol-let-7atherapy modulatedras/RAS abundance in HCC orthotopic xenografts. A: Let-7a expression in HepG2 orthotopic xenografts was examined by quantitative real-time PCR. Relative quantification of let-7a was calculated using the comparative cycle threshold (CT) method (2 ΔΔct) with let-7a normalized to U6. The results shown represent the mean and standard error from 3 independent experiments, *p <0.05, **p <0.01 in comparison with controls. T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.008; Chol-let-7a vs. blank, p = 0.013; Chol-miRCtrl vs. blank, p = 0.128. B: RAS protein expression in xenografts examined by western blotting. KRAS, HRAS, and NRAS protein expression was measured in xenografts by western blotting. Representative data are shown from 2 experiments. C: qRT-PCR analysis of ras mRNA in xenografts. Relative quantification of target genes was calculated using the comparative cycle threshold (CT) method (2 ΔΔct) with genes normalized to GAPDH. The results shown represent the mean and standard error from 3 independent experiments.*p <0.05, ** p <0.01 in comparison with controls. Analysis revealed deregulated expression of k-ras, h-ras, and n-ras mRNA. T-test: n-ras (Chol-let-7a vs. Chol-miRCtrl, p = 0.002; Chol-let-7a vs. blank, p = 0.002; Chol-miRCtrl vs. blank, p = 0.837); h-ras (Chol-let-7a vs. Chol-miRCtrl, p = 0.016; Chol-let-7a vs. blank, p = 0.033; Chol-miRCtrl vs. blank, p = 0.801); and k-ras (Chol-let-7a vs. Chol-miRCtrl, p = 0.041; Chol-let-7a vs. blank, p = 0.005; Chol-miRCtrl vs. blank, p = 0.84).
Mentions: To confirm that Chol-let-7a effectively carried let-7a to target tumours in vivo, we measured let-7a abundance in HepG2 orthotopic xenografts by qRT-PCR after systemic therapy. Using miRNA-specific primers, we found significant increases in let-7a miRNA abundance in treated xenografts (Figure 4A) (Chol-let-7a vs. Chol-miRCtrl, p = 0.008; Chol-let-7a vs. blank, p = 0.013; Chol-miRCtrl vs. blank, p =0.128). These results verified that Chol-let-7a successfully reached tumour tissues.Figure 4

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