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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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Related in: MedlinePlus

Chol-let-7ainhibited HCC cell growth and cell viability in vitro.A,B: MTT assays show the effects of Chol-let-7a and Chol-miRCtrl on the growth of HepG2 and SMMC7721 cells. The figure shows the growth curve of tumour cells from days 1–5. blank, parental cells; Chol-miRCtrl, Chol-miRCtrl-transfected cells; Chol-let-7a, Chol-let-7a-transfected cells. C: A chamber-based cell migration assay showing that Chol-let-7a inhibits the migration of HCC cells. HepG2: Chol-let-7a vs. Chol-miRCtrl vs. blank, 148.3 ± 7.02 vs. 203.0 ± 5.29 vs. 214.67 ± 11.67; T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.001; Chol-let-7a vs. blank, p = 0.005; Chol-miRCtrl vs. blank, p = 0.207. SMMC7721: Chol-let-7a vs. Chol-miRCtrl vs. blank, 155.67 ± 6.66 vs. 218.33 ± 9.45 vs.230.67 ± 7.02; T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.021; Chol-let-7a vs. blank, p = 0.01; Chol-miRCtrl vs. blank, p = 0.066. Magnification: 20× D: The inhibitory effect of Chol-let-7a on cell invasion ability in the Boyden chamber invasion assay. HepG2: Chol-let-7a vs. Chol-miRCtrl vs. blank, 66.33 ± 4.73vs. 91.33 ± 3.21 vs. 97.00 ± 5.29; T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.005; Chol-let-7a vs. blank, p = 0.026; Chol-miRCtrl vs. blank, p = 0.362. SMMC7721 cells: Chol-let-7a vs. Chol-miRCtrl vs. blank: 73.00 ± 5.29 vs. 91.33 ± 3.21 vs.103.33 ± 4.73. T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.019; Chol-let-7a vs. blank, p =0.016; Chol-miRCtrl vs. blank, p = 0.408. Magnification: 20×.
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Fig1: Chol-let-7ainhibited HCC cell growth and cell viability in vitro.A,B: MTT assays show the effects of Chol-let-7a and Chol-miRCtrl on the growth of HepG2 and SMMC7721 cells. The figure shows the growth curve of tumour cells from days 1–5. blank, parental cells; Chol-miRCtrl, Chol-miRCtrl-transfected cells; Chol-let-7a, Chol-let-7a-transfected cells. C: A chamber-based cell migration assay showing that Chol-let-7a inhibits the migration of HCC cells. HepG2: Chol-let-7a vs. Chol-miRCtrl vs. blank, 148.3 ± 7.02 vs. 203.0 ± 5.29 vs. 214.67 ± 11.67; T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.001; Chol-let-7a vs. blank, p = 0.005; Chol-miRCtrl vs. blank, p = 0.207. SMMC7721: Chol-let-7a vs. Chol-miRCtrl vs. blank, 155.67 ± 6.66 vs. 218.33 ± 9.45 vs.230.67 ± 7.02; T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.021; Chol-let-7a vs. blank, p = 0.01; Chol-miRCtrl vs. blank, p = 0.066. Magnification: 20× D: The inhibitory effect of Chol-let-7a on cell invasion ability in the Boyden chamber invasion assay. HepG2: Chol-let-7a vs. Chol-miRCtrl vs. blank, 66.33 ± 4.73vs. 91.33 ± 3.21 vs. 97.00 ± 5.29; T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.005; Chol-let-7a vs. blank, p = 0.026; Chol-miRCtrl vs. blank, p = 0.362. SMMC7721 cells: Chol-let-7a vs. Chol-miRCtrl vs. blank: 73.00 ± 5.29 vs. 91.33 ± 3.21 vs.103.33 ± 4.73. T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.019; Chol-let-7a vs. blank, p =0.016; Chol-miRCtrl vs. blank, p = 0.408. Magnification: 20×.

Mentions: The growth curves of HepG2 and SMMC7721 cells during the MTT assay are shown in Figure 1A and B, respectively. After 5 days, Chol-let-7a decreased HepG2 and SMMC7721 viability by 37.7% and 36.6%, respectively, in comparison with the parental cells (blank) (p <0.05). No significant differences in growth were observed among the 2 control HCC cell lines, the negative control miRNA mimic (Chol-miRCtrl)-treated cells, and the parental cells (p >0.05). These results verified that Chol-let-7a inhibited HCC cell growth in vitro.Figure 1


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)

Chol-let-7ainhibited HCC cell growth and cell viability in vitro.A,B: MTT assays show the effects of Chol-let-7a and Chol-miRCtrl on the growth of HepG2 and SMMC7721 cells. The figure shows the growth curve of tumour cells from days 1–5. blank, parental cells; Chol-miRCtrl, Chol-miRCtrl-transfected cells; Chol-let-7a, Chol-let-7a-transfected cells. C: A chamber-based cell migration assay showing that Chol-let-7a inhibits the migration of HCC cells. HepG2: Chol-let-7a vs. Chol-miRCtrl vs. blank, 148.3 ± 7.02 vs. 203.0 ± 5.29 vs. 214.67 ± 11.67; T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.001; Chol-let-7a vs. blank, p = 0.005; Chol-miRCtrl vs. blank, p = 0.207. SMMC7721: Chol-let-7a vs. Chol-miRCtrl vs. blank, 155.67 ± 6.66 vs. 218.33 ± 9.45 vs.230.67 ± 7.02; T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.021; Chol-let-7a vs. blank, p = 0.01; Chol-miRCtrl vs. blank, p = 0.066. Magnification: 20× D: The inhibitory effect of Chol-let-7a on cell invasion ability in the Boyden chamber invasion assay. HepG2: Chol-let-7a vs. Chol-miRCtrl vs. blank, 66.33 ± 4.73vs. 91.33 ± 3.21 vs. 97.00 ± 5.29; T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.005; Chol-let-7a vs. blank, p = 0.026; Chol-miRCtrl vs. blank, p = 0.362. SMMC7721 cells: Chol-let-7a vs. Chol-miRCtrl vs. blank: 73.00 ± 5.29 vs. 91.33 ± 3.21 vs.103.33 ± 4.73. T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.019; Chol-let-7a vs. blank, p =0.016; Chol-miRCtrl vs. blank, p = 0.408. Magnification: 20×.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Fig1: Chol-let-7ainhibited HCC cell growth and cell viability in vitro.A,B: MTT assays show the effects of Chol-let-7a and Chol-miRCtrl on the growth of HepG2 and SMMC7721 cells. The figure shows the growth curve of tumour cells from days 1–5. blank, parental cells; Chol-miRCtrl, Chol-miRCtrl-transfected cells; Chol-let-7a, Chol-let-7a-transfected cells. C: A chamber-based cell migration assay showing that Chol-let-7a inhibits the migration of HCC cells. HepG2: Chol-let-7a vs. Chol-miRCtrl vs. blank, 148.3 ± 7.02 vs. 203.0 ± 5.29 vs. 214.67 ± 11.67; T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.001; Chol-let-7a vs. blank, p = 0.005; Chol-miRCtrl vs. blank, p = 0.207. SMMC7721: Chol-let-7a vs. Chol-miRCtrl vs. blank, 155.67 ± 6.66 vs. 218.33 ± 9.45 vs.230.67 ± 7.02; T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.021; Chol-let-7a vs. blank, p = 0.01; Chol-miRCtrl vs. blank, p = 0.066. Magnification: 20× D: The inhibitory effect of Chol-let-7a on cell invasion ability in the Boyden chamber invasion assay. HepG2: Chol-let-7a vs. Chol-miRCtrl vs. blank, 66.33 ± 4.73vs. 91.33 ± 3.21 vs. 97.00 ± 5.29; T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.005; Chol-let-7a vs. blank, p = 0.026; Chol-miRCtrl vs. blank, p = 0.362. SMMC7721 cells: Chol-let-7a vs. Chol-miRCtrl vs. blank: 73.00 ± 5.29 vs. 91.33 ± 3.21 vs.103.33 ± 4.73. T-test: Chol-let-7a vs. Chol-miRCtrl, p = 0.019; Chol-let-7a vs. blank, p =0.016; Chol-miRCtrl vs. blank, p = 0.408. Magnification: 20×.
Mentions: The growth curves of HepG2 and SMMC7721 cells during the MTT assay are shown in Figure 1A and B, respectively. After 5 days, Chol-let-7a decreased HepG2 and SMMC7721 viability by 37.7% and 36.6%, respectively, in comparison with the parental cells (blank) (p <0.05). No significant differences in growth were observed among the 2 control HCC cell lines, the negative control miRNA mimic (Chol-miRCtrl)-treated cells, and the parental cells (p >0.05). These results verified that Chol-let-7a inhibited HCC cell growth in vitro.Figure 1

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