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MicroRNA-21 induces resistance to the anti-tumour effect of interferon-α/5-fluorouracil in hepatocellular carcinoma cells.

Tomimaru Y, Eguchi H, Nagano H, Wada H, Tomokuni A, Kobayashi S, Marubashi S, Takeda Y, Tanemura M, Umeshita K, Doki Y, Mori M - Br. J. Cancer (2010)

Bottom Line: The aim of this study was to investigate the anti-tumour effects of microRNA (miR)-21 on the sensitivity of HCC cells to IFN-α/5-FU and whether miR-21 can be used as a predictor of the response to such therapy in HCC.Annexin V assay showed that the percentage of apoptotic cells was significantly lower in cells transfected with pre-miR-21 than control cells.The miR-21 in HCC cell lines and clinical HCC samples is a significant modulator of the anti-tumour effect of IFN-α and 5-FU.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Graduate School of Medicine, Osaka University, Suita, 2-2 Yamadaoka E-2, Osaka 565-0871, Japan.

ABSTRACT

Background: We reported recently the clinical efficiency of interferon (IFN)-α/5-fluorouracil (5-FU) combination therapy in advanced hepatocellular carcinoma (HCC). However, prediction of the response to the combination therapy remains unsatisfactory. The aim of this study was to investigate the anti-tumour effects of microRNA (miR)-21 on the sensitivity of HCC cells to IFN-α/5-FU and whether miR-21 can be used as a predictor of the response to such therapy in HCC.

Methods: Changes in the sensitivity of HCC cells (PLC/PRF/5 and HepG2) to IFN-α/5-FU were examined after transfection with pre-miR-21 or anti-miR-21. The correlation between miR-21 expression level, evaluated by qRT-PCR, and response to the therapy was also investigated in clinical HCC specimens.

Results: Hepatocellular carcinoma cells transfected with pre-miR-21 were significantly resistant to IFN-α/5-FU. Annexin V assay showed that the percentage of apoptotic cells was significantly lower in cells transfected with pre-miR-21 than control cells. Transfection of anti-miR-21 rendered HCC cells sensitive to IFN-α/5-FU, and such sensitivity was weakened by transfection of siRNAs of target molecules, PETN and PDCD4. miR-21 expression in clinical HCC specimens was significantly associated with the clinical response to the IFN-α/5-FU combination therapy and survival rate.

Conclusions: The miR-21 in HCC cell lines and clinical HCC samples is a significant modulator of the anti-tumour effect of IFN-α and 5-FU. This suggests that miR-21 is a potentially suitable marker for the prediction of the clinical response to the IFN-α/5-FU combination therapy.

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Transfection of pre-miR-21 into PLC/PRF/5 and HepG2. (A) qRT–PCR showed significant overexpression of miR-21 in the transfected cells compared with control cells (*P<0.05). (B) MTT assay showed that the anti-tumour effects of the combination of IFN-α and 5-FU in the miR-21 upregulated cells was significantly lower than in control cells (*P<0.05). (C and D) MTT assay revealed that the anti-tumour effects of IFN-α (C) and 5-FU (D) in the miR-21 upregulated cells was significantly less profound than in control cells (*P<0.05). (E) The percentage of early apoptotic cells induced by 1000 IU per ml IFN-α or 1.0 μg per ml 5-FU among miR-21 upregulated cells was significantly lower than in control cells (*P<0.05). Data are mean±s.d. of three experiments.
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fig2: Transfection of pre-miR-21 into PLC/PRF/5 and HepG2. (A) qRT–PCR showed significant overexpression of miR-21 in the transfected cells compared with control cells (*P<0.05). (B) MTT assay showed that the anti-tumour effects of the combination of IFN-α and 5-FU in the miR-21 upregulated cells was significantly lower than in control cells (*P<0.05). (C and D) MTT assay revealed that the anti-tumour effects of IFN-α (C) and 5-FU (D) in the miR-21 upregulated cells was significantly less profound than in control cells (*P<0.05). (E) The percentage of early apoptotic cells induced by 1000 IU per ml IFN-α or 1.0 μg per ml 5-FU among miR-21 upregulated cells was significantly lower than in control cells (*P<0.05). Data are mean±s.d. of three experiments.

Mentions: To evaluate the effect of miR-21 on the response to IFN-α and 5-FU, we transfected pre-miR-21 into PLC/PRF/5 and HepG2, which showed the highest and lowest expression level of miR-21 among the five cell lines, respectively. The expression of miR-21 was confirmed to be significantly increased in the transfected cells by qRT–PCR (Figure 2A). The MTT assay showed that cells overexpressing miR-21 were significantly more resistant to the combination therapy of IFN-α and 5-FU than the control cells (Figure 2B). Next, we investigated the effect of transfection of pre-miR-21 on the separate growth-inhibitory effect of each of IFN-α and 5-FU. The result showed that transfection of pre-miR-21 significantly weakened the growth-inhibitory effect of both IFN-α and 5-FU in the two cancer cell lines compared with the control cells (Figure 2C and D). We also evaluated the extent of apoptosis of these cells at 24 h induced by treatment with 1000 IU per ml IFN-α or 1.0 μg per ml 5-FU by the Annexin V assay. The percentage of early apoptotic cells was significantly lower in the two cancer cell lines transfected with pre-miR-21 than in control cells (Figure 2E).


MicroRNA-21 induces resistance to the anti-tumour effect of interferon-α/5-fluorouracil in hepatocellular carcinoma cells.

Tomimaru Y, Eguchi H, Nagano H, Wada H, Tomokuni A, Kobayashi S, Marubashi S, Takeda Y, Tanemura M, Umeshita K, Doki Y, Mori M - Br. J. Cancer (2010)

Transfection of pre-miR-21 into PLC/PRF/5 and HepG2. (A) qRT–PCR showed significant overexpression of miR-21 in the transfected cells compared with control cells (*P<0.05). (B) MTT assay showed that the anti-tumour effects of the combination of IFN-α and 5-FU in the miR-21 upregulated cells was significantly lower than in control cells (*P<0.05). (C and D) MTT assay revealed that the anti-tumour effects of IFN-α (C) and 5-FU (D) in the miR-21 upregulated cells was significantly less profound than in control cells (*P<0.05). (E) The percentage of early apoptotic cells induced by 1000 IU per ml IFN-α or 1.0 μg per ml 5-FU among miR-21 upregulated cells was significantly lower than in control cells (*P<0.05). Data are mean±s.d. of three experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2990590&req=5

fig2: Transfection of pre-miR-21 into PLC/PRF/5 and HepG2. (A) qRT–PCR showed significant overexpression of miR-21 in the transfected cells compared with control cells (*P<0.05). (B) MTT assay showed that the anti-tumour effects of the combination of IFN-α and 5-FU in the miR-21 upregulated cells was significantly lower than in control cells (*P<0.05). (C and D) MTT assay revealed that the anti-tumour effects of IFN-α (C) and 5-FU (D) in the miR-21 upregulated cells was significantly less profound than in control cells (*P<0.05). (E) The percentage of early apoptotic cells induced by 1000 IU per ml IFN-α or 1.0 μg per ml 5-FU among miR-21 upregulated cells was significantly lower than in control cells (*P<0.05). Data are mean±s.d. of three experiments.
Mentions: To evaluate the effect of miR-21 on the response to IFN-α and 5-FU, we transfected pre-miR-21 into PLC/PRF/5 and HepG2, which showed the highest and lowest expression level of miR-21 among the five cell lines, respectively. The expression of miR-21 was confirmed to be significantly increased in the transfected cells by qRT–PCR (Figure 2A). The MTT assay showed that cells overexpressing miR-21 were significantly more resistant to the combination therapy of IFN-α and 5-FU than the control cells (Figure 2B). Next, we investigated the effect of transfection of pre-miR-21 on the separate growth-inhibitory effect of each of IFN-α and 5-FU. The result showed that transfection of pre-miR-21 significantly weakened the growth-inhibitory effect of both IFN-α and 5-FU in the two cancer cell lines compared with the control cells (Figure 2C and D). We also evaluated the extent of apoptosis of these cells at 24 h induced by treatment with 1000 IU per ml IFN-α or 1.0 μg per ml 5-FU by the Annexin V assay. The percentage of early apoptotic cells was significantly lower in the two cancer cell lines transfected with pre-miR-21 than in control cells (Figure 2E).

Bottom Line: The aim of this study was to investigate the anti-tumour effects of microRNA (miR)-21 on the sensitivity of HCC cells to IFN-α/5-FU and whether miR-21 can be used as a predictor of the response to such therapy in HCC.Annexin V assay showed that the percentage of apoptotic cells was significantly lower in cells transfected with pre-miR-21 than control cells.The miR-21 in HCC cell lines and clinical HCC samples is a significant modulator of the anti-tumour effect of IFN-α and 5-FU.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Graduate School of Medicine, Osaka University, Suita, 2-2 Yamadaoka E-2, Osaka 565-0871, Japan.

ABSTRACT

Background: We reported recently the clinical efficiency of interferon (IFN)-α/5-fluorouracil (5-FU) combination therapy in advanced hepatocellular carcinoma (HCC). However, prediction of the response to the combination therapy remains unsatisfactory. The aim of this study was to investigate the anti-tumour effects of microRNA (miR)-21 on the sensitivity of HCC cells to IFN-α/5-FU and whether miR-21 can be used as a predictor of the response to such therapy in HCC.

Methods: Changes in the sensitivity of HCC cells (PLC/PRF/5 and HepG2) to IFN-α/5-FU were examined after transfection with pre-miR-21 or anti-miR-21. The correlation between miR-21 expression level, evaluated by qRT-PCR, and response to the therapy was also investigated in clinical HCC specimens.

Results: Hepatocellular carcinoma cells transfected with pre-miR-21 were significantly resistant to IFN-α/5-FU. Annexin V assay showed that the percentage of apoptotic cells was significantly lower in cells transfected with pre-miR-21 than control cells. Transfection of anti-miR-21 rendered HCC cells sensitive to IFN-α/5-FU, and such sensitivity was weakened by transfection of siRNAs of target molecules, PETN and PDCD4. miR-21 expression in clinical HCC specimens was significantly associated with the clinical response to the IFN-α/5-FU combination therapy and survival rate.

Conclusions: The miR-21 in HCC cell lines and clinical HCC samples is a significant modulator of the anti-tumour effect of IFN-α and 5-FU. This suggests that miR-21 is a potentially suitable marker for the prediction of the clinical response to the IFN-α/5-FU combination therapy.

Show MeSH
Related in: MedlinePlus