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Targeting the microRNA-21/AP1 axis by 5-fluorouracil and pirarubicin in human hepatocellular carcinoma.

He X, Li J, Guo W, Liu W, Yu J, Song W, Dong L, Wang F, Yu S, Zheng Y, Chen S, Kong Y, Liu C - Oncotarget (2015)

Bottom Line: We demonstrated that microRNA-21 suppression in combination with 5-fluorouracil and pirarubicin treatment inhibited tumor growth in subcutaneous xenograft mice models.Mechanistically, the AP-1 and microRNA-21-mediated axis was verified to be a therapeutic target of cytotoxic drugs and deregulation of this axis led to an enhanced cell growth in HCC.Targeting microRNA-21 enhances the effect of chemotherapeutic drugs, thereby suggesting that microRNA-21 suppression in combination with HAIC may be a novel approach for HCC treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, PR China.

ABSTRACT
MicroRNAs function as oncomiRs and tumor suppressors in diverse cancers. However, the utility of specific microRNAs in predicting the clinical benefit of chemotherapy has not been well-established. Here, we investigated the correlation between microRNA-21 expression and hepatic arterial infusion chemotherapy with 5-fluorouracil and pirarubicin (HAIC) for hepatocellular carcinoma (HCC). We found that HCC patients with low microRNA-21 levels in tumors tended to have a longer time to recurrence and disease-free survival. We demonstrated that microRNA-21 suppression in combination with 5-fluorouracil and pirarubicin treatment inhibited tumor growth in subcutaneous xenograft mice models. Mechanistically, the AP-1 and microRNA-21-mediated axis was verified to be a therapeutic target of cytotoxic drugs and deregulation of this axis led to an enhanced cell growth in HCC. Taken together, our findings demonstrate that microRNA-21 is a chemotherapy responsive microRNA and can serve as a prognostic biomarker for HCC patients undergoing HAIC. Targeting microRNA-21 enhances the effect of chemotherapeutic drugs, thereby suggesting that microRNA-21 suppression in combination with HAIC may be a novel approach for HCC treatment.

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Related in: MedlinePlus

The miR-21-mediated program is modified by 5-fluorouracil and pirarubicin treatment in HCC cellsA: The correlation between miR-21 and its targets was validated in Hep3b cells via luciferase reporter assay (i) and immunoblots (ii) analysis. GAPDH was included as a loading control, and the data were normalized to scr or anti-control treated cells. The numbers below the panels represent the normalized protein expression levels. B: The protein levels of miR-21 target proteins in Hep3b and SMMC7721 cells after treatment with 5-fluorouracil (i) or pirarubicin (ii) as measured through immunoblot analysis. GAPDH served as a loading control. C: Modulation of miR-21 targets expression in 5-fluorouracil-treated Hep3b (i), SMMC7721 (ii) cells, and pirarubicin-treated Hep3b (iii), SMMC7721 (iv) cells was performed by transfection with miR-21 mimic. The target proteins level was measured through immunoblot analysis. GAPDH served as a loading control. The numbers below the panels represent the normalized protein expression levels. D: HCC cell growth was determined using CCK-8 at 36 h on different treatment conditions as described as (C). E: Immunoblot analyses of p-AKT, AKT, p-GSK-3β, GSK-3β, CDK2, and CDK4 were performed using Hep3b and SMMC7721 cells with 5-fluorouracil (i) and pirarubicin (ii) treatment. GAPDH served as the loading control. F: 5-fluorouracil and pirarubicin have clinical benefit for HCC treatment through modulating the AP-1 and miR-21-mediated axis.
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Figure 5: The miR-21-mediated program is modified by 5-fluorouracil and pirarubicin treatment in HCC cellsA: The correlation between miR-21 and its targets was validated in Hep3b cells via luciferase reporter assay (i) and immunoblots (ii) analysis. GAPDH was included as a loading control, and the data were normalized to scr or anti-control treated cells. The numbers below the panels represent the normalized protein expression levels. B: The protein levels of miR-21 target proteins in Hep3b and SMMC7721 cells after treatment with 5-fluorouracil (i) or pirarubicin (ii) as measured through immunoblot analysis. GAPDH served as a loading control. C: Modulation of miR-21 targets expression in 5-fluorouracil-treated Hep3b (i), SMMC7721 (ii) cells, and pirarubicin-treated Hep3b (iii), SMMC7721 (iv) cells was performed by transfection with miR-21 mimic. The target proteins level was measured through immunoblot analysis. GAPDH served as a loading control. The numbers below the panels represent the normalized protein expression levels. D: HCC cell growth was determined using CCK-8 at 36 h on different treatment conditions as described as (C). E: Immunoblot analyses of p-AKT, AKT, p-GSK-3β, GSK-3β, CDK2, and CDK4 were performed using Hep3b and SMMC7721 cells with 5-fluorouracil (i) and pirarubicin (ii) treatment. GAPDH served as the loading control. F: 5-fluorouracil and pirarubicin have clinical benefit for HCC treatment through modulating the AP-1 and miR-21-mediated axis.

Mentions: Because an miR-21-mediated PTEN/PDCD4/RECK program has been identified in HepG2 and SMMC7721 HCC cells, we next investigated whether this program was modified by 5-fluorouracil and pirarubicin treatment. The correlation between miR-21 and their targets was further validated in Hep3b cells, indicating that PTEN, PDCD4, and RECK were direct effectors of miR-21 in this cell line (Figure. 5A). We next conducted immunoblot analyses to evaluate the protein levels of PTEN, PDCD4, and RECK in Hep3b and SMMC7721 cells after 5-fluorouracil and pirarubicin treatment and we showed that the tested drugs increased the levels of these proteins, as compared to control-treated cells (Figure. 5B). To further probe the correlation between cell phenotypic alterations and drugs-mediated AP-1/miR-21 axis, we performed a rescue assay that increased and then decreased the level of miR-21 targets via drugs treatment in combination with miR-21 overexpression in HCC cells. Immunoblotting was used to evaluate PTEN, PDCD4, and RECK expression and demonstrated that the level of these proteins was altered under different treatment conditions in Hep3b and SMMC7721 cells (Figure. 5C). Furthermore, cell growth analysis was conducted to determine the alterations in cell viability corresponding with the expression level variations of targeted proteins, and we observed that miR-21 overexpression prevent the suppressive impact of 5-fluorouracil (Figure. 5D-i) and pirarubicin (Figure. 5D-ii) treatment on HCC cell growth.


Targeting the microRNA-21/AP1 axis by 5-fluorouracil and pirarubicin in human hepatocellular carcinoma.

He X, Li J, Guo W, Liu W, Yu J, Song W, Dong L, Wang F, Yu S, Zheng Y, Chen S, Kong Y, Liu C - Oncotarget (2015)

The miR-21-mediated program is modified by 5-fluorouracil and pirarubicin treatment in HCC cellsA: The correlation between miR-21 and its targets was validated in Hep3b cells via luciferase reporter assay (i) and immunoblots (ii) analysis. GAPDH was included as a loading control, and the data were normalized to scr or anti-control treated cells. The numbers below the panels represent the normalized protein expression levels. B: The protein levels of miR-21 target proteins in Hep3b and SMMC7721 cells after treatment with 5-fluorouracil (i) or pirarubicin (ii) as measured through immunoblot analysis. GAPDH served as a loading control. C: Modulation of miR-21 targets expression in 5-fluorouracil-treated Hep3b (i), SMMC7721 (ii) cells, and pirarubicin-treated Hep3b (iii), SMMC7721 (iv) cells was performed by transfection with miR-21 mimic. The target proteins level was measured through immunoblot analysis. GAPDH served as a loading control. The numbers below the panels represent the normalized protein expression levels. D: HCC cell growth was determined using CCK-8 at 36 h on different treatment conditions as described as (C). E: Immunoblot analyses of p-AKT, AKT, p-GSK-3β, GSK-3β, CDK2, and CDK4 were performed using Hep3b and SMMC7721 cells with 5-fluorouracil (i) and pirarubicin (ii) treatment. GAPDH served as the loading control. F: 5-fluorouracil and pirarubicin have clinical benefit for HCC treatment through modulating the AP-1 and miR-21-mediated axis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: The miR-21-mediated program is modified by 5-fluorouracil and pirarubicin treatment in HCC cellsA: The correlation between miR-21 and its targets was validated in Hep3b cells via luciferase reporter assay (i) and immunoblots (ii) analysis. GAPDH was included as a loading control, and the data were normalized to scr or anti-control treated cells. The numbers below the panels represent the normalized protein expression levels. B: The protein levels of miR-21 target proteins in Hep3b and SMMC7721 cells after treatment with 5-fluorouracil (i) or pirarubicin (ii) as measured through immunoblot analysis. GAPDH served as a loading control. C: Modulation of miR-21 targets expression in 5-fluorouracil-treated Hep3b (i), SMMC7721 (ii) cells, and pirarubicin-treated Hep3b (iii), SMMC7721 (iv) cells was performed by transfection with miR-21 mimic. The target proteins level was measured through immunoblot analysis. GAPDH served as a loading control. The numbers below the panels represent the normalized protein expression levels. D: HCC cell growth was determined using CCK-8 at 36 h on different treatment conditions as described as (C). E: Immunoblot analyses of p-AKT, AKT, p-GSK-3β, GSK-3β, CDK2, and CDK4 were performed using Hep3b and SMMC7721 cells with 5-fluorouracil (i) and pirarubicin (ii) treatment. GAPDH served as the loading control. F: 5-fluorouracil and pirarubicin have clinical benefit for HCC treatment through modulating the AP-1 and miR-21-mediated axis.
Mentions: Because an miR-21-mediated PTEN/PDCD4/RECK program has been identified in HepG2 and SMMC7721 HCC cells, we next investigated whether this program was modified by 5-fluorouracil and pirarubicin treatment. The correlation between miR-21 and their targets was further validated in Hep3b cells, indicating that PTEN, PDCD4, and RECK were direct effectors of miR-21 in this cell line (Figure. 5A). We next conducted immunoblot analyses to evaluate the protein levels of PTEN, PDCD4, and RECK in Hep3b and SMMC7721 cells after 5-fluorouracil and pirarubicin treatment and we showed that the tested drugs increased the levels of these proteins, as compared to control-treated cells (Figure. 5B). To further probe the correlation between cell phenotypic alterations and drugs-mediated AP-1/miR-21 axis, we performed a rescue assay that increased and then decreased the level of miR-21 targets via drugs treatment in combination with miR-21 overexpression in HCC cells. Immunoblotting was used to evaluate PTEN, PDCD4, and RECK expression and demonstrated that the level of these proteins was altered under different treatment conditions in Hep3b and SMMC7721 cells (Figure. 5C). Furthermore, cell growth analysis was conducted to determine the alterations in cell viability corresponding with the expression level variations of targeted proteins, and we observed that miR-21 overexpression prevent the suppressive impact of 5-fluorouracil (Figure. 5D-i) and pirarubicin (Figure. 5D-ii) treatment on HCC cell growth.

Bottom Line: We demonstrated that microRNA-21 suppression in combination with 5-fluorouracil and pirarubicin treatment inhibited tumor growth in subcutaneous xenograft mice models.Mechanistically, the AP-1 and microRNA-21-mediated axis was verified to be a therapeutic target of cytotoxic drugs and deregulation of this axis led to an enhanced cell growth in HCC.Targeting microRNA-21 enhances the effect of chemotherapeutic drugs, thereby suggesting that microRNA-21 suppression in combination with HAIC may be a novel approach for HCC treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, PR China.

ABSTRACT
MicroRNAs function as oncomiRs and tumor suppressors in diverse cancers. However, the utility of specific microRNAs in predicting the clinical benefit of chemotherapy has not been well-established. Here, we investigated the correlation between microRNA-21 expression and hepatic arterial infusion chemotherapy with 5-fluorouracil and pirarubicin (HAIC) for hepatocellular carcinoma (HCC). We found that HCC patients with low microRNA-21 levels in tumors tended to have a longer time to recurrence and disease-free survival. We demonstrated that microRNA-21 suppression in combination with 5-fluorouracil and pirarubicin treatment inhibited tumor growth in subcutaneous xenograft mice models. Mechanistically, the AP-1 and microRNA-21-mediated axis was verified to be a therapeutic target of cytotoxic drugs and deregulation of this axis led to an enhanced cell growth in HCC. Taken together, our findings demonstrate that microRNA-21 is a chemotherapy responsive microRNA and can serve as a prognostic biomarker for HCC patients undergoing HAIC. Targeting microRNA-21 enhances the effect of chemotherapeutic drugs, thereby suggesting that microRNA-21 suppression in combination with HAIC may be a novel approach for HCC treatment.

Show MeSH
Related in: MedlinePlus