摘要
BACKGROUND Hepatocellular carcinoma(HCC) is the third leading cause of death from malignant tumors worldwide. More than 50% of HCC cases occur in China. The prognosis remains poor and overall efficacy is still unsatisfactory. Chemotherapy resistance is the most important reason for the poor outcome. Much progress has been made in the study of chemotherapy resistance of HCC;however, the specific mechanisms of progression of HCC have still only been partially established.Therefore, the mechanism of chemotherapy resistance in HCC requires more research.AIM To investigate the effect of miR-34 a expression on the growth inhibition of HepG2 cells by doxorubicin.METHODS A recombinant lentiviral vector containing miR-34 a was constructed and transfected into HepG2 cells. The expression of miR-34 a was detected by reverse transcription-polymerase chain reaction(commonly known as RT-PCR) before and after transfection. Cells were exposed to 2 μM doxorubicin or phosphatebuffered saline before and after transfection. Cell viability in each group was detected by MTT assay, and cell cycle and apoptosis were detected by flow cytometry. Changes in expression levels of phospho(p)-p53, sirtuin(SIRT) 1,cyclin D1, cyclin-dependent kinase(CDK) 4, CDK6, BCL-2, multidrug resistance protein(MDR) 1/P glycoprotein(P-gp), and AXL were detected by Western blotting.RESULTS Recombinant lentiviral vector LV-hsa-mir-34 a was successfully constructed by restriction endonuclease digestion and sequencing. RT-PCR showed that expression of miR-34 a in HepG2 cells was significantly upregulated after transfection(P < 0.01). MTT assay showed that growth of HepG2 cells was inhibited after upregulation of miR-34 a, and viability was significantly decreased after combined treatment with doxorubicin(P < 0.01). Flow cytometry showed that the number of HepG2 cells in G1 phase increased, and G1 phase arrest was more obvious after intervention with doxorubicin(P < 0.01). The apoptosis rate of HepG2 cells was increased after upregulation of miR-34 a, and became more obvious after intervention with doxorubicin(P < 0.01). Western blotting showed that upregulation of miR-34 a combined with treatment with doxorubicin caused significant changes in the expression levels of p-p53, SIRT1, cyclin D1, CDK4,CDK6, BCL-2, MDR1/P-gp and AXL proteins(P < 0.01).CONCLUSION MiR-34 a may enhance the inhibitory effect of doxorubicin by downregulating MDR1/P-gp and AXL, which may be related to p53 expression.
BACKGROUND Hepatocellular carcinoma(HCC) is the third leading cause of death from malignant tumors worldwide. More than 50% of HCC cases occur in China. The prognosis remains poor and overall efficacy is still unsatisfactory. Chemotherapy resistance is the most important reason for the poor outcome. Much progress has been made in the study of chemotherapy resistance of HCC; however, the specific mechanisms of progression of HCC have still only been partially established.Therefore, the mechanism of chemotherapy resistance in HCC requires more research.AIM To investigate the effect of miR-34 a expression on the growth inhibition of HepG2 cells by doxorubicin.METHODS A recombinant lentiviral vector containing miR-34 a was constructed and transfected into HepG2 cells. The expression of miR-34 a was detected by reverse transcription-polymerase chain reaction(commonly known as RT-PCR) before and after transfection. Cells were exposed to 2 μM doxorubicin or phosphatebuffered saline before and after transfection. Cell viability in each group was detected by MTT assay, and cell cycle and apoptosis were detected by flow cytometry. Changes in expression levels of phospho(p)-p53, sirtuin(SIRT) 1,cyclin D1, cyclin-dependent kinase(CDK) 4, CDK6, BCL-2, multidrug resistance protein(MDR) 1/P glycoprotein(P-gp), and AXL were detected by Western blotting.RESULTS Recombinant lentiviral vector LV-hsa-mir-34 a was successfully constructed by restriction endonuclease digestion and sequencing. RT-PCR showed that expression of miR-34 a in HepG2 cells was significantly upregulated after transfection(P < 0.01). MTT assay showed that growth of HepG2 cells was inhibited after upregulation of miR-34 a, and viability was significantly decreased after combined treatment with doxorubicin(P < 0.01). Flow cytometry showed that the number of HepG2 cells in G1 phase increased, and G1 phase arrest was more obvious after intervention with doxorubicin(P < 0.01). The apoptosis rate of HepG2 cells was increased after upregulation of miR-34 a, and became more obvious after intervention with doxorubicin(P < 0.01). Western blotting showed that upregulation of miR-34 a combined with treatment with doxorubicin caused significant changes in the expression levels of p-p53, SIRT1, cyclin D1, CDK4,CDK6, BCL-2, MDR1/P-gp and AXL proteins(P < 0.01).CONCLUSION MiR-34 a may enhance the inhibitory effect of doxorubicin by downregulating MDR1/P-gp and AXL, which may be related to p53 expression.
基金
the National Natural Science Foundation of China,No.81302124
Shandong Key R and D Program No.2017GSF218038
Shandong Provincial Natural Science Foundation,No.ZR2014HP065
