1-Methylhydantoin Inhibits The Secretion Of Growth Hormone In Rabbits

Objective: To investigate the mechanism of resistance to TRAIL in hepatocellular carcinoma cell lines, To explore the role of ISG15 and mi R-138 in the mechanism of drug resistance in TRAIL, in To further enhance the TRAIL-induced apoptosis in cancer cells.Methods: We examined TRAIL sensitivity of different human HCC cell lines: LH86, Huh7, HLCZ01, Huh7. Treatment of four HCC cell lines(4h, 6h, 12 h, 8h) with different concentrations of TRAIL(1ng/ml, 5ng/ml,10ng/ml, 20ng/ml), TRAIL-induced apoptosis was tested by MTT assay.Then use the quantitative real-time PCR assay and western blot assay to showed the different expression of ISG15 and mi R-138 in TRAIL-sensitive cells and TRAIL-resistant cells. We established Plasmid construction and transfected into cells with Lipofectamine 2000, overexpression ISG15 in TRAIL-sensitive cells, in contrast, silenced ISG15 in TRAIL-resistant cells.And verify the effect of ISG15 in TRAIL induced cell apoptosis by western blot assay. We performed bioinformatics search(Targetscan) and found that ISG15 is a putative target of mi R-138.And we overexpression mi R-138 by transfected mi R-138 mimics into TRAIL-sensitive cells and TRAIL-resistant cells. We use qt-PCR assay to detected the effect of mi R-138 to the expression of ISG15 and verify the effect of ISG15 in TRAIL induced cell apoptosis by western blot assay.Results: 1. The cytotoxic effect of TRAIL on human HCC,and LH86 and SMMC-7721 is sensitive for TRAIL treatment, in contrast, the Huh7 and HLCZ01 cells is TRAIL-resistant.Use 10ng/ml of TRAIL and treat for 8h is the most suitable way to liver cancer cell lives.2.The expression of ISG15 in sensitive cells is lower than in sensitive cells,and the expression of mi R-138 in sensitive cells is higher than in sensitive cells.3.Overexpression ISG15 in TRAIL-sensitive cells caused a corresponding loss of sensitization to TRAIL-induced apoptosis. in contrast, silenced ISG15 in TRAIL-resistant cells increase the TRAIL-induced apoptosis. These data suggested that ISG15 regulates the sensitivity of cancer cells to TRAIL induced apoptosis in vitro.4. Overexpression of mi R-138 in TRAIL-sensitive and TRAIL-resistant cells can inhibit the expression of ISG15, which indicates that ISG15 is the target gene of mi R-138.5. Overexpression of mi R-138 in TRAIL-sensitive cells and TRAIL-resistant cells can increase the TRAIL-induced apoptosis. These data suggested that Mi R-138 modulates the sensitivity of cancer cells to TRAIL-induced apoptosis.6. Forced expression of mi R-138 and ISG15 at the same time in TRAIL-sensitive cells showed that changed the TRAIL sensitive phenotype to a resistant one. These data suggested that mi R-138 modulates the sensitivity of cancer cells to TRAIL-induced apoptosis by targeting ISG15.Conclusions:1. ISG15 regulates the sensitivity of cancer cells to TRAIL treatment.2. Mi R-138 inhibiting the expression of ISG15 to regulate the sensitivity of hepatocellular carcinoma cells to TRAIL.