Yuen PW, Lam KY, Chan AC, Wei WI, Lam LK

Yuen PW, Lam KY, Chan AC, Wei WI, Lam LK. in vitro and in vivo were determined by RNA-Seq, qRT-PCR, western blots, transmission electron microscopy, and circulation cytometry, fluorescence, CCK8, Transwell, and wound healing assays. < 0.05, **< 0.01, ***< 0.001. Open in a separate window Physique 2 Docetaxel resistance in HSC-3 cells (HSC-3DR) was associated with EMT and elevated drug efflux. (A) Migration ability of HSC-3 and HSC-3DR cells was determined by wound healing assays (level bars = 100 m). (B) The expressions of EMT-associated proteins in HSC-3DR cells were determined by western blots. (C) The expression of nuclear KR1_HHV11 antibody -H2AX of HSC-3 and HSC-3DR cells was determined by fluorescence assays (level bars = 10 m). Data are offered as mean SD. *< 0.05, **< 0.01, ***< 0.001. Downregulation of miR-200c was essential for DTX resistance in HSC-3 cells In this study, we performed RNA-Seq analysis to Papain Inhibitor determine the differential miRNA expression profile between HSC-3 and HSC-3DR cells, and the results were plotted in the volcano plot (Physique 3A). Papain Inhibitor Then, we used qRT-PCR assay to verify the expressions of miRNAs that were found to be decreased in RNA-Seq analysis (Physique 3B). The results exhibited that miR-200c was one of significantly decreased miRNA in HSC-3DR cells compared with HSC-3 cells. MiR-200c has been demonstrated to be essential for chemoresistance in several malignancy types [25, 28]. Thus, we focused on the role of miR-200c in DTX resistance in TSCC. Next, we examined the expression of miR-200c in five TSCC cell lines and the results revealed that the level of miR-200c was lower in all five carcinoma cell lines relative to NTECs, but the HSC-3 cell collection had higher expression of miR-200c than the other cell lines (Physique 3C). Also, the expression of miR-200c was significantly lower in HSC-3DR cells compared to HSC-3 cells (Physique 3D). To further investigate the function of miR-200c in DTX resistance, we overexpressed miR-200c through the miR-200c-encoding lentiviral vector (LV-200c). After transfection with LV-200c, the level of miR-200c was markedly increased in HSC-3DR cells (Physique 3E). In a series of Papain Inhibitor functional experiments, forced expression of miR-200c resulted in lower cell viability (Physique 3F), elevated apoptosis (Physique 3G), and inhibited abilities of migration and invasion (Physique 3H, ?,3I),3I), as well as reduced motility (Physique 4A). Furthermore, overexpression of miR-200c reversed the effect of DTX resistance around the expressions of EMT-associated proteins (Physique 4B) which led to more DNA damage in HSC-3DR cells (Physique 4C). Moreover, we investigated the effect of miR-200c on DTX in vivo by subcutaneously injecting LV-200c-transfected HSC-3DR cells into nude mice, followed by DTX treatment. The results showed that overexpression of miR-200c reduced DTX resistance in HSC-3DR cells in response to DTX treatment in vivo and mice treated with LV-200c-transfected HSC-3DR cells and DTX displayed the slowest tumor growth (Physique 4D, ?,4E).4E). Therefore, these results together exhibited that forced expression of miR-200c could sensitize HSC-3DR cells to DTX in both in vitro and in vivo. Open in a separate window Physique 3 Downregulation of miR-200c was involved in docetaxel resistance in HSC-3 cells (HSC-3DR). (A) volcano plot of RNA-Seq analysis. Red and green points symbolize significantly upregulated and downregulated miRNAs, respectively, according to fold switch > 2 and adjusted <.