(I) IC50 values of PTX were calculated by MTT assay in transfected MGC-803/PTX and AGS/PTX cells. Our results showed that circ-PVT1 expression was up-regulated in PTX-resistant GC tissues and cells. Circ-PVT1 down-regulation enhanced PTX sensitivity in PTX-resistant GC cells by negatively regulating miR-124-3p. ZEB1 served as a direct target of miR-124-3p. Circ-PVT1 enhanced ZEB1 expression by sponging miR-124-3p. Circ-PVT1 knockdown increased PTX sensitivity of GC Taken together, our studies disclosed that circ-PVT1 facilitated PTX resistance by up-regulating ZEB1 mediated via miR-124-3p, suggesting an underlying therapeutic strategy for GC. = 6 per group) were collected from Hubei Research Center of Laboratory Animal (Wuhan, China). MGC-803/PTX cells were stably transfected with sh-circ-PVT1 or sh-control. Whereafter, the left flank of the nude mice was subcutaneously injected 4 ICEC0942 HCl 106 transduced cells. At 7 days after injection, sh-control + PBS ICEC0942 HCl and sh-circ-PVT1 + PBS groups were intraperitoneally administrated PBS, sh-control + PTX and sh-circ-PVT1 + PTX groups were intraperitoneally administrated 3 mg/kg PTX (Bristol-Myers Squibb, Shanghai, China) every 4 days. Tumor volume was examined every 4 days after the first injection. Twenty-seven days later, tumors were excised and weighed. Also, the circ-PVT1 expression level in xenograft tumors was detected by RT-qPCR assay. Statistical analysis SPSS 17.0 software (IBM, Chicago, IL, U.S.A.) and Graph pad Prism 5.0 were employed for all statistical analysis. The difference of data between two groups or among multiple groups was analyzed with Students value 0.05. Results Circ-PVT1 was highly expressed in PTX-resistant GC tissues and cells Firstly, we investigated the expression level of circ-PVT1 in PTX-resistant GC tissues (= 30) and adjacent PTX-sensitive GC tissues (= 30) obtained from patients with GC. As displayed in Physique 1A, compared with PTX-sensitive tissues, circ-PVT1 expression was apparently increased in PTX-resistant GC tissues. Then, we also confirmed that circ-PVT1 was highly expressed in GC cell lines (MKN-45, HGC-27, MGC-803, and AGS) relative to normal human gastric epithelial cell line GES-1, particularly in MGC-803 and AGS cells (Physique 1B). Therefore, we selected H460 and A549 cells for the subsequent analyses. Furthermore, circ-PVT1 level in PTX-resistant GC cells (MGC-803/PTX and AGS/PTX) was further explored. Similarly, higher expression of circ-PVT1 was observed in PTX-resistant GC cells in contrast with their parental cells MGC-803 and AGS (Physique 1C). In a word, these results suggested that circ-PVT1 might be associated with PTX resistance in GC cells. Open in a separate window Physique 1 Circ-PVT1 was highly expressed in PTX-resistant GC tissues and cells(A) Expression of circPVT1 in 30 pairs of PTX-resistant GC tissues Rabbit Polyclonal to DYNLL2 and adjacent PTX-sensitive GC tissues. (B) RT-qPCR assay was employed to detect the expression of circPVT1 in GC cell lines (MKN-45, HGC-27, MGC-803, and AGS) and normal human gastric epithelial cell line (GES-1). (C) Expression level of circPVT1 in PTX-resistant GC cells was measured by RT-qPCR assay; * 0.05. ICEC0942 HCl Circ-PVT1 knockdown enhanced PTX sensitivity in PTX-resistant GC cells Next, to identify the effect of circ-PVT1 on PTX-resistant GC cells, IC50 value of PTX was first measured by MTT assay. As illustrated in Physique 2A, IC50 value of PTX was evidently elevated in MGC-803/PTX and AGS/PTX cells in comparison with MGC-803 and AGS cells, suggesting the production of PTX resistance in MGC-803/PTX and AGS/PTX cells. Then, to identify the effect of circPVT1 on PTX-resistant GC cells, the knockdown short hairpin RNA (shRNA) of circ-PVT1 was synthesized. The transfection efficiency was detected and presented in Physique 2B. Moreover, some studies have indicated that certain proteins were related to multidrug resistance (MDR), such as P-gp and GST- [24,25]. Thus, we used the knockdown system to further explore the impact of circPVT1 around the levels of P-gp and GST-. RT-qPCR analysis disclosed that mRNA levels of P-gp and GST- were obviously repressed after down-regulation of circ-PVT1 in MGC-803/PTX and AGS/PTX cells (Physique 2C,D). Similarly, deletion of circ-PVT1 obviously also suppressed the protein levels of P-gp and GST- in MGC-803/PTX and AGS/PTX cells (Physique 2E,F). Besides, IC50 determination displayed that knockdown of circ-PVT1 resulted in an evident decrease in PTX resistance in MGC-803/PTX and AGS/PTX cells (Physique 2G). Subsequently, to further probe the influence of circPVT1 around the progression of PTX-resistant GC cells, cell apoptosis and invasion were assessed. The results of flow cytometry.