PRPF6 promotes metastasis and paclitaxel resistance of ovarian cancer via SNHG16/CEBPB/GATA3 axis

Metastasis and paclitaxel(PTX)resistance are the main reason for the poor prognosis of ovarian cancer(OC).Evidence showed that RNA-binding proteins(RBPs)and long noncoding RNAs(lncRNAs)can modulate post-transcriptional regulation.The aim of this study was to determine the relationship among RBP,lncRNA and OC and to further guide clinical therapy.Immunohistochemistry revealed that pre-mRNA processing factor 6(PRPF6)was upregulated in OC chemoresistant tissues and was closely related to advanced(Federation of International of Gynecologists and Obstetricians)FIGO stages and chemo-resistance.PRPF6 promoted progression,and PTX resistance in vitro and in vivo.And the transcripts of small nucleolar RNA host gene SNHG16-L/S were differentially expressed in OC cells and tissues as detected through real-time PCR(RT-PCR).SNHG16-L/S had opposite effects on progression and PTX resistance in OC.Mechanistically,SNHG16-L inhibited GATA-binding protein 3(GATA3)transcription by binding to CCAAT/enhancer-binding protein B(CEBPB).Moreover,PRPF6 induced the alternative splicing of SNHG16,causing downregulation of SNHG16-L and,leading to the upregulation of GATA3 expression to further promote metastasis and PTX-resistance in OC.Totally,these data unveiled that PRPF6 promotes metastasis and PTX resistance of OC through SNHG16-L/CEBPB/GATA3 axis,which provides a new direction for OC treatment.

Targeting of p38 Mitogen-activated Protein Kinases to Early Growth Response gene 1 (EGR-1) in the Human Paclitaxel-resistance Ovarian Carcinoma Cells

To investigate the relationship between the expression of early growth response gene 1 （EGR-1） and p38MAPK pathway in the paclitaxel resistance of ovarian carcinoma cells, the effect of p38MAPK inhibitor SB203580 on cell apoptosis was examined by using Hoechst 33258 staining. The intracellular Rh123 （Rhodamine 123） accumulation was detected by the flow cytometry （FCM）. The 50% inhibition concentration （IC50） of paclitaxel for A2780/Taxol cells was determined by MTT method. Electrophoretic motility shift assay （EMSA） was employed to examine the EGR-1DNA binding activity. MDR1 and EGR-1 mRNA were assessed by RT-PCR. The expressed of p-gp, phos- phorylated p53 and p38 were detected by Western blotting. SB203580 could remarkably promote the apoptosis of A2780/Taxol cells, and the cell apoptosis was in a time-dependent manner. Cellular Rh123 accumulation was increased, and the IC50 of paclitaxel for A2780/Taxol cells was decreased significantly. A2780/Taxol cell line after SB203580 treatment was shown to have a significantly higher level of EGR-1 DNA binding activity. SB203580 down-regulated the activity of p38MAPK pathway, but up-regulated EGR-1 expression. SB203580 significantly increased the level of cellular phosphorylated p53 protein, but decreased the p-gp protein level and MDR1 mRNA level in A2780/Taxol cells. There existed a close relationship between p38MAPK pathway and the paclitaxel resistance of ovarian carcinoma cells. The expression of EGR-1 mediated by p38MAPK pathway plays a critical role in paclitaxel resistance of ovarian carcinoma cells.

Upregulation of miR-34c after silencing E2F transcription factor 1 inhibits paclitaxel combined with cisplatin resistance in gastric cancer cells

BACKGROUND MicroRNA 34c(miR-34c)has been reported to be associated with malignant types of cancer,however,it remains unknown whether miR-34c is involved in chemoresistance in gastric cancer(GC).AIM To investigate the effect of miR-34c and its upstream transcription factor E2F1 on paclitaxel combined with cisplatin resistance in GC cells.METHODS Paired GC tissues and adjacent normal tissues were randomly sampled from 74 GC patients.miR-34c and E2F1 were detected by real-time quantitative PCR(qPCR)and Western blot.In addition,the drug resistance of GC cells to paclitaxel and cisplatin was induced by concentration gradient increasing methods,and changes in miR-34c and E2F1 during this process were measured.Furthermore,E2F1 and miR-34c overexpression or underexpression vectors were constructed and transfected into drug-resistant GC cells.MTT was employed to test the sensitivity of cells to paclitaxel combined with cisplatin,qPCR was adopted to detect the expression of miR-34c,Western blot was applied to detect the expression levels of E2F1,drug resistance-related proteins and apoptosis-related proteins,and flow cytometry was used for the determination of cell apoptosis and cell cycle status.RESULTS E2F1 was overexpressed while miR-34c was underexpressed in GC.After inducing GC cells to be resistant to paclitaxel and cisplatin,E2F1 expression increased while miR-34c expression decreased.Both silencing E2F1 and overexpressing miR-34c could increase the sensitivity of drug-resistant GC cells to paclitaxel combined with cisplatin,promote cell apoptosis and inhibit cell proliferation.Among which,silencing E2F1 could reduce the expression of drug resistance-related proteins and apoptosis-related proteins,while over-expression of miR-34c could upregulate the expression of apoptosis-related proteins without affecting the expression of MDR-1,MRP and other drug resistance-related proteins.Rescue experiments demonstrated that inhibiting miR-34c could significantly weaken the sensitization of drug resistant cells,and Si E2F1 to paclitaxel combined with cisplatin.CONCLUSION E2F1 inhibits miR-34c to promote the proliferation of GC cells and enhance the resistance to paclitaxel combined with cisplatin,and silencing E2F1 is conducive to improving the efficacy of paclitaxel combined with cisplatin in GC cells.

Effect of Mad2 on Paclitaxel-induced Cell Death in Ovarian Cancer Cells

In this article, the status of spindle assembly checkpoint and the alteration of its major component, Mad2 protein level were examined in A2780 and SKOV3 ovarian cancer cell lines.Recombinant eukaryotic expression plasmid pEGFP-Mad2 was transfected into paclitaxel-resistant SKOV3 cells and Mad2 protein was knocked down by Mad2-specific siRNA in paclitaxel-sensitive A2780 cells.Then the expression level of Mad2 gene was detected by Western blotting.Flow cytometry revealed that SKOV3 cells were not fully arrested in G2/M phase in contrast to A2780 cells in the presence of paclitaxel.However, paclitaxel sensitivity assay showed that sensitivity to paclitaxel was reversed after the transfection in both cell lines in terms of number of cells arrested at G2/M phase and the expression of Bcl-2 was significantly changed.These results suggest that weakened spindle checkpoint with reduced expression of Mad2 is associated with resistance to paclitaxel in ovarian cells and Bcl-2 may be involved in this process.