| Objective:Ovarian cancer is one of the most common malignant tumors of the female reproductive system and is highly aggressive and lethal.Due to the deep ovarian location,atypical early symptoms,and unreliable screening methods,most ovarian cancer patients are diagnosed at an advanced stage.The first-line treatment for ovarian cancer includes surgical resection combined with paclitaxel(PTX)chemotherapy.Although the median survival rate has improved in recent years,the ensuing phenomenon of paclitaxel resistance has become a major cause of treatment failure in ovarian cancer patients,with a 5-year survival rate of only 15%-30%.Studies have shown that RAB small GTPases and related regulatory proteins and effectors are involved in the developmental process of many cancers,and as a member of the RAB family,RAB17 plays an important role in regulating the metastasis and progression of numerous cancers,but,as of today,the specific mechanism of RAB17 involvement in tumor drug resistance remains unclear.CircRNAs(circRNAs)are a class of non-codingRNAs without 5'-cap or 3'-polyadenosine tails and with covalent closed-loop structures,which have structural stability,good conservation,tissue specificity,and different developmental stages in different species with Expression specificity and other properties have made circRNAs a hot research topic in recent years.circRNAs can act as competitive endogenousRNAs(ceRNAs)or miRNA sponges that bind to miRNAs through miRNA response elements(MREs)and negatively regulate their activities.miRNAs are an abundant class of small non-codingRNAs,about 22 nucleotides.They act as negative regulators of gene expression at the post-transcriptional level through incomplete base pairing with the respective 3'-untranslated region(3'-UTR).As miRNA sponges,circRNAs can function as oncogenes or tumor suppressor genes,and a growing number of studies have shown that dysregulation of circRNAs is associated with the progression of numerous cancers.Nevertheless,the functional role of circRNAs in paclitaxel resistance in ovarian cancer remains unclear.In this study,we found that RAB17 was highly expressed in paclitaxel-resistant ovarian cancer cells A2780/PTX,confirming the important role of RAB17 in paclitaxel resistance,cell proliferation,and cell cycle,and the role of RAB17 and its non-codingRNA network hsa?circ?0000714/miR-370-3p/RAB17 in paclitaxel resistance in ovarian cancer and its This will help to improve the understanding of paclitaxel resistance and identify potential biomarkers of paclitaxel resistance in ovarian cancer.Methods:1?The RAB family proteins with significantly different expression in paclitaxelresistant ovarian cancer cells A2780/PTX and paclitaxel-sensitive cells A2780 were screened by gene microarray,and the RAB17 with high expression in A2780/PTX cells and low expression in A2780 with the largest difference ploidy was selected for study by combining the difference ploidy and P-value.2?The paclitaxel resistance index of ovarian cancer paclitaxel-sensitive cells SKOV3,A2780 and drug-resistant cells SKOV3/PTX,A2780/PTX was detected by CCK8 assay.The expression of RAB17 mRNA and protein levels in SKOV3,A2780,and SKOV3/PTX,A2780/PTX cells were detected by quantitative real-time PCR(qRT-PCR)combined with Western blotting.3?The transfection efficiency was detected by qRT-PCR and Western blotting after transfection of A2780/PTX cells with RAB17 interference plasmid and A2780 cells with RAB17 overexpression virus,respectively.CCK8 was used to determine the paclitaxel resistance index after A2780/PTX interference with RAB17 expression and A2780 overexpression of RAB17.Clonal proliferation assay was performed to detect the clonal proliferation ability of cells after RAB17 expression change.Flow cytometry was used to analyze cell cycle changes.western blotting was used to detect changes in the expression of associated apoptotic proteins,anti-apoptotic proteins,adhesion proteins,and related signaling pathways.4?A combination of bioinformatics database Star Base(http://starbase.sysu.edu.cn/)and dual-luciferase reporter gene assay was used to screen miRNA miR-370-3p complementary to the 3'-UTR region of RAB17 and to validate the relationship between RAB17 and miR-370-3p targeting relationship.Screening of circRNA hsa?circ?0000714,which is low expressed in A2780 and high expressed in A2780/PTX and can be used as a molecular sponge for miR-370-3p,was performed by Star Base analysis of bioinformatics database,and the localization of hsa?circ?0000714 was detected by cell nucleoplasm separation assay,and dual luciferase assay to verify the targeting relationship between miR-370-3p and hsa?circ?0000714.QRT-PCR and Western blotting to detect RAB17 expression after transfection of A2780/PTX cells with miR-370-3p mimics or interference with hsa?circ?0000714,CCK8 assay changes in cellular paclitaxel resistance index.A clonogenic proliferation assay was performed to detect cell proliferation Flow cytometry was used to detect cell cycle changes.western blotting was used to detect changes in related signaling pathway proteins.Results:1?RAB17 was significantly highly expressed in paclitaxel-resistant ovarian cancer cells A2780/PTX,while it was lowly expressed in corresponding ovarian cancer paclitaxelsensitive cells A2780.2?Knockdown of RAB17 expression in A2780/PTX cells significantly increased cell sensitivity to paclitaxel,decreased cell paclitaxel resistance index,significantly inhibited cell proliferation,increased G1 phase and decreased S phase in A2780/PTX cells,blocked more cells in G1 phase,inhibited cell cycle,and increased expression of corresponding adhesion and apoptosis proteins,and decreased expression of anti-apoptotic proteins.3?Overexpression of RAB17 in A2780 cells decreased the sensitivity of A2780 cells to paclitaxel,increased the paclitaxel resistance index of A2780 cells,promoted cell proliferation,decreased cell G1 phase and increased S phase,accelerated cell G1-S phase transition,promoted cell cycle,and decreased the expression of corresponding adhesion and apoptotic proteins,and increased the expression of anti-apoptotic proteins.4?Bioinformatics database Star Base analysis of the 3'-UTR region of RAB17 has a potential binding site for miR-370-3p.The results of the dual-luciferase assay showed that when miR-370-3p mimics were cotransfected with RAB17-WT,the luciferase activity was significantly reduced,indicating that RAB17 is a direct target gene of miR-370-3p.5 ? A2780/PTX cells transfected with miR-370-3p mimics showed significant inhibition of RAB17 expression,decreased cellular paclitaxel resistance index,diminished clonal proliferation ability,increased cellular G1 phase,and decreased S-phase resulting in cell cycle arrest in G1 phase and cell cycle inhibition.6?Hsa?circ?0000714 was highly expressed in drug-resistant cells A2780/PTX and low expressed in sensitive cells A2780.Nucleoplasmic separation assay showed that hsa?circ?0000714 was mainly expressed in the cytoplasm,which provides the possibility that hsa?circ?0000714 exerts a molecular sponge.dual luciferase assay confirmed the interaction between miR-370-3p and hsa?circ?0000714.hsa?circ?0000714 was expressed in A2780/PTX cells when hsa?circ?0000714 expression was disturbed when qRT-PCR showed that miR-370-3p expression was increased while RAB17 expression was suppressed,confirming that hsa?circ?0000714 can act as a molecular sponge for miR-370-3p to further regulate the expression of the target gene RAB17.7?When the siRNA plasmid interfered with hsa?circ?0000714 expression,the paclitaxel resistance index of A2780/PTX cells decreased,cell clonal proliferation ability was weakened,cell G1 phase increased while S phase decreased,blocking more cells in the G1 phase and inhibiting cell cycle.8 ? Hsa?circ?0000714/miR-370-3p regulates RAB17 expression,which affects paclitaxel resistance in ovarian cancer cells A2780/PTX by activating CDK6/RB signaling pathway.Conclusion:The hsa?circ?0000714/miR-370-3p/RAB17 regulatory network exists in ovarian cancer paclitaxel-resistant cells A2780/PTX.hsa?circ?0000714 acts as a molecular sponge for miR-370-3p to regulate the target gene RAB17 expression through activating the CDK6/RB signaling pathway in ovarian cancer paclitaxel resistance progression. |
PDF Full Text Request