Exosomal LncRNAH19 Promotes Paclitaxel Resistance By Regulating MiR-340-3p In Breast Cancer

Objective: To investigate the molecular mechanism of whether lnc RNA H19,derived from drug-resistant cells,regulates miR-340-3p to induce paclitaxel resistance in breast cancer and changes in its biological activity.Methods: 1.Gradient centrifugation was used to separate exosomes from cell supernatant;2.The shape of exosomes was captured by transmission electron microscopy;3.Western Blot analysis of exosome marker proteins(CD63,TSG101);4.The uptake of exosomes by the recipient cells was observed by laser confocal method;5.Quantitative real time polymerase chain reaction(q RT-PCR)and Western Blot were used to detect the expression of drug resistance indexes(P-gp,BCRP,MRP)after co-culture of SKBR-3/PR-derived exosomes with SKBR-3 receptor cells;6.Transwell and cell scratches were used to verify the effects of co-culture of exosomes with recipient cells on cell invasion and migration;7.Expression of miR-340-3p and lnc RNA H19 in SKBR-3 cells and SKBR-3/PR cells was analyzed by q RT-PCR;8.Efficiency of knockdown or overexpression of lnc RNA H19 and transfection with miR-340-3p mimics/inhibitor was analyzed by q RT-PCR;9.The expression of miR-340-3p after knockdown and overexpression of lnc RNA H19 was analyzed by q RT-PCR;10.CCK-8,flow cytometry,Transwell and cell scratch were used to detect the effects of lnc RNA H19 and miR-340-3p on cell invasion,proliferation,migration and apoptosis;11.The expression changes of knockdown or overexpression of lnc RNA H19 and miR-340-3p mimics/inhibitor(p-gp,BCRP,MRP)after transfection were detected by q RT-PCR and Western Blot;12.Bioinformatics database analysis showed that lnc RNA H19 and miR-340-3p had complementary binding sequences,and dual luciferase assay further verified the structural complementarity of the two;13.Two-way complementary tests were conducted in combination with miR-340-3p mimics or inhibitor for overexpression or interference of lnc RNA H19,respectively.Transwell and cell Nick were used to detect cell invasion and migration ability,and q RT-PCR and Western Blot were used to detect the expression of EMT-related indicators(E-cadherin,Vimentin and snail).Results: 1.Expression of lnc RNA H19 in exosomes from SKBR-3/PR cells(SKBR-3/PR-exo)was higher than that from SKBR-3 cells(SKBR-3-exo);2.The expression of lnc RNA H19 was up-regulated after SKBR-3/ PR-Exo co-culture with SKBR-3;3.Coculture of SKBR-3 cells with SKBR-3/ PR-Exo can enhance cell invasion and migration;4.After SKBR-3 cells were co-cultured with SKBR-3/ PR-Exo,the protein and m RNA expression levels of drug resistance related indexes P-gp,BCRP and MRP were upregulated;5.Expression of lnc RNA H19 in SKBR-3 cells was lower than that in SKBR-3/PR cells,and expression of miR-340-3p in SKBR-3 cells was higher than that in SKBR-3/PR cells;6.After overexpression of lnc RNA H19,the expression of miR-340-3p decreased;However,knockdown of lnc RNA H19 could up-regulate the expression of miR-340-3p;7.Overexpression of lnc RNA H19 enhanced the ability of cell migration,proliferation and invasion,and decreased the rate of cell apoptosis;However,knockdown of lnc RNA H19 resulted in the opposite biological effect;8.Overexpression of lnc RNA H19 resulted in down-regulated protein and m RNA expressions of drug-resistance related indicators E-cadherin,and up-regulated protein and m RNA expressions of Vimentin and Snail.Knocking down lnc RNA H19 resulted in up-regulation of the protein and m RNA expressions of E-cadherin and down-regulation of the protein and m RNA expressions of Vimentin and Snail;9.After transfection with miR-340-3p inhibitor,the ability of cell migration,proliferation and invasion was enhanced,and the number of cell apoptosis was decreased;10.After transfection with miR-340-3p inhibitor,the protein and m RNA expression levels of drug-resistant indicators E-cadherin were down-regulated,while the protein and m RNA expression levels of Vimentin and Snail were up-regulated.However,miR-340-3p mimics up-regulated the protein and m RNA expression levels of E-cadherin,while down-regulated the protein and m RNA expression levels of Vimentin and Snail;11.The fluorescence of miR-340-3p mimics transfected into lnc RNA H19 mutant vector cells remained unchanged,while the fluorescence of miR-340-3p mimics transfected into lnc RNA H19 wild-type vector cells decreased;12.When lnc RNA H19 was knocked down or overexpressed,the effect of miR-340-3p inhibitor/mimics could be reversed by transfection respectively;13.Overexpression or knockdown of lnc RNA H19 affected EMT phenotype.However,transfection of miR-340-3p inhibitor/mimics at the same time of knockdown or overexpression of lnc RNA H19 reversed its effect on EMT.Conclusion: 1.Drug resistant exosomes can deliver lnc RNA H19 to recipient cells;2.Exosome lnc RNA H19 regulates miR-340-3p to induce paclitaxel resistance in breast cancer cells,enhancing cell migration,invasion,proliferation and reducing apoptosis.