Study On Paclitaxel Resistance Induced By Exosomal LncRNA SNHG15 Regulating MiR-141-3p In Breast Cancer

Objective: To investigate the mechanism of drug-resistant cell-derived exosome lnc RNA SNHG15 regulating the miR-141-3p /CXCL12 pathway to induce paclitaxel resistance and change of biological activity in breast cancer.Methods: 1.Exosomes of cell supernatant were extracted by kit method;2.Exosomes were photographed by transmission electron microscopy;3.Western Blot analysis of exosome marker proteins（CD63,TSG101）;4.Observation of exosome uptake by recipient cells by fluorescence microscope;5.SKBR-3 /PR cell-derived exosomes were co-cultured with recipient SKBR-3 cells,and the expression of drug resistance indicators（P-gp and BCRP）were detected by q RT-PCR and Western Blot.Transwell and cell scratch assay were used to detect cell migration;6.Expression of lnc RNA SNHG15 and miR-141-3p in SKBR-3 and SKBR-3/PR were detected by q RT-PCR;7. qRT-PCR was used to detect the expression efficiency of knockdown and overexpressed lnc RNA SNHG15 and miR-141-3p mimics/inhibitor transfection;8.qRT-PCR was used to detect the expression efficiency of miR-141-3p after knockdown and overexpression of lnc RNA SNHG15;9.Transwell and scratch assay were used to detect the effects of lnc RNA SNHG15 and miR-141-3p on cell migration.Western Blot was used to detect the expression of drug resistance indicators（P-gp BCRP）;10.The binding sequence of lnc RNA SNHG15 and miR-141-3p was analyzed by bioinformatics software,and the regulatory relationship was verified by dual luciferase assay;11.Lnc RNA SNHG15 overexpression/knockdown combined with miR-141-3p mimics/inhibitor bidirectional complementary assay,respectively.Transwell and cell nicks assay were used to detect cell migration ability.Western Blot was used to detect the expression of drug resistance indicators（P-gp,BCRP）;12.miR-141-3p mimics was combined with CXCL12 overexpression,and the expression of drug resistance indicators（P-gp and BCRP）was detected by Western Blot;13.miR-141-3p mimics/inhibitor was combined with AKT phosphorylation inhibitor,and p-Akt expression was detected by Western Blot;14.Animal models of paclitaxel resistant mice were established to observe the interference of SNHG15 expression.CXCL12 expression level in lung tissues of mice was detected by immunohistochemical staining through tumor tissue volume and weight,and drug sensitivity of paclitaxel was analyzed.Results: 1.Compared with SKBR-3 cell-derived exosomes（SKBR-3/ PR-EXO）,lnc RNA SNHG15 expression was higher in SKBR-3/ PR-EXO;2.After SKBR-3 /PR-EXO co-cultured with SKBR-3 cells,the expression of lnc RNA SNHG15 increased,the migration and invasion ability of SKBR-3 cells increased,and the expression levels of drug resistance indicators P-gp and BCRP increased;3.Lnc RNA SNHG15 expression in SKBR-3 cells was lower than that in SKBR-3/PR cells,and miR-141-3p expression in SKBR-3 cells was higher than that in SKBR-3/PR cells;4.After knockdown of lnc RNA SNHG15,the expression level of miR-141-3p was up-regulated,while after overexpression of lnc RNA SNHG15,the expression level of miR-141-3p was decreased;5.After knockdown lnc RNA SNHG15,the cell migration ability were weakened,and the expression levels of drug resistance index proteins P-gp and BCRP were decreased.Overexpression of lnc RNA SNHG15 enhanced the cell migration ability,and the expression levels of P-gp and BCRP were up-regulated;6.After transfection with miR-141-3p inhibitor,cell migration and invasion ability were enhanced,and the expression levels of drug resistance indicator proteins P-gp and BCRP were increased;However,after transfection with miR-141-3p mimics,the cell migration ability were weakened,and the expression levels of P-gp and BCRP,drug resistance indicators,were decreased;7.The luciferase activity of wild-type lnc RNA SNHG15 vector was decreased after transfection with miR-141-3p mimics,while the luciferase activity of mutant lnc RNA SNHG15 vector was not significantly changed after transfection with miR-141-3p mimics;8.Compared with the lnc RNA SNHG15 knockdown group,the expression of CXCL12 induced by lnc RNA SNHG15 knockdown was reversed in the combined group with miR-141-3p inhibitor,and the migration and invasion ability of cells were weakened.The expression levels of P-gp and BCRP were down-regulated.Compared with the overexpressed lnc RNA SNHG15 group,the combination group with miR-141-3p mimics reversed the up-regulation of CXCL12 expression caused by overexpression of lnc RNA SNHG15,and enhanced the migration ability of cells,as well as the up-regulation of the expression levels of drug resistance indicator proteins P-gp and BCRP;9.Compared with the control group,interfering CXCL12 expression could inhibit the migration ability of cells and down-regulate the expression of drug resistance index proteins P-gp and BCRP;10.Compared with the control group,overexpression of CXCL12 enhanced the migration and invasion ability of breast cancer cells and up-regulated the expression of drug resistance indicators,while miR-141-3p mimics reversed this phenomenon;11.Compared with the control group,the expression level of P-Akt increased after transfection with miR-141-3p inhibitor,while decreased after transfection with miR-141-3p mimics;12.Compared with the control group,the tumor tissue volume of si-SNHG15+PTX group decreased,and the expression level of CXCL12 in lung tissue decreased.Conclusions: 1.Lnc RNA SNHG15 can be delivered to recipient cells by exosomes from drug-resistant cells.2.Lnc RNA SNHG15 induces drug resistance of breast cancer cells by regulating the expression of miR-141-3p /CXCL12,and enhances the ability of cell migration,invasion and proliferation.