| Objective:To investigate the changes of long non-coding RNA(LncRNA)and messenger RNA(mRNA)expression profiles in paclitaxel-resistant cells(MCF-7 /PR)of breast cancer relative to parental cells(MCF-7).To explore the role of lncRNA LINC00173 in the induction of paclitaxel resistance in breast cancer.Methods: 1.Construction of paclitaxel-resistant cells MCF-7 /PR and SK-BR-3 /PR of human breast cancer cells MCF-7 and SK-BR-3 by concentration gradient method.2.Expression of lncRNA and mRNA in MCF-7 /PR relative to MCF-7 cells by Arraystar lncRNA microarray.3.Screening differentially expressed LncRNA and mRNA using Agilent GeneSpring GX v12.1 software,and performing GO and KEGG pathway analysis on differential mRNAs to obtain biological pathways in which mRNAs participate.4.Differential expression of LINC00173 in breast normal epithelial cells MCF10 A and breast cancer cells MCF-7,SK-BR-3,MDA-MB-231 and MDA-MB-468 by qRT-PCR.5.Analysis of the localization of LINC00173 in the nucleus and cytoplasm by qRT-PCR.6.Differential expression of LINC00173 in MCF-7,SK-BR-3 and MCF-7/PR,SK-BR-3 /PR cells by qRT-PCR.7.qRT-PCR detection and screening of the best interference of LINC00173 Fragments and identification of the constructed LINC00173 lentiviral overexpressing cell line.8.CCK-8,plate cloning assay to detect interference and overexpression of LINC00173 on breast cancer cell lines MCF-7,MCF-7/PR,SK-BR-3 and SK-BR-3/PR proliferative capacity.9.The effect of LINC00173 overexpression in MCF-7 /PR and SK-BR-3 /PR on the sensitivity of paclitaxel was detected by CCK-8.10.Effects of interference and overexpression of LINC00173 on migration and invasion of MCF-7,MCF-7 /PR,SK-BR-3 and SK-BR-3 /PR in breast cancer cells detected by scratch test and transwell test.11.qRT-PCR and Western blot were used to detect the effects of interference and overexpression of LINC00173 on the expression of P-gp,CyclinD1 and ?-catenin mRNA and protein in breast cancer cell lines MCF-7,MCF-7/PR,SK-BR-3 and SK-BR-3/PR.Results: 1.The extracted total RNA was detected by spectrophotometer NanoDrop ND-1000 to obtain RNA samples of the required purity,and the total RNA integrity was determined by denaturing agarose gel electrophoresis.The spectrophotometer NanoDropND-1000 detects the quality of fluorescently labeled cRNA,and the sample can be subjected to chip hybridization experiments.2.There were 1504 LncRNA and 2264 mRNAs differentially expressed by MCF-7/PR cells compared with MCF-7 parental cells(P <0.05;difference fold > 2.0).3.Analysis by GO and KEGG showed: Up-regulation of mRNA and down-regulation of mRNA are involved in a variety of different biological processes and pathways.4.LINC00173 expression in breast cancer cell lines was lower than normal breast epithelial cells(P<0.05).5.LINC00173 was mainly located in the nucleus.6.The expression of LINC00173 in breast cancer resistant cell lines MCF-7/PR and SK-BR-3/PR was lower than that of parental cell lines MCF-7 and SK-BR-3(P<0.05).7.The interference effect of LINC00173 interference fragment 3 was the best(P<0.05),and the overexpressed cell line was successfully constructed.8.Overexpression of LINC00173,cell proliferation,invasion and metastasis decreased(P<0.05);interfered with the expression of LINC00173,cell proliferation,invasion and metastasis capacity increased(P<0.05).9.Overexpression of LINC00173 in breast cancer resistant cell lines increased the sensitivity of paclitaxel(P<0.05).10.Overexpression of LINC00173 down-regulated the expression of P-gp,CyclinD1,?-catenin,and interfered with the up-regulation of LINC00173 Expression of P-gp,CyclinD1 and ?-catenin(P<0.05).Conclutions: 1.This study found that the expression of LncRNA and mRNA in paclitaxel-resistant cells of breast cancer is different.Differentially expressed genes can provide clues for finding new sensitive targets or biomarkers for chemotherapy.2.The expression of LINC00173 was down-regulated in breast cancer tissues and paclitaxelresistant cell lines,and down-regulation of LINC00173 induced paclitaxel resistance.Overexpression of LINC00173 inhibited breast cancer cell proliferation,invasion and metastasis,and improved paclitaxel drug sensitivity.3.LINC00173 may be involved in the paclitaxel resistance process by regulating the expression of ?-catenin. |
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