| Objective: Gemcitabine is considered as an essential first-line chemotherapy drug for patients with advanced triple-negative breast cancer(TNBC),and resistance to gemcitabine will result in tumor progression.The applicant has constructed human cell lines of gemcitabine-resistance in TNBC,and found that mi R-484-CDA axis played a part in regulating the chemo-sensitivity of breast cancer cells to gemcitabine.In addition,using the Gene Ontology analysis,it was revealed that differential expression genes were related to drug metabolism processes,also may modulate the sensitivity to gemcitabine by multiple pathways.How to efficiently and high throughout explore the gene related to gemcitabine resistance is an important basis to solve clinical gemcitabine-resistance.Methods: 1.To validate the stability of Gemcitabine-resistant cell that have been done,the Half Maximal Inhibitory Concentration of gemcitabine on MDA-231-Gem was detected by cytotoxicity assay.2.Expression profile changes of m RNA between the gemcitabine-resistance and gemcitabine-sensitivity cell was analyzed by gene ontology analysis,and the resistance-related candidate genes were screened.3.With the DNABarcode library,human opening reading frame library,plasmids library which overexpress gemcitabine resistance-related gene of triple negative breast cancer cell,and lentivirus system,we established a breast cancer cell line which stable overexpresses resistance-related candidate genes.4.After Administrated gemcitabine,the gemcitabine-resistance target genes were screened by the next generation sequencing analysis platform and bioinformatics technique.5.Real-time quantitative PCR and cytotoxicity were used to validate the target gene.SPSS22.0 statistical software was used to analyze the data.Results: 1.CCK-8 Cytotoxicity assay detected and validated the resistance stability of MDA-231-Gem.The IC50 of MDA-231-Gem was 21.85 n M,and it is more than 12 times of IC50 of MDA-231(P <0.05).2.The analysis of expression profile chip showed that there were 829 resistance-related genes.And GO analysis showed that there were40 gene sets which P <0.01 and 228 genes which constituted a set of resistance-related candidate genes.3.We constructed p DEST-Barcode library,and amplified 145 resistant-related candidate genes from ORF library and followed constructed plasmids which overexpress gemcitabine resistance-related genes,then mixed plasmids in equimolar volume.4.We constructed the stable expression cell lines were named as MDA-231-Lib and MDA-231-Con,and IC50 of them was 66.96 n M and 3.47 n M,respectively(P<0.05).The correlation of candidate gene between gemcitabine resistances was strong.5.The results of the next-generation sequencing showed that cumulative distribution curve of the number of Barcode reads in the day14 control group and the day14 experimental group were slightly separated from each other.It revealed that the distribution of the equal number of each candidate gene cells was changed.Correlation analysis showed that correlation of the day7 control group between the day7 experimental group and the experimental day14 group and correlation of the day14 control group between the day7 experimental group and the day14 experimental group were less.A total of 22 candidate target genes were screened from the day14 experimental group to day0 group,and 33 candidate genes were screened in the day14 experimental group to the day14 control group.6.The results of quantitative PCR showed that expression of most of the candidate target genes in MDA-231-Gem were higher than which in MDA-231.Cytotoxicity assay showed that KCNN4,BCL2A1 and TNFRSF11 B were screened by our screening system.The IC50 of cells which express these three genes were 60.27 n M,30.20 n M and 15.95 n M,respectively.Conclusions: 1.The plasmid library which overexpress gemcitabine resistance-related genes was constructed,and the breast cancer cell line MDA-231-Lib which overexpress gemcitabine resistance-related genes was obtained.2.Analysis of potential gemcitabine-resistance target genes using next-generation sequencing showed that the target genes,KCNN4,BCL2A1 and TNFRSF11 B were obtained.3.We demonstrated that the p Dest-Barcode library have ability to high-throughput screening of functional genes. |
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