Screening Drug-resistant Breast Cancer Cell Line Using Genome-wide Small Regulatory Library And Mechanical Exploring

Object: Breast cancer is the most malignant tumors,which is of the highest mortality and morbidity in women.Breast cancer drug resistance remains a thorny problem faced in the clinical treatment.HER2 is high expressed in 20 to 30% of breast cancer patient;what is more,the expression of HER2 is in strong correlation with poor prognosis in HER2 breast cancer patient.Laptinib is currently one of the main drugs that treat for Her2 positive breast cancer patients,but the clinical drug resistance still be a serious problem.Emerging evidence shows that shows that: short regulatory RNA(srRNA)plays an important regulating role in mediating the drug-resistance of tumor cells.Considering of this,we proposed to build a randomized small regulatory(srRNA)library screening system which can quickly get Laptinib drug-resistant cell model.Taking best advantage of this drug resistance research model,we can explore the drug resistant molecular mechanism of HER2 positive breast cancer,with aims to find molecular targets for clinical intervention.Method: Using retrovirus system pSOKD plasmid as the carrier,via U6/H1 double promoters precise control system,19 random oligonucleotides were assembled in the middle of the two promoters by Gibson Assembly.And making best use of Gibson Assembly's efficiency,we built srRNA plasmid library,successfully.The plasmid library was packaged via retrovirus system,and then,the viruses were collected and used to infect SKBR3.Through drug screening,Laptinib resistant breast cancer cell research model is established.Based on this model,enriched N19 fragments can be got through the analysis of high-throughput DNA sequencing data.Through RNA-seq analysis,differential expressed genes and the regulation mechanism will also be explored.Results: Through Gibson Assembly efficient way of recombination,we successfully built a large scale library containing about 1.08 x106 srRNAs.Using this library,we quickly got Laptinib resistant breast cancer cell model(SKBR3LN19).Through the genomic DNA extraction and amplification of the model cells;we successfully got enriched N19 fragments' sequence information.Through rebuilding stable cell lines with individual N19 fragments of top15,we got 5 single fragments(LTR1,LTR3,LTR6,LTR8,LTR10),which can significantly regulating Laptinib resistance in SKBR3.Through RNA-seq analysis,we found 821 dysregulated genes(upregulated: 450 and downregulated: 371)and KEGG showed that these dysregulated genes participated in cell metabolism signaling pathways.Conclusion: Using SKBR3 cells(HER2+)as the research model,through infecting with srRNA library,drug resistance screening,high-throughput DNA sequencing,RNA seq,and bioinfomatic analysis of its target genes,we got 5 single fragments,which can significantly regulating Laptinib resistance in SKBR3 separately.We also found 821 dysregulated genes(upregulated: 450 and downregulated: 371)and KEGG showed that these dysregulated genes participated in cell metabolism signaling pathways.This study is promising for clarifying the Laptinib drug resistance mechanisms of HER2 positive breast cancer and searching for a new method of breast cancer treatment.