Identification Of Key Genes Responsible For Epirubicin And Paclitaxel Resistance In Breast Cancer Cells

Background: Breast cancer is the most common malignant female cancer worldwide,it is also one of the leading cause of cancer mortality.Chemotherapy is an important means of treatment of breast cancer,Epirubicin and Paclitaxel are two of the most commonly used chemotherapy drugs with genetic toxicity,however,various reasons causing tumor cells drug resistance therefore led to chemotherapy failure.From another point of view,a large number of patients got excessive treatment,take the risk of toxicity of adjuvant chemotherapy whereas has no significant treatment effect.So to explore the specific mechanism of chemo resistance and to predict the patient's outcome is particularly important.Epirubicin is anthracycline antibiotics,can insert to the double-stranded DNA and topoisomerase? to inhibit the synthesis of DNA and RNA,trigger DNA degradation,activating pathways leading to cell death.So far,the mechanisms of Epirubicin resistance include multi-drug resistant protein overexpression,topoisomerase mutation,P53 and related proteins mutation,Bcl-2 overexpression,C-myc and receptor tyrosine kinase(AXL)overexpression And DNA damage associated proteins overexpression like FOXM1.Paclitaxel is extracted from the Pacific yew tree,has antitumor activity,it can combine with tubulin,interfering tubulin dimer and soluble polymer dynamic balance,and inhibit microtubule disassembly,thereby inhibit cell division.The reports of Paclitaxel resistance mechanism mainly include,tubulin mutant or expression reduced,composition of different subtypes,Multi-drug resistant protein overexpression,chromosome instability,apoptosis related proteins overexpression such as BIM,Spleen tyrosine kinase overexpression leading to phosphorylation of microtubules and microtubule associated proteins.The expression change of various proteins after chemo resistance is the result of a variety reactions,genes involved in these reactions have complicated interactions.Finding the most important gene therefore is the key to solve the problem of drug resistance.This project want to find out the key genes responsible to epirubicin and paclitaxel resistance.Methods:(1)RNA-seq to reveal the differential expression genes of normal breast cancer cells and drug resistant cells.Among them,select the genes that is significantly different in two cell lines.(2)Plasmid overexpression and shRNA knockdown of the candidate genes to change gene's expression.(3)MTT experiment to get cell survival rate under drug treatment,compare each gene's resistant ability,then select the key genes in which cause drug resistance.Results:(1)Screening the genes we got from RNA-seq result,the differential candidate genes between Epirubicin resistant cell and normal MCF-7 cell is 187,up regulated 124,down regulated 63.The differential candidate genes between Paclitaxel resistant cell and normal MCF-7 cell is 163,up regulated 103,down regulated 60.Overlapping genes is 142.(2)Using the plasmids in which contain shRNA to construct20 single gene knock down stable cell lines,each gene choose 2 different shRNAs.Using the plasmids in which contain the cDNA of candidate genes to construct 35 overexpression stable cell lines.(3)By the means of cell survival experiment MTT to got the drug effect curve,each line's area under curve are compared with those of the normal and resistant cells,after change expression of LPXN,DUSP4,RB1,HIST1H3 F and DUSP6,the resistant ability change significantly,especially LPXN.Conclusion: LPXN,DUSP4,RB1,HIST1H3 F and DUSP6 may play key roles in the process of drug resistant.