Gene Expression Microarray Data Based Biomarker Screening For Breast Cancer With Malignant Phenotype

Background:Breast cancer is the second leading cause of death in women worldwide,with one in eight women developing this disease,and the most diagnosed cancer in females.The most important barrier for this scientific interest is that we lack a complete picture of it,which encompasses a large number of entities showing different morphological features and having multiple clinical behaviors.During the last 15 years,with the application of gene expression profiling,breast cancer has been classified into five categories:luminal A,luminal B,human epidermal growth factor receptor 2(HER2 or ERBB2)-enriched,basal-like(BL),and claudinlow.Among them,basal-like breast cancer has long been a research hotspot since it accounts for about 85%of triple negative breast cancer which is a biologically aggressive neoplasia with a strong association with many malignant phenotypes,including early onset,higher histological grade,increased distant recurrence and visceral metastases,insensitive to endocrine therapy and target therapy and poor prognosis.Because of the inconsistent diagnostic criteria for basal-like breast cancer and the controversial research results,although currently used prognostic and treatment predictive markers for breast cancer have markedly improved tratment decisions,there is still urgent for the discovery of more specific molecular markers.The rapid development of high throughput gene expression microarray analysis has accelerated the research of the disease mechanism and made a breakthrough in the field of tumor related research.With the wide application of gene expression microarray technology,a large amount of data is uploaded to the public database including the original or primary and the secondary database for global sharing.Because of their multicenter,large patient cohorts,comprehensive clinico-pathological features and extensive follow-up prognosis sources,these data can be used for many independent analyses and are particularly important in the light of the vast literature on potential biomarkers that are often tested on very limited patient cohorts and are not validated on independent data sets.To this end,indepth excavation and make full use of the existing microarray database will be much beneficial for the field of cancer research.Objective:(1)To screen out and validate the breast cancer malignant phenotype related immunosuppressive factors between luminal-and basal-like breast cancer cell lines and tissue samples from online shared cDNA expression microarray datasets;(2)To screen out and validate the breast cancer malignant phenotype related miRNA;(3)To study effective function mechanism of "miRNA pair" and look for the common targets.Methods:(1)The mRNA expression microarrays raw data,GSE40057,GSE1561,GSE40056 were downloaded,remodeled and differentially expressed genes(DEGs)between lunminal-and basal-like breast cancer cell lines and tissue samples were identified in R using Bioconductor and associated packages.Enrichment analyses were performed for different expression genes using the DAVID functional annotation clustering tool(2)qRT-PCR,western blot and immunohistochemistry techniques were used to validate the expression of immunosuppressive factors,miRNAs and their targets;(3)The GeneChip? Human Gene 1.0 ST Array was used for the whole-transcript profiling analysis in the miRNA mimic transfected 4 aggressive breast cancer cells.(4)U6,miR-200c and miR-205 in-situ hybridization on breast disease related tissue array.Results:(1)2189 and 1963 genes have statistically different expression pattern between breast cancer cell lines and tissue samples of different malignancy,respectively.Gene ontology GO term and KEGG pathway analysis based on DEGs reveal different molecular mechanism for breast cancer cell lines and tissue samples and most immunity related genes are upregulated in basal-like cell lines and tissue samples.(2)GOBO analysis showed that IL8 expression is comparatively consistent between luminal-and basal-like breast cancer cell lines with a higher average expression in basal-like and TN cell lines.(3)qRT-PCR and western blot showed that expression of CD274 and IL8 were all upregulated in BT549 and MDA-MB-231,compared to MCF7 and T47D cell lines.(4)Kaplan Meier Plotter showed that lower CXCL12,CXCL5,IDO2 and PTGS2 mRNA expression had comparatively lower survival rate(p<0.01),while lower MIF and VEGFA had higher survival rate(p<0.01).An analysis on 18 mRNA profiling microarray datasets her confirmed both lower MIF and VEGFA correspondent with higher 50/60-month survival rate.(5)Immunohistochemical detection on tissue microarray showed that the MIF expression increased dramatically in the metastasis group(p<0.05),and the VEGFA expression positively correlated with the pathologic grade of the tumor(p<0.05).2.(1)28 miRNAs have statistically different expression in miRNA expression microarray,include miR-200c and miR205;(2)qRT-PCR results showed a consistent expression for miR200c and miR-205 was found in most breast(lower expression in aggressive breast cancer)and non-breast cancer cell lines.And most interestingly,miR-200c and miR-205 have a similar expression pattern through most tissue samples detected by tissue microarray;(3)The microarray data showed that a large number of genes were differentially modulated compared with mimic control group.1174 and 2216 coDGs have statistically different expression pattern between miR200c and miR-205 mimic transfected cell lines,respectively.GO-MF analysis showed almost same molecular function for coDGs of miR-200c and miR-205,but an intersection of two modules was very small;(4)92 and 55 targets gene have been screened to miR-200c and miR205,the number of intersection targets is 13,include QKI;(5)Immunohistochemical detection on tissue microarray showed that the QKI expression positively correlated with the pathologic grade of the tumor(p=0.014)and increased dramatically in the TNBC(P<0.001).Conclusion:1.During the formation and development of breast cancer malignancy,immunity related genes were activated and immunosuppressive factors,CD274,IL8,MIF and VEGFA were upregulated,which could be used as biomarkers for breast cancer prognosis prediction.However,based upon the dual potential role of the majority of immunity related factors,the substantial relationship between immunosuppressive factors and the malignant phenotype of breast cancer and the feasibility of their application as drug targets in clinical practice remains to be further elucidated.2.A consistent expression for miR-200c and miR-205 was found in most breast and non-breast cancer cell lines,and may be function as tumor suppressors in triple negative breast cancer.miR-200c and miR-205 co-downregulated genes have little intersection but showed almost same molecular function and a larger coverage of the same KEGG pathway.RNA binding protein QKI had been identified as the co-targets of miR-200c and miR-205,and detected increased dramatically in the TNBC,but it’s action mechanism remains to be further study.