Bioinformatics Analysis Of Potential Signaling Pathways On Aberrant Expression Of MicroRNAs In Breast Cancers With Different PR Status And Its Preliminary Experimental Verification

BackgroundBreast cancer is one of the most common cancers in adult females,Incidence of various malignant tumors accounted for the body of7%to10%,second only to cervicalcancer in women. For a long time, based on the traditional histological type, clinical and pathological staging, different breast cancer treatment options including surgical treatment, chemotherapy, radiotherapy, endocrine therapy or molecularly targeted treatment are selected. Understanding of the molecular taxonomy of breast caner stems form classic mRNA profiling studies, which broadly sub-classified breast cancer into5major groups:Luminal A, Luminal B, Basal-like, HER2-positive, and normal-like tumors. Except the different gene expression patterns, there are significant difference in biological behavior, growth rate and the activity of specific signaling pathways between breast cancer molecular subtypes.Through a complex network,miRNAs plays an important regulatory role in human different sub-type breast cancers.microRNA(miRNAs) are the class of approximately21-25nuleotide long non-coding RNAs,which exists in a wide range of eukaryotes.miRNAs gene transcription product of the primary(pri-miRNA)in the nucleus has been cut RNAse Drosha to become precursor miRNA(pre-miRNA),pre-miRNA in the transporter protein of the role of exportin-5by the nucleus to the cytoplasm,then by a further cut RNaseⅢDicer have mature miRNA.These mature miRNA components and other proteins with RISC(RNA-induced silencing complex),which regulate target genes post-transcriptionally by inhibiting the translation through imperfect base-pairing interaction with the3’-untranslated regions(3’-UTR)of their respective target genes,or degrading their target mRNAs through perfect or near-perfect base pairing.Large number of studies show that, miRNAs have important roles in many biological processes,such as proliferation, differentiation,cell death,development,metabolize and disease.And increasing evidences show that mutation and differential expression of some specific miRNAs are associated with the formation and progress of many types of cancers,including breast cancer,lung cancer,hepatocellular carcinoma,colorectal neoplasia,gastric cancer,chronic lymphocytic leukemia,etc.Objective1. To learn the current research situations of breast cancer endocrine sensitivity related candidate genes and the hub genes from interactive gene network to guide the scientific research.2. To perform bioinformatic analysis on aberrant expression of microRNAs in PR(+)/PR(-) breast cancers, so as to explore the possible regulatory signaling pathways of different progesterone receptor status.3. The purpose of this study was to verify whether CARM1was targeted by miRNA through experimental approaches.MethodsPart1We documented all literatures marked out inclusion and exclusion reasons on every literature and got the needed information with the help of Endnote X3software. We exported the information in Endnote X3using reference list transformator. Finally, the included papers were analyzed on publication year, country, journal, research institute, authors and genes. Part2Target genes of related microRNAs retrieved through literature mining were predicted by the biosoftwares of TargetScan, then, an analysis on gene sets was carried out by GO overrepresentation and pathway analysis using DAVID database.Part3A luciferase gene expression vector containing3’UTR translated region(3’UTR) of CARM1gene was constructed. The resulting luciferase expression plasmids were named psi-CHECK-2-CARM1-9and psi-CHECK-2-CARM1-103. The firefly luciferase expression plasmid psi-CHECK-2-control and the renilla luciferase construct psi-CHEK-2-CARM1-3’UTR with miRNA mimics were cotransfected into HELA cells. Then, the firefly and renilla luciferase activity were detectedby using the dual glo luciferase assay system.Results:1. A total of168papers about this subject were obtained, involving267genes. This study is gaining more and more attention. USA was the most active country. This subject articles mainly concentrates in the journey of Breast Cancer Research and Treatmen and Clinical Cancer Research. Erasmus MC, Josephine Nefkens Institute from Netherlands offered the most literatures. Bioinformatics analysis demonstrated that the interactive network of related genes can be constructed to predict candidate genes.2. Three datasets of aberrant expressed microRNAs were obtained. The gene ontology category indicated that these targetcandidates mainly participate in intracellular signaling cascade, protein amino acid phosphorylation, protein methyltransferase activity, transcription factor activity and so on. Pathway analysis showed that these target genes were mainly involved in3KEGG signaling pathways and3BIOCARTA pathways, correlating with different PR status.3. The recombinant vector psi-CHECK-2-CARM1-3’UTR was verified by sequencing. Luciferase activity was significantly reduced to42.54%in HELA cells transfected with psi-CHECK-2-CARM1-3’UTR and psi-CHECK-2control in the presence of miRNA mimics.Conclusion:1. Trough the literature metrology analysis, we clearly understand the current research situation on this subject which lays foundation for further studies on candidate predictors of breast cancer endocrine therapy sensitivity.2. This study analyze the bioinformatics of cellular functions and pathways in different PR status affected by differently expressed microRNAs, which lays foundation for further studies on biological interpretation of microRNAs profiling data in human different sub-type breast cancers.3. The microRNA plasmid psi-CHECK-2-CARM1-3’UTR(miR-9and miR-103) was constructed successfully. CARM1is indeed a target of miR-103.