Genome-Wide Pathway Analysis Reveals Different Signaling Pathways between Secreted Lactoferrin and Intracellular Delta-Lactoferrin

Abstract

Human lactoferrin (LF) is a multifunctional protein involved in immunomodulation, cellular growth, and differentiation. In addition to its secreted form (sLF), an alternative form (ΔLF) lacking the signal sequence has been found to be downregulated in cancer. Although the signaling pathways mediated by LF have been studied in a few cell models, there have been no relevant systemic approaches. Therefore, this study was carried out to identify and compare signaling networks provoked by the two LF isoforms. For this, the two forms were overexpressed in HEK293 cells using the Flp-In T-Rex system, after which genome-wide expression analysis of 18,367 genes was conducted. Pathway analysis of the genes showing altered expression identified pathways which are responsible for cell survival and apoptosis. In addition, the pathways mediated by the two LF forms were within distantly related networks. GPCR, PI3K complex, and POU5F1, which are involved in receptor-mediated pathways, were centered in the sLF network, whereas RIF1, NOS3, and RNPS1, which are involved in intracellular signaling, were centered in the ΔLF network. These results suggest that structural differences between the LF isoforms, mainly glycosylation, determine the fate of LF signaling. Furthermore, these findings provide information relating to the role of ΔLF which is downregulated during carcinogenesis.

Transcriptome-Wide Identification of RNA Targets of Arabidopsis SERINE/ARGININE-RICH45 Uncovers the Unexpected Roles of This RNA Binding Protein in RNA Processing

“…Briefly, the fastq data were first evaluated for their quality using FastQC_0.10.1(multi-file). The high quality fastq data were then mapped to the TAIR10 genome using TopHat2-SE and the following parameter settings: reference genome, Arabidopsis thaliana [Mouse-ear cress] (Ensembl 14); reference annotations, Arabidopsis thaliana (Mouse-ear cress) (Ensembl 14); minimum length of read segments, 20; minimum isoform fraction, 0.15; Bowtie 2 speed and sensitivity, sensitive; maximum intron length that may be found during split-segment search, 10,000; minimum intron length, 50; Bowtie version, 2.1.0; anchor length, 8; number of mismatches allowed in each segment alignment for reads mapped independently, 2; maximum intron length, 5000; maximum number of mismatches that can appear in the anchor region of spliced alignment, 0; maximum number of alignments to be allowed, 20; minimum intron length that may be found during split-segment search, 50; TopHat version, 2.0.9 (Kim et al, 2013). The bam files generated from TopHat analysis were further processed to remove the duplicate reads and reads aligned to multiple genomic positions using R scripts with Bioconductor packages as follows: the R code: > library(GenomicAlignments); > library(GenomicRanges); > library(Rsamtools); > library(rtracklayer); > flag0 <-scanBamFlag(isDuplicate=FALSE, isNotPassingQualityControls=FALSE); > param0 <-ScanBamParam(flag=flag0, what="seq"); > ; > x <-readGAlignments("input.bam", use.names=TRUE, param=param0); > dup <-duplicated(mcols(x)$seq); > table(dup); > y <-x[!dup]; > export(y, BamFile("output.bam")).…”

Variability of lysozyme and lactoferrin bioactive protein concentrations in equine milk in relation to LYZ and LTF gene polymorphisms and expression

“…Owing to its antiproliferative activity, delta-lactoferrin is probably an important antitumoral agent. 11 The concentration of LTF in mare's milk is around 1 g L −1 , but some differences between independent investigations are noticed (within the range 0.6-1.3 g L −1 ). Generally, the LTF content in equine milk is placed between an average concentration of lactoferrin observed for human breast milk and for cow's milk.…”

“…in cell cycle regulation. Owing to its antiproliferative activity, delta‐lactoferrin is probably an important antitumoral agent . The concentration of LTF in mare's milk is around 1 g L −1 , but some differences between independent investigations are noticed (within the range 0.6–1.3 g L −1 ).…”

SILAC-Based Proteomic Profiling of the Human MDA-MB-231 Metastatic Breast Cancer Cell Line in Response to the Two Antitumoral Lactoferrin Isoforms: The Secreted Lactoferrin and the Intracellular Delta-Lactoferrin

“…Recently, a genome-wide pathway analysis which compared the different signaling pathways triggered by Lf and ΔLf in HEK 293 cells treated with Lf or expressing ΔLf has generated a considerable body of information on the molecular features of the re-introduction of Lf isoforms in cancerous cells [48]. Data showed significant up and down-regulation respectively of 74 and 125 genes in Lf-treated cells and 327 and 256 genes in ΔLf-expressing cells.…”