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

<div><p>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.</p> </div

RT-PCR analysis of selected genes induced by sLF.

<p>Real-time RT-PCR analysis of ten selected genes displaying altered expression in response to sLF in HEK293 cells. Gray and black bars denote the expression of the indicated genes for non-treated and treated with tetracycline in the sLF cells, respectively. Each sample was analyzed in duplicate, and the average relative expression level of 15 clones is presented. *Significant (P<0.05; Student's <i>t</i>-test).</p

Genome-wide expression analysis in LF-overexpressing HEK293 cells.

<p>Expression histogram of control (X-axis) vs. sLF (Y-axis) (A) and control vs. ΔLF (B). Expression levels of 18,367 genes were measured by Phalanx Human 32K microarray and are presented on a log scale. Best fit and two-fold difference lines were added. Altered expression was observed more often in ΔLF cells than in sLF cells, indicating that ΔLF generally regulated more genes.</p

Highest confidence network of genes regulated by sLF.

<p>Highest confidence network of genes displaying altered expression levels in response to secreted sLF in HEK293 cells. According to IPA, the network is relevant to ‘Genetic Disorder, Hematological Disease, Metabolic Disease’. Genes that were upregulated are presented in red, whereas those that were downregulated are presented in green, where the intensity of the color reflects the magnitude of the expression change, as indicated in the scale bar. Each interaction is supported by at least one literature reference, with solid lines representing direct interactions and dashed lines representing indirect interactions.</p

RT-PCR analysis of selected genes induced by ΔLF.

<p>Gray and black bars denote the expression of the indicated genes for non-treated and treated with tetracycline in the ΔLF cells, respectively. Each sample was analyzed in duplicate, and the average relative expression level of 15 clones is presented. *Significant (P<0.05; Student's <i>t</i>-test).</p

Pre-mRNA levels in ΔLF-overexpressing cells.

<p>Real-time RT-PCR was carried out to measure the pre-mRNA (A) and mature-mRNA levels (B) of the three selected genes. Primers spanning an exon and its downstream intron were used for pre-mRNA and primers spanning two exons were used for mature-mRNA. Gray and black bars denote the expression of the indicated genes for non-treated and treated with tetracycline in the ΔLF cells, respectively. Each clone was analyzed in duplicate, and the average relative expression level of 15 clones is presented with the standard error. *Significant (P<0.05; Student's <i>t</i>-test).</p

Overexpression of human LF in HEK293 cells using Flp-In T-Rex system.

<p>(A) End-point RT-PCR analysis of LF. RT-PCR was conducted for RNAs from HEK293 cells transfected with T-Rex LF expression vectors with signal sequence (sLF) or without signal sequence and 26 N-terminal amino acid residues (ΔLF), non-treated with (Tet(−), top panel) or treated with tetracycline (Tet(+), bottom panel) for 24 or 36 h. C, vector alone. M, size marker. (B) Real-time RT-PCR analysis of LF. Each reaction per clone was carried out in duplicate, and the average of 15 clones is presented along with the standard error. (C) Immunoblot analysis of LF. LF protein expressed from recombinant vector was immunobloted using anti-LF antibody from cell lysate and culture media. LF, commercially available sLF (80 kDa). C, vector alone. LFs expressed from vectors with and without the signal sequence and 26 N-terminal amino acid residues are marked by arrows. Signals below the LF bands appearing in all samples in the medium are non-specifically-reacted serum proteins. The result of ELISA for the LF in medium is shown at the bottom.</p

The regression analysis using with generalized equation estimating of outcome variable: Cesarean delivery, medical cost, length of stay.

<p>The regression analysis using with generalized equation estimating of outcome variable: Cesarean delivery, medical cost, length of stay.</p

General characteristics of participants and hospital.

<p>General characteristics of participants and hospital.</p

Subgroup analysis of outcome variable by age group.

<p>Subgroup analysis of outcome variable by age group.</p