Unknown,Transcriptomics,Genomics,Proteomics

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Differential miRNA expression in gingival epithelial cells infected with Porphyromonas gingivalis

ABSTRACT: Porphyromonas gingivalis is a pathogen in severe periodontal disease. Able to exploit an intracellular lifestyle within primary gingival epithelial cells (GECs), a reservoir of P. gingivalis can persist within the gingival epithelia. This process is facilitated by manipulation of the host cell signal transduction cascades which can impact cell cycle, cell death and cytokine responses. Using microarrays, we investigated the ability of P. gingivalis 33277 to regulate microRNA (miRNA) expression in GECs. One of several miRNAs differentially regulated by GECs in the presence of P. gingivalis was miR-203, which was upregulated 4-fold compared with uninfected controls. Differential regulation of miR-203 was confirmed by qRT-PCR. Putative targets of miR-203, suppressors of cytokine signaling (SOCS) 3 and 6, were evaluated by qRT-PCR. SOCS3 and SOCS6 mRNA levels were reduced >5-fold and >2-fold, respectively, in P. gingivalis-infected GECs compared with controls. Silencing miR-203 using a si-RNA construct reversed the inhibition of SOCS3 expression. A dual luciferase assay confirmed binding of miR-203 to the putative target binding site of SOCS3 3’ UTR. Western blot analysis demonstrated that activation of Stat3, a downstream target of SOCS, was diminished following miR-203 silencing. This study shows that induction of miRNAs by P. gingivalis can modulate important host signaling responses. MicroRNAs differentially expressed in human gingival epithelial cells in response to P. gingivalis infection were identified. P. gingivalis were reacted with primary gingival epithelial cells at an MOI of 100 for 6 hours at 37C in 5% CO2. Co-cultures were carried out in triplicate. The GECs were lysed with Trizol (Invitrogen) prior to RNA extraction and total RNA isolation. The microarray assay was performed using a service provider (LC Sciences). The assay started from 4 to 8 µg total RNA sample which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore) and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining. Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies). The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 uL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C. After RNA hybridization, tag-conjugating Cy3 dyes were circulated through the microfluidic chip for dye staining. Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics). Microarray data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (Locally-weighted Regression). For a transcript to be listed as detectable it had a signal intensity higher than 3×(background standard deviation) and spot coefficient of variance (CV) < 0.5. CV was calculated by (standard deviation)/(signal intensity). When repeating probes are present on an array, a transcript is listed as detectable only if the signals from at least 50% of the repeating probes are above detection level. t-Test is performed between “control” and “test” sample groups. T-values are calculated for each miRNA, and p-values are computed from the theoretical t-distribution. miRNAs with p-values below a critical p-value (typically 0.01) are selected for cluster analysis. The clustering is done using hierarchical method and is performed with average linkage and Euclidean distance metric. This experiment includes a total of 6 samples which are divided in to 2 groups, each with 3 biological repeats: 3 untreated normal samples as controls and 3 infected samples as the treated condition.

ORGANISM(S): Homo sapiens

SUBMITTER: Richard Lamont

PROVIDER: E-GEOD-28635 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: There is a limited capacity to repair damage in the mammalian heart after birth, which is primarily due to the inability of cardiomyocytes to proliferate after birth. This is in contrast to zebrafish and salamander, in which cardiomyocytes retain the ability to proliferate throughout life and can regenerate their heart after significant damage. Recent studies in zebrafish and rodents implicate microRNAs (miRNAs) in the regulation of genes responsible for cardiac cell cycle progression and regeneration, in particular, miR-133a, the miR-15 family, miR-199a and miR-590. However, the significance of these miRNAs and miRNA in general in the regulation of cardiomyocyte proliferation in large mammals, including humans, where the timing of heart development relative to birth is very different than in rodents, is unclear. To determine the involvement of miRNAs in the down-regulation of cardiomyocyte proliferation occurring before birth in large mammals, we investigated miRNA and target gene expression in sheep hearts before and after birth. The experimental approach included targeted transcriptional profiling of miRNA and target mRNA previously identified in rodent studies as well as genome-wide miRNA profiling using microarrays. Results: The cardiac expression of miR-133a increased and its target gene IGF1R decreased with increasing age, reaching their respective maximum and minimum abundance when the majority of ovine cardiomyocytes were quiescent. The expression of the miR-15 family members was variable with age, however, four of their target genes decreased with age. These latter profiles are inconsistent with the direct involvement of this family of miRNA in cardiomyocyte quiescence in late gestation sheep. The expression patterns of ‘pro-proliferative’ miR-199a and miR-590 were also inconsistent with their involvement in cardiomyocyte quiescence. Consequently, miRNA microarray analysis was undertaken, which identified six discrete clusters of miRNA with characteristic developmental profiles. The functions of predicted target genes for the miRNA in four of the six clusters were enriched for aspects of cell division and regulation of cell proliferation suggesting a potential role of these miRNA in regulating cardiomyocyte proliferation. Conclusion: The results of this study show that the expression of miR-133a and one of its target genes is consistent with it being involved in the suppression of cardiomyocyte proliferation, which occurs across the last third of gestation in sheep. The expression patterns of the miR-15 family, miR-199a and miR-590 were inconsistent with direct involvement in the regulation cardiomyocyte proliferation in sheep, despite studies in rodents demonstrating that their manipulation can influence the degree of cardiomyocyte proliferation. miRNA microarray analysis suggests a coordinated and potentially more complex role of multiple miRNA in the regulation of cardiomyocyte quiescence and highlights significant differences between species that may reflect their substantial differences in the timing of this developmental process. RNA, (2 µg) from three heart samples from each of the 91 d (fetus), 141 d (fetus), 5 d (postnatal) and 173 d (postnatal) groups, was used for analysis according to standard procedures. The experimental design included three biological replicates from each of the four normal developmental ages in sheep and thus a total of 12 samples was analyzed.

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Differential miRNA expression in gingival epithelial cells infected with Porphyromonas gingivalis

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