Characterization of the MG828507 lncRNA Located Upstream of the FLT1 Gene as an Etiology for Pre-Eclampsia

Abstract

Background: FLT1 is one of the significantly overexpressed genes found in a pre-eclamptic placenta and is involved with the etiology of this disease.

Methods: We conducted genome-wide expression profiling by RNA-seq of placentas from women with pre-eclampsia and those with normotensive pregnancy.

Results: We identified a lncRNA gene, MG828507, located ~80 kb upstream of the FLT1 gene in a head-to-head orientation, which was overexpressed in the pre-eclamptic placenta. MG828507 and FLT1 are located within the same topologically associated domain in the genome. The MG828507 mRNA level correlated with that of the FLT1 in placentas from pre-eclamptic women as well as in samples from uncomplicated pregnancies. However, neither the overexpression nor knockdown of MG828507 affected the expression of FLT1. Analysis of pre-eclampsia-linking genetic variants at this locus suggested that the placental genotype of one variant was associated with the expression of MG828507. The MG828507 transcript level was not found to be associated with maternal blood pressure, but showed a relationship with birth and placental weights, suggesting that this lncRNA might be one of the pivotal placental factors in pre-eclampsia.

Conclusion: Further characterization of the MG828507 gene may elucidate the etiological roles of the MG828507 and FLT1 genes in pre-eclampsia in a genomic context.

Keywords: FLT1; lncRNA; placenta; pre-eclampsia.

Figure 1

Genomic structure of the FLT1 upstream region. (A) Location of the MG828507 and FLT1 genes. The horizontal arrows indicate their transcription direction. The extent of the TAD that incorporates these genes is indicated by the large box. The vertical arrows indicate the location of the three variants tested in this study. The STR, s149427560, is underlined. (B) RNA-seq of MG828507. The horizontal bar indicates the MG828507 gene registered in the public database with its exon-intron structure. The upper panel indicates the position of the cDNA for overexpression experiments as well as the position targeted by the siRNA oligonucleotide used in this study.

Figure 2

MG828507 and FLT1 expression in pre-eclamptic and normotensive placentas. (A) Differential expression of MG828507 and FLT1 in uncomplicated pregnancies (left) and in the pre-eclampsia samples (right). The boxes indicate the 25th and 75th percentiles, whilst the bands near the middle indicate the median values. The bars indicate the 1.5 interquartile ranges, with the outliers specifically marked. (B) Analysis of MG828507 and FLT1 correlations. Open circles indicate the uncomplicated pregnancy controls, and open triangles indicate pre-eclampsia. Regression lines are shown with correlation coefficients and p values.

Figure 3

Analysis of the overexpression and knockdown of MG828507. (A) Expression of MG828507 and FLT1 after the exogenous overexpression of MG828507. HTR-8 trophoblast cells were transfected with an MG828507 expression vector. The MG828507 and FLT1 transcript levels were analyzed by qRT-PCR. The vertical axis indicates the ratio relative to the data of cells transfected with control vector. (B) Expression of MG828507 and FLT1 after the knockdown of MG828507. HTR-8 trophoblast cells were transfected with an siRNA targeting MG828507. The MG828507 and FLT1 transcript levels were again analyzed by qRT-PCR. The siRNA for FLT1 as well as a scrambled siRNA control were also used independently as control transfection experiments. The vertical axis indicates the ratio relative to the data of cells transfected with control oligonucleotides.

Figure 4

Correlations between MG828507 expression and the indicated genetic variants. (A) Comparison of the placental mRNA levels by variant genotype. Data for rs4769613 (left), rs12050029 (center), and rs149427560 (right) are shown. In each panel, data for the normotensive placental controls are shown on the left, and those from pre-eclamptic cases are shown on the right. (B) Comparison of the placental mRNA levels by variant allele type. The boxes indicate the 25th and 75th percentiles. The bands near the middle indicate the median values. The bars indicate the 1.5 interquartile ranges, with the outliers specifically marked. Sample numbers and p values are shown in each panel.

Figure 5

Correlations between MG828507 expression and systolic blood pressure (A), diastolic blood pressure (B), normalized birth weight (C), or placental weight (D). Open circles indicate control uncomplicated pregnancies, and open triangles indicate pre-eclampsia. Regression lines are shown with correlation coefficients and p values.