The Analysis of Platelet-Derived circRNA Repertoire as Potential Diagnostic Biomarker for Non-Small Cell Lung Cancer.

Tumor-educated Platelets (TEPs) have emerged as rich biosources of cancer-related RNA profiles in liquid biopsies applicable for cancer detection. Although human blood platelets have been found to be enriched in circular RNA (circRNA), no studies have investigated the potential of circRNA as platelet-derived biomarkers for cancer. In this proof-of-concept study, we examine whether the circRNA signature of blood platelets can be used as a liquid biopsy biomarker for the detection of non-small cell lung cancer (NSCLC). We analyzed the total RNA, extracted from the platelet samples collected from NSCLC patients and asymptomatic individuals, using RNA sequencing (RNA-Seq). Identification and quantification of known and novel circRNAs were performed using the accurate CircRNA finder suite (ACFS), followed by the differential transcript expression analysis using a modified version of our thromboSeq software. Out of 4732 detected circRNAs, we identified 411 circRNAs that are significantly (p-value < 0.05) differentially expressed between asymptomatic individuals and NSCLC patients. Using the false discovery rate (FDR) of 0.05 as cutoff, we selected the nuclear receptor-interacting protein 1 (NRIP1) circRNA (circNRIP1) as a potential biomarker candidate for further validation by reverse transcription-quantitative PCR (RT-qPCR). This analysis was performed on an independent cohort of platelet samples. The RT-qPCR results confirmed the RNA-Seq data analysis, with significant downregulation of circNRIP1 in platelets derived from NSCLC patients. Our findings suggest that circRNAs found in blood platelets may hold diagnostic biomarkers potential for the detection of NSCLC using liquid biopsies.

Circular RNA Sequencing of Maternal Platelets: A Novel Tool for the Identification of Pregnancy-Specific Biomarkers.

BACKGROUND: In the first trimester of pregnancy, the maternal platelet is directly involved in a positive feedback mechanism that facilitates invasion of the extravillous trophoblast into the maternal spiral arteries. Dysfunctional trophoblast invasion with defective deep placentation is primordial in the etiology of the "great obstetrical syndromes." METHODS: In this proof-of-concept study, using transcriptome analysis of circular RNA (circRNA) following RNA sequencing of maternal platelets, we tested whether pregnancy-specific circRNA markers could be identified in the first trimester of normal pregnancies. Differential transcript expression analysis of circRNAs, as predicted by Accurate CircRNA Finder Suite, CircRNA Identifier (version 2), and Known and Novel Isoform Explorer, was done using thromboSeq.R with variation of multiple settings. Test performance was checked for (a) de novo circRNA identification using the novel platelet-specific Plt-circR4 as a positive control, (b) complete segregation of groups (pregnant vs nonpregnant) after heat map-dendrogram clustering, (c) identification of pregnancy-specific circRNA markers at a false discovery rate (FDR) <0.05, and (d) confirmation of differentially expressed circRNA markers with an FDR <0.05 by an independent method, reverse transcription-quantitative PCR. RESULTS: Of the differentially expressed circRNAs with P values <0.05, 41 circRNAs were upregulated (logFC >2), and 52 circRNAs were downregulated (logFC less than -2) in first-trimester platelet RNA. Of these, nuclear receptor-interacting protein 1 circRNA covering exons 2 and 3 of the 5'-untranslated region was pregnancy specific with upregulation in first-trimester maternal platelets compared to nonpregnant controls. CONCLUSION: CircRNA sequencing of first-trimester maternal platelets permits the identification of novel pregnancy-specific RNA biomarkers. Future use could include the assessment of maternal and fetal well-being.

The bivariate NRIP1/ZEB2 RNA marker permits non-invasive presymptomatic screening of pre-eclampsia.

Using genome-wide transcriptome analysis by RNA sequencing of first trimester plasma RNA, we tested whether the identification of pregnancies at risk of developing pre-eclampsia with or without preterm birth or growth restriction is possible between weeks 9-14, prior to the appearance of clinical symptoms. We implemented a metaheuristic approach in the self-learning SVM algorithm for differential gene expression analysis of normal pregnancies (n = 108), affected pregnancies (n = 34) and non-pregnant controls (n = 19). Presymptomatic candidate markers for affected pregnancies were validated by RT-qPCR in first trimester samples (n = 34) from an independent cohort. PRKG1 was significantly downregulated in a subset of pregnancies with birth weights below the 10thpercentile as shared symptom. The NRIP1/ZEB2 ratio was found to be upregulated in pregnancies with pre-eclampsia or trisomy 21. Complementary quantitative analysis of both the linear and circular forms of NRIP1 permitted discrimination between pre-eclampsia and trisomy 21. Pre-eclamptic pregnancies showed an increase in linear NRIP1 compared to circular NRIP1, while trisomy 21 pregnancies did not.

Noncoding RNA-regulated gain-of-function of STOX2 in Finnish pre-eclamptic families.

The familial forms of early onset pre-eclampsia and related syndromes (HELLP) present with hypertension and proteinuria in the mother and growth restriction of the fetus. Genetically, these clinically similar entities are caused by different founder-dependent, placentally-expressed paralogous genes. All susceptibility genes (STOX1, lincHELLP, INO80B) identified so far are master control genes that regulate an essential trophoblast differentiation pathway, but act at different entry points. Many genes remain to be identified. Here we demonstrate that a long non-coding RNA (lncRNA) within intron 3 of the STOX2 gene on 4q35.1 acts as a permissive cis-acting regulator of alternative splicing of STOX2. When this lncRNA is mutated or absent, an alternative exon (3B) of STOX2 is included. This introduces a stop codon resulting in the deletion of a highly conserved domain of 64 amino acids in the C-terminal of the STOX2 protein. A mutation present within a regulatory region within intron 1 of STOX2 has the same effect after blocking with CRISPR technology: transcripts with exon 3B are upregulated. This proces appears related to transcriptional control by a chromatin-splicing adaptor complex as described for FGFR2. For STOX2, CHD5, coding for a chromodomain helicase DNA binding protein, qualifies as the chromatin modifier in this process.

Genome-Wide Identification of Epigenetic Hotspots Potentially Related to Cardiovascular Risk in Adult Women after a Complicated Pregnancy.

BACKGROUND: The physiological demands of pregnancy on the maternal cardiovascular system can catapult women into a metabolic syndrome that predisposes to atherosclerosis in later life. We sought to identify the nature of the epigenomic changes associated with the increased cardiovascular disease (CVD) risk in adult women following pre-eclampsia. FINDINGS: We assessed the genome wide epigenetic profile by methyl-C sequencing of monozygotic parous twin sister pairs discordant for a severe variant of pre-eclampsia. In the adult twin sisters at risk for CVD as a consequence of a complicated pregnancy, a set of 12 differentially methylated regions with at least 50% difference in methylation percentage and the same directional change was found to be shared between the affected twin sisters and significantly different compared to their unaffected monozygous sisters. CONCLUSION: The current epigenetic marker set will permit targeted analysis of differentially methylated regions potentially related to CVD risk in large cohorts of adult women following complicated pregnancies.

Mutations within the LINC-HELLP non-coding RNA differentially bind ribosomal and RNA splicing complexes and negatively affect trophoblast differentiation.

LINC-HELLP, showing chromosomal linkage with the pregnancy-specific HELLP syndrome in Dutch families, reduces differentiation from a proliferative to an invasive phenotype of first-trimester extravillous trophoblasts. Here we show that mutations in LINC-HELLP identified in HELLP families negatively affect this trophoblast differentiation either by inducing proliferation rate or by causing cell cycle exit as shown by a decrease in both proliferation and invasion. As LincRNAs predominantly function through interactions with proteins, we identified the directly interacting proteins using chromatin isolation by RNA purification followed by protein mass spectrometry. We found 22 proteins predominantly clustering in two functional networks, i.e. RNA splicing and the ribosome. YBX1, PCBP1, PCBP2, RPS6 and RPL7 were validated, and binding to these proteins was influenced by the HELLP mutations carried. Finally, we show that the LINC-HELLP transcript levels are significantly upregulated in plasma of women in their first trimester of pregnancy compared with non-pregnant women, whereas this upregulation seems absent in a pilot set of patients later developing pregnancy complications, indicative of its functional significance in vivo.

Susceptibility allele-specific loss of miR-1324-mediated silencing of the INO80B chromatin-assembly complex gene in pre-eclampsia.

In humans, the elucidation of the genetics underlying multifactorial diseases such as pre-eclampsia remains complex. Given the current day availability of genome-wide linkage- and expression data pools, we applied pathway-guided genome-wide meta-analysis guided by the premise that the functional network underlying these multifactorial syndromes is under selective genetic pressure. This approach drastically reduced the genomic region of interest, i.e. 2p13 linked with pre-eclampsia in Icelandic families, from 8 679 641 bp (region with linkage) to 45 264 bp (coding exons of prioritized genes) (0.83%). Mutation screening of the candidate genes (n = 13) rapidly reduced the minimal critical region and showed the INO80B gene, encoding a novel winged helix domain (pfam14465) and part of the chromatin-remodeling complex, to be linked to pre-eclampsia. The functional defect in placental cells involved a susceptibility allele-dependent loss-of-gene silencing due to increased INO80B RNA stability as a consequence of differential binding of miR-1324 to the susceptibility allele of rs34174194. This risk allele is located at position 1 in an absolutely conserved 7-mer (UUGUCUG) in the 3-UTR of INO80B immediately downstream of a variant Pumillio Recognition Element (UGUANAAG). These data support that pre-eclampsia genes affect a conserved fundamental mechanism that evolved as a consequence of hemochorial placentation. Functionally, this involves founder-dependent, placentally expressed paralogous genes that regulate an essential trophoblast differentiation pathway but act at different entry points.

HELLP babies link a novel lincRNA to the trophoblast cell cycle.

The HELLP syndrome is a pregnancy-associated disease inducing hemolysis, elevated liver enzymes, and low platelets in the mother. Although the HELLP symptoms occur in the third trimester in the mother, the origin of the disease can be found in the first trimester fetal placenta. A locus for the HELLP syndrome is present on chromosome 12q23 near PAH. Here, by multipoint nonparametric linkage, pedigree structure allele sharing, and haplotype association analysis of affected sisters and cousins, we demonstrate that the HELLP locus is in an intergenic region on 12q23.2 between PMCH and IGF1. We identified a novel long intergenic noncoding RNA (lincRNA) transcript of 205,012 bases with (peri)nuclear expression in the extravillous trophoblast using strand-specific RT-PCR complemented with RACE and FISH. siRNA-mediated knockdown followed by RNA-sequencing, revealed that the HELLP lincRNA activated a large set of genes that are involved in the cell cycle. Furthermore, blocking potential mutation sites identified in HELLP families decreased the invasion capacity of extravillous trophoblasts. This is the first large noncoding gene to be linked to a Mendelian disorder with autosomal-recessive inheritance.

Naturally occurring variation in trophoblast invasion as a source of novel (epigenetic) biomarkers.

During the first trimester of pregnancy fetal trophoblasts invade the maternal decidua, thereby remodeling the maternal spiral arteries. This process of trophoblast invasion is very similar to cancer cell invasion, with multiple signaling pathways shared between the two. Pregnancy-related diseases, e.g., pre-eclampsia, and cancer metastasis start with a decrease or increase in cellular invasion, respectively. Here, we investigate if first trimester placental explants can be used to identify epigenetic factors associated with changes in cellular invasion and their potential use as biomarkers. We show that the outgrowth potential of first trimester explants significantly correlates with promoter methylation of PRKCDBP and MMP2, two genes known to be differentially methylated in both placenta and cancer. The increase in methylation percentage of placental cells coincides with an increase in invasion potential. Subsequently, as a non-invasive marker must be detectable in blood, plasma samples of pregnant and non-pregnant women were analyzed. The MMP2 promoter showed high methylation levels in non-pregnant plasma samples, which decreased in pregnant plasma samples which also contain placental DNA. The decrease in methylated plasma DNA during pregnancy is most likely due to the fractional increase in unmethylated placental DNA. This suggests that the level of unmethylated DNA has the potential to be used as an invasion marker, where higher levels of unmethylated DNA indicate a lower invasion potential of trophoblasts. These proof of principle data provide evidence that human first trimester placental explants are an excellent ex vivo model system to identify (epigenetic) factors and thus potential biomarkers associated with changes in cellular invasion, e.g., to detect pregnancy-related diseases or cancer metastasis. To identify novel biomarkers the next step is to correlate naturally occurring variation in invasion potential to changes in (epigenetic) factors by genome-wide approaches such as massively parallel sequencing.

The pre-eclampsia gene STOX1 controls a conserved pathway in placenta and brain upregulated in late-onset Alzheimer's disease.

Pre-eclampsia and late-onset Alzheimer's disease (LOAD) share no clinical features. In contrast to these clinical dissimilarities, striking parallels exist between the (epi)genetic features associated with pre-eclampsia and LOAD for the genes located on 10q22. The parallels in identity between the 10q22 genes involved and active in the organs (placenta, brain) primarily affected in the respective diseases led us to explore, if the pre-eclampsia susceptibility gene STOX1 is functionally involved in LOAD. We demonstrate that isoform A of STOX1 is abundantly expressed in the brain, correlates with severity of disease, and selectively transactivates LRRTM3 in neural cells with increased amyloid-beta protein precursor processing. Similar in vitro results were seen in trophoblast. Our data indicate that STOX1 controls a conserved pathway shared between placenta and brain with overexpression in LOAD.

Maternal segregation of the Dutch preeclampsia locus at 10q22 with a new member of the winged helix gene family.

Preeclampsia is a pregnancy-associated disease with maternal symptoms but placental origin. Epigenetic inheritance is involved in some populations. By sequence analysis of 17 genes in the 10q22 region with maternal effects, we narrowed the minimal critical region linked with preeclampsia in the Netherlands to 444 kb. All but one gene in this region, which lies within a female-specific recombination hotspot, encode DNA- or RNA-binding proteins. One gene, STOX1 (also called C10orf24), contained five different missense mutations, identical between affected sisters, cosegregating with the preeclamptic phenotype and following matrilineal inheritance. Four STOX1 transcripts are expressed in early placenta, including invasive extravillus trophoblast, generating three different isoforms. All contain a winged helix domain related to the forkhead (FOX) family. The largest STOX1 isoform has exclusive nuclear or cytoplasmic expression, indicating activation and inactivation, respectively, of the PI3K-Akt-FOX pathway. Because all 38 FOX proteins and all 8 STOX1 homologs have either tyrosine or phenylalanine at position 153, the predominant Y153H variation is highly mutagenic by conservation criteria but subject to incomplete penetrance. STOX1 is a candidate for preeclampsia controlling polyploidization of extravillus trophoblast.

C2360, a nuclear protein expressed in human proliferative cytotrophoblasts, is a representative member of a novel protein family with a conserved coiled coil-helix-coiled coil-helix domain.

In this study, we describe the identification of nine novel genes isolated from a unique human first-trimester cDNA library generated from the placental bed. One of these clones, called C2360 and located on chromosome 10q22, was selected as it showed restricted expression in placental bed tissue as well as in JEG3 choriocarcinoma cells with absent expression in adult tissues. We show that the expression is restricted to first-trimester proliferative trophoblasts of the proximal column and show that C2360 is a nuclear protein. No detectable transactivation potential was observed for different domains of the protein. Secondary structure prediction showed that C2360 is a representative member of a eukaryotic family of proteins with a low conservation at the amino acid level, but with strong conservation at the structural level, sharing the general domain (coiled coil 1)-(helix 1)-(coiled coil 2)-(helix 2), or CHCH domain. Each alpha-helix within this domain contains two cysteine amino acids, and these intrahelical cysteines are separated by nine amino acids (C-X(9)-C motif). The fixed position within each helix indicated that both helices could form a hairpin structure stabilized by two interhelical disulfide bonds. Other proteins belonging to the family include estrogen-induced gene 2 and the ethanol-induced 6 protein. The conserved motif was found in yeast, plant, Drosophila, Caenorhabditis elegans, mouse, and human proteins, indicating that the ancestor of this protein family is of eukaryotic origin. These results indicate that C2360 is a representative member of a multifamily of proteins, sharing a protein domain that is conserved in eukaryotes.

NEUROD1 acts in vitro as an upstream regulator of NEUROD2 in trophoblast cells.

The basic helix-loop-helix (bHLH) transcription factors NEUROD1, NEUROD2 and ATH2 are expressed during first trimester human placental development. We determined the transactivation potential of each of these factors in trophoblasts by measuring changes in the endogenous gene activity using absolute quantification by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) after transient transfection. In these assays, NEUROD1 was found to transiently transactivate NEUROD2 in trophoblast cells. Promotor truncation assays, using luciferase constructs, showed the presence of two domains in the NEUROD2 promotor, which showed increased activity after NeuroD1 transfection. Each of these NeuroD1-responsive domains contains an E-box sequence. The NEUROD2 transactivation data fit with the spatial expression pattern of NEUROD1 and NEUROD2, since they are expressed in endovascular trophoblasts. This expression pattern, as well as the present transactivation results, might suggest the presence of a NEUROD differentiation cascade during first trimester human placental development.

Quantitative reverse transcription-polymerase chain reaction measurement of HASH1 (ASCL1), a marker for small cell lung carcinomas with neuroendocrine features.

PURPOSE: The Human Achaete-Scute homologue 1 (HASH1, ASCL1), a lineage-specific basic helix-loop-helix member of the achaete-scute family, is essential for the generation of pulmonary neuroendocrine (NE) cells during lung development. In small cell lung cancer (SCLC), the most lethal form of lung cancer, the gene is highly expressed and the expression of HASH1 correlates with NE features found in SCLCs. Here we describe a highly sensitive reverse transcription-PCR method for quantifying HASH1 mRNA in clinical samples, using real-time fluorescence resonance energy transfer technology (LightCycler). EXPERIMENTAL DESIGN: The HASH1-positive NE cell line NCI-H187 was compared with the non-NE cell line NCI-N417 by quantitative reverse transcription-PCR. Signals were normalized using the housekeeping gene PBGD, which is pseudogene free. Subsequently, HASH1 expression in RNA isolated from biopsies from SCLC patients (n = 4) was compared with biopsies from non-SCLC (NSCLC) patients (n = 2) or normal bronchus (n = 2). RESULTS: The HASH1-positive NE cell line NCI-H187 showed 50,000-fold higher normalized expression of HASH1 than did the non-NE cell line NCI-N417, indicating that the method is applicable over a wide dynamic range. Normalized average mRNA expression levels in SCLC clinical samples were 1,000-fold higher than in the NSCLC samples. Expression in normal bronchus was comparable to the expression levels in the NSCLC. CONCLUSIONS: These results show that marked and measurable differences exist between SCLCs and other lung tissues (either NSCLC or normal bronchus). We show that the method is applicable to small biopsy samples and can discriminate SCLC from NSCLC. This method could contribute to diagnosis based on molecular profiling of tumors.

The proneural genes NEUROD1 and NEUROD2 are expressed during human trophoblast invasion.

During early human pregnancy, extravillous trophoblast cells invade the maternal tissue of the uterus in a way similar to invasion by cancer cells. However, the process of trophoblast invasion is regulated in a time and place restricted way, in contrast to cancer invasion. We screened first trimester placental tissue enriched by extravillous invasive trophoblasts for the expression of proneural basic helix-loop-helix (bHLH) transcription factors, which are important controllers of cell fate. Surprisingly, the presence of NEUROD1, NEUROD2 and ATH2 transcripts was found by reverse transcriptase polymerase chain reaction (RT-PCR) analysis in first trimester placentabed. Of these genes, the proneural genes NEUROD1 and NEUROD2 are expressed in different subsets of invasive trophoblasts. NEUROD1 expression is found in interstitial and endovascular invasive cells, while NEUROD2 expression is observed mainly in endovascular invasive cells, respectively. These data suggest that in addition to the involvement of proneural genes in neuron, neurendocrine and pancreas differentiation, these genes are involved in trophoblast differentation during progression of invasion.