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Other frequently methylated genes in HCC include RIZ1 (45.2%), CDKN2A (69.7%), SCARA5 (30%), EFEMP1 (50%), TIP30 (47%), WIFI, FBLN1 (50%), DLEC1 (70.6%), FBP1 (80%), ITGA4 (23%), KLK10 (94%), LIFR (47.9%), MTIG (60.4%), HHIP (53.6%), HINT1 (55%), SYK (12%), and TAT (54%) [18, 19].
Out of these eight genes, amphiregulin (AREG), fibulin 1 (FBLN1), and claudin 6 (CLDN6) were the most differentially expressed.
Assay-on-Demand Gene Expression Product (Applied Biosystems, Foster City, CA, USA, 4331182), consisting of unlabeled PCR primers and a TaqMan MGB probe (FAM dye-labeled) optimized to work with the TaqMan Universal PCR Master Mix (P/N 4304437) in an ABI Prism 7700 system (Perkin Elmer Life Sciences, Boston, MA, USA), was employed to quantitatively measure AREG, FBLN1, CLDN6, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression.
Out of these eight genes, AREG, FBLN1, and CLDN6 were strongly expressed after treatment with both [T.sub.3] and [E.sub.2].
Out of these eight genes, AREG, FBLN1, and CLDN6 were strongly expressed by both [T.sub.3] and [E.sub.2] treatment.
We performed quantitative real-time PCR (q-RT-PCR) using random hexamer primers and Taqman gene expression assays (Applied Biosystems Prism 7700) for von Willebrand factor (VWF)12 (Hs01109460_m1); leptin (LEP) (Hs00174877_m1); matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase) (MMP2) (Hs00234422_m1); fibulin 1 (FBLN1) (Hs00242546_m1); and fibulin 2 (FBLN2) (Hs01002054_m1).
Among the upregulated genes, FBLN1 displayed the highest level of fold change.
 Human genes: VWF, von Willebrand factor; LEP, leptin; MMP2, matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase); FBLN1, fibulin 1; FBLN2, fibulin 2; ACTS, actin, beta; CRISPLD2, Cysteine-rich secretory protein LCCL domain containing 2; SERPINF1, Serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1; ITM2A, Integral membrane protein 2A; LEPR, leptin receptor; ZFP36L2, zinc finger protein 36, C3H type-like 2; ELN, elastin.
Moreover, maternal smoking has been shown to alter the expression of genes related to neuropeptide signaling and signal transduction, as well as development, particularly tachykinin 3 (TAC3), left-right determination factor 2 (LEFTY2), heparin-binding EGF-like growth factor (HBEGF), mitochondrial fission factor (MFF), and fibulin1 (FBLN1), via mechanisms possibly related to genome-wide changes in DNA methylation .
ENSMUSG00000086859 targeted genes like Efna1, Fbln1, and Fbln2.
A series of positively regulated target genes were significantly enriched in the pathways of cholesterol biosynthesis (e.g., Cyp51 and Fdps) and elastic fibre formation (e.g., Fbln1, Fbln2, and Fbln5) (Table 2).
The DEGs positively regulated by DE-lncRNAs were mainly enriched in the functions about the development of blood vessel (e.g., Efna1 and Efnb2), as well as the pathways of cholesterol biosynthesis (e.g., Cyp51 and Fdps) and elastic fibre formation (e.g., Fbln1, Fbln2, and Fbln5).
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