Time course of rat GRP78 mRNA, rno-miR-144, rno-miR-376a, and rno-miR-451 expression in rat ovaries induced by PMSG and hCG.

<p>Female 21-day-old rats injected subcutaneously with PMSG (30 IU/rat), followed by hCG (20 IU/rat) 48 h later, were sacrificed at the indicated times. The ovaries were removed, and total RNA was isolated. Rat GRP78 mRNA (A), rno-miR-144 (B), rno-miR-376a (C), and rno-miR-451 (D) expression levels were measured using real-time RT-PCR as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108997#s2" target="_blank">Materials and Methods</a>. The amount of rat GRP78 mRNA, rno-miR-144, rno-miR-376a, and rno-miR-451 in the hCG 0 h group was set at 1. Data were normalized to 18S rRNA (for GRP78 mRNA) and 4.5S RNA(H) (for rno-miR-144, rno-miR-376a, and rno-miR451) levels in each sample and represent the mean ±SE of three independent experiments. *, significantly different from the control value at hCG 0 h, <i>P</i><0.05.</p

MicroRNA-376a Regulates 78-Kilodalton Glucose-Regulated Protein Expression in Rat Granulosa Cells

<div><p>The 78-kilodalton glucose-regulated protein (GRP78) is a molecular chaperone that assists in protein assembly, folding, and translocation. Recently, our laboratory reported that GRP78 regulates the expression of luteinizing hormone-human chorionic gonadotropin receptor (LHR) in the early stage of corpus luteum formation. In this study, we investigated whether microRNAs (miRNAs), which post-transcriptionally regulate mRNA, are involved in the regulation mechanism of GRP78 in the ovary. A miRNA microarray was performed to analyze the overall miRNA expression profile, and the results indicated that 44 miRNAs were expressed highly after ovulation was induced. The results from a bio-informative database analysis and <i>in vitro</i> granulosa cell culture studies led us to focus on rno-miR-376a for further analysis. In both <i>in vivo</i> and <i>in vitro</i> studies, rno-miR-376a levels increased 12 h after human chorionic gonadotropin (hCG) administration. To elucidate whether rno-miR-376a induced mRNA destabilization or translational repression of GRP78, rno-miR-376a was transfected into cultured granulosa cells, resulting in decreased GPR78 protein levels without an alteration in GRP78 mRNA levels. To confirm that rno-miR-376a binds to GRP78 mRNA, we cloned the 3′-end of GRP78 mRNA (nucleotides 2439–2459) into a reporter vector that contained a Renilla luciferase coding region upstream of the cloning site. The luciferase assays revealed that rno-miR-376a bound to the 3′-end of GRP78 mRNA. From these data, we conclude that rno-miR-376a potentially negatively regulates GRP78 protein expression through translational repression at an early stage transition from the follicular phase to luteinization.</p></div

Effects of rno-miR-376a on GRP78 protein in granulosa cells.

<p>Primary rat granulosa cells were prepared, and the indicated reagents were added to the medium after 24 h of culture. The cells were incubated with FSH (30 ng/mL) and estradiol (10 nM) for 48 h. Negative control (A), precursor (B) or inhibitor (C) was transfected into the cells, and 30 ng/mL hCG was added 12 h later. Cells were harvested 24 h after the addition of hCG, and GRP78 protein levels were quantified using western blot analysis. (D) Levels of GRP78 protein were quantified by densitometric scanning. The expression of GRP78 protein in the control (hCG 0 h) was set at 1. Values represent the mean ±SE of three independent experiments. *, significantly different from the control value, <i>P</i><0.05.</p

Rat GRP78 mRNA, rno-miR-144, rno-miR-376a, and rno-miR-451 expression in primary rat granulosa cells induced by FSH and hCG.

<p>Primary rat granulosa cells were prepared, and the indicated reagents were added to the medium after 24 h of culture. Cells were then incubated with FSH (30 ng/mL) and estradiol (10 nM) for 48 h. Subsequently, hCG (30 ng/mL) was added to the culture medium, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108997#s2" target="_blank">Materials and Methods</a>. Total RNA was isolated, and GRP78 mRNA (A), rno-miR-144 (B), rno-miR-376a (C), and rno-miR-451 (D) expression levels were measured using real-time RT-PCR as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108997#s2" target="_blank">Materials and Methods</a>. The amounts of GRP78 mRNA, rno-miR-144, rno-miR-376a, and rno-miR-451 in the hCG 0 h group were set at 1. Data were normalized for 18S rRNA (for GRP78 mRNA) and 4.5S RNA(H) (for rno-miR-144, rno-miR-376a, and rno-miR-451) levels in each sample and represent the mean ±SE of 3 independent experiments. *, significantly different from the control value at hCG 0 h, <i>P</i><0.05.</p

Effects of Pre-miR-376a (precursor) and Anti-miR-376a (inhibitor) transfection on rat GRP78 mRNA expression in primary rat granulosa cells.

<p>Primary rat granulosa cells were prepared, and the indicated reagents were added to the medium after 24 h of culture. The cells were incubated with FSH (30 ng/mL) and estradiol (10 nM) for 48 h. Pre-miR-376a (precursor) or Anti-miR-376a (inhibitor) was transfected into the cells, and 30 ng/mL hCG was added 12 h later. The effects of precursor and inhibitor on the expression of GRP78 mRNA were measured using real-time RT-PCR, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108997#s2" target="_blank">Materials and Methods</a>. The expression of GRP78 mRNA at hCG 0 h in the control was set at 1. Each value represents the mean ±SE of three independent experiments.</p

Luciferase assays for the identification of the rno-miR-376a-binding site in the 3′-UTR of GRP78 mRNA.

<p>(A) Arrangement of rno-miR-376a and GRP78 mRNA and a schematic drawing of the predicted rno-miR-376a-binding site in the 3′-UTR of GRP78 mRNA. (B) Schematic drawings of the pMIR-REPORT luciferase vectors used in our experiment. To identify the rno-miR-376a-binding site in the 3′-UTR of GRP78 mRNA, luciferase reporter vectors were generated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108997#s2" target="_blank">Materials and Methods</a>. (C) Luciferase activity was measured to identify the rno-miR-376a-binding site in the 3′-UTR of GRP78 mRNA. HEK293 cells were prepared, and the cells were transfected with 200 ng of each reporter vector with 50 nM Pre-miR-376a (precursor) or Anti-miR-376a (inhibitor) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108997#s2" target="_blank">Materials and Methods</a>. For transfection normalization, the cells were also transfected with the pMIR-REPORT βgal vector. Luciferase activity was measured 24 h after transfection. The activity of the control (empty vector) was set at 1. Each value represents the mean ±SE of three independent experiments. *, significantly different from the control value, <i>P</i><0.05.</p

Rat GRP78 mRNA, rno-miR-376a expression in primary rat granulosa cells induced by FSH.

<p>Primary rat granulosa cells were prepared, and the indicated reagents were added to the medium after 24 h of culture. Cells were then incubated with FSH (30 ng/mL) and estradiol (10 nM) for 48 h in the same way as described in Fig. 2. The time after 48 h of incubation with FSH and estradiol was considered “0 h.” Total RNA was isolated, and rno-miR-376a expression levels was determined using real-time RT-PCR at the indicated time. The amounts of rno-miR-376a in the 0 h group were set at 1. Data were normalized for 4.5S RNA(H) levels in each sample and represent the mean ±SE of 3 independent experiments.</p

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Additional file 1 of Utility of vaginal vault cytology in the local recurrence of cervical cancer

Additional file 1

Visual Abstract - Follistatin-Like 1 Regulates Hypertrophy in Heart Failure With Preserved Ejection Fraction: 10.1016/j.jacbts.2016.04.002

• Fstl1, also known as transforming growth factor-β–stimulated clone 36, is an extra-cellular glycoprotein implicated in the pathophysiology of cardiac disease.• Fstl1 acts in a noncanonical manner relative to other follistatin family members, but its functions remain poorly understood.• Circulating Flst1 levels are increased in humans with chronic stable HFpEF.• Fstl1 treatment modulates cardiomyocyte hypertrophy in vitro and in vivo.• Cardiac myocyte deletion of Fstl1 worsens the HFpEF phenotype in mice.• These studies indicate that Fstl1 may be therapeutically effective in HFpEF by modulating cardiac hypertrophy and improving parameters of diastolic dysfunction.</div