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Small RNA analysis of epidermis uncovered miRNAs involved in cuticle biosynthesis in Brassica napus

ABSTRACT: To investigate the function of small RNAs in cuticle biosynthesis in B. napus, we constructed four small RNA libraries from stem epidermis of wax-deficient and wild-type materials for sequencing. Subsequently, a total of 43,840,451 clean reads were generated and 24nt sequences represented the dominant percentage. Totally, 300 unique conserved miRNAs were identified and 8 of them showed differentially expressed. In addition, the expression of six novel miRNAs were also changed. After target prediction and function annotation, three miRNAs (aly-miR165a-5p, aly-miR408-5p, and novel-mir2) might influence cuticle morphogenesis not only by repressing the biosynthesis of cuticular substrates and components, but also blocking the transmembrane transport processes. The expression level of aly-miR408-5p and aly-miR165a-5p were increased in glossy materials examined by stem-loop qRT-PCR, while their predicted target genes (BnaC04g29170D, BnaA01g25630D, BnaA06g40560D) were down-regulated. These results suggested that miRNAs might influence cuticle biosynthesis by repressing cuticle-related genes expression in B. napus. Both the biosynthesis and secretion processes of cuticlular components might be influenced by miRNAs. These results will promote the study of post-transcriptional regulation mechanisms of cuticle biosynthesis in B. napus and provide new direction for further research.

ORGANISM(S): Brassica napus

PROVIDER: GSE115073 | GEO | 2018/11/30

REPOSITORIES: GEO

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Project description:We tested the hypothesis that a panel of placental mammal-specific miRNAs and their targets play important to establish receptivity to implantation and their dysregulated expression may be a feature in women with early pregnancy loss. Relative expression levels of miR-340-5p, −542-3p, and −671-5p all increased following treatment of Ishikawa cells with progesterone (10 μg/ml) for 24 hrs (p < 0.05). RNA sequencing of these P4-treated cells identified co-ordinate changes to 6,367 transcripts of which 1713 were predicted targets of miR-340-5p, 670 of miR-542-3p, and 618 of miR-671-5p. Quantitative proteomic analysis of Ishikawa cells transfected with mimic or inhibitor (48 hrs: n=3 biological replicates) for each of the P4-regulated miRNAs was carried out to identify targets of these miRNAs. Excluding off target effects, mir-340-5p mimic altered 1,369 proteins while inhibition changed expression of 376 proteins (p < 0.05) of which, 72 were common to both treatments. A total of 280 proteins were identified between predicted (mirDB) and confirmed (in vitro) targets. In total, 171 proteins predicted to be targets by mirDB were altered in vitro by treatment with miR-340-5p mimic or inhibitor and were also altered by treatment of endometrial epithelial cells with P4. In vitro targets of miR-542-3p identified 1,378 proteins altered by mimic while inhibition altered 975 a core of 200 proteins were changed by both. 100 protein targets were predicted and only 46 proteins were P4 regulated. miR-671-mimic altered 1,252 proteins with inhibition changing 492 proteins of which 97 were common to both, 95 were miDB predicted targets and 46 were also P4-regulated. All miRNAs were detected in endometrial biopsies taken from patients during the luteal phase of their cycle, irrespective of prior or future pregnancy outcomes Expression of mir-340-5p showed an overall increase in patients who had previously suffered a miscarriage and had a subsequent miscarriage, as compared to those who had infertility or previous miscarriage and subsequently went on to have a life birth outcome. The regulation of these miRNAs and their protein targets regulate the function of transport and secretion, and adhesion of the endometrial epithelia required for successful implantation in humans. Dysfunction of these miRNAs (and therefore the targets they regulate) may contribute to endometrial-derived recurrent pregnancy loss in women.

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Small RNA analysis of epidermis uncovered miRNAs involved in cuticle biosynthesis in Brassica napus

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