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Deep-sequencing identification of differentially expressed miRNAs in decidua and villus of recurrent miscarriage patients.

Wang JM, Gu Y, Zhang Y, Yang Q, Zhang X, Yin L, Wang J - Arch. Gynecol. Obstet. (2016)

Bottom Line: In the decidual of RM patients, expression of hsa-miR-516a-5p, -517a-3p, -519a-3p and -519d were significantly up-regulated compared to normal pregnancy.In the villi of RM patients, expression of hsa-miR-100 and -146a-5p were significantly higher, while hsa-miR-1 and -372 were significantly lower compared to normal pregnancy.These data suggested that the pathogenic process of RM might be associated with the alteration of miRNAs expression profiles in decidua and villus.

View Article: PubMed Central - PubMed

Affiliation: The Second Hospital of Tianjin Medical University, Tianjin, China.

ABSTRACT

Purpose: MicroRNAs (miRNAs) are small non-coding RNA molecules that play critical roles in post-transcriptional gene expression regulation. The aim of this study was to identify differentially expressed miRNAs in decidua and villus of recurrent miscarriage (RM) patients.

Methods: Participants were recruited at the outpatient Department of Gynecology and Obstetrics, The Second Hospital of Tianjin Medical University, China. Decidua and villus tissues were collected by curettage from recruited RM patients and normal pregnant women with their informed consent. MiRNAs expression profiles in decidua or villus were respectively determined by the deep-sequencing analysis. The predicated target genes of these differentially expressed miRNAs were analyzed by miRWalk. The differential expressions of four miRNAs in decidua and four miRNAs in villus between the six pairs of RM patients and normal pregnant women were confirmed by qRT-PCR analysis. The expression patterns of two predicated target genes, Bcl-2 and Pten, in the same six pairs of decidual or villus tissues were detected by Western blotting analysis, respectively.

Results: Totally 18 RM patients and 15 normal pregnant women were recruited. Thirty-two miRNAs in decidua and four miRNAs in villus of RM patients were screened out to be significantly up-regulated compared to that of normal pregnant women, and five miRNAs in villus of RM patients were screened out to be remarkably down-regulated compared to that of normal pregnant women (P value < 0.05 and Fold change >2). These differentially expressed miRNAs were predicted to target a large number of genes that involved in cell apoptosis, p53 signaling pathway, cell cycle and other cellular bio-functions. Differential expressions of hsa-miR-516a-5p, -517a-3p, -519a-3p and -519d in decidua, as well as hsa-miR-1, -372, -100-5p and -146a-5p in villus, were validated by qRT-PCR analysis. In the decidual of RM patients, expression of hsa-miR-516a-5p, -517a-3p, -519a-3p and -519d were significantly up-regulated compared to normal pregnancy. In the villi of RM patients, expression of hsa-miR-100 and -146a-5p were significantly higher, while hsa-miR-1 and -372 were significantly lower compared to normal pregnancy. Furthermore, the expression of Bcl-2 and Pten, a predicated target gene of hsa-miR-1 or hsa-miR-372 respectively, was significantly up-regulated in the villi of RM patients.

Conclusions: These data suggested that the pathogenic process of RM might be associated with the alteration of miRNAs expression profiles in decidua and villus. Especially, the aberrant placental expression of hsa-miR-1 and -372 might be involved in the progression of RM, but need to be further investigated by larger studies in the future.

No MeSH data available.


Related in: MedlinePlus

Expression profile of miRNAs in villus and decidua of RM and normal pregnancy. a Heatmap of differentially expressed miRNAs in decidua. b Log–log scatter plot comparing global miRNAs expression profiles in decidua of RM patients with those of normal pregnancy. c Heatmap of differentially expressed miRNAs in villus. d Log–log scatter plot comparing global miRNAs expression profiles in villus of RM patients with those of normal pregnancy
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Fig1: Expression profile of miRNAs in villus and decidua of RM and normal pregnancy. a Heatmap of differentially expressed miRNAs in decidua. b Log–log scatter plot comparing global miRNAs expression profiles in decidua of RM patients with those of normal pregnancy. c Heatmap of differentially expressed miRNAs in villus. d Log–log scatter plot comparing global miRNAs expression profiles in villus of RM patients with those of normal pregnancy

Mentions: Human decidua and villus tissues were obtained from 18 RM patients and 15 normal pregnant women. No significant differences were observed in the age and gestational period between the two groups (Table 1). Total RNA product was respectively extracted from each tissue sample. After checking the RNA integrity, all of 18 RM decidua RNA samples and 15 normal pregnancy decidua RNA samples were qualified for subsequent analysis; however, only three RM villus RNA samples (4, 10 and 12 V) and four normal pregnancy villus RNA samples (C2 V, C4 V, C7 V and C10 V) were qualified to construct cDNA libraries for deep sequencing [RNA integrities (RIN) ≥ 7 and 28 s/18 s ≥ 0.7]. Differentially expressed miRNAs were screened out by meeting our designated criteria: P < 0.05 and fold change >2. A total of 32 miRNAs were screened to be significantly up-regulated in decidua of RM patients, whereas a total of nine miRNAs was differentially expressed in villus of RM patients, including four up-regulated and five down-regulated miRNAs (Fig. 1; Table 2). To validate the reliability of the deep-sequencing data, we selected four miRNAs from decidua (hsa-miR-516a-5p, 517a-3p, 519a-3p and 519d) and four miRNAs from villus (hsa-miR-1, 372, 100-5p and 146a-5p) to confirm their differential expressions in decidua or villus between RM and normal pregnancy group by qRT-PCR analysis. As the villous RNA sample sizes used for deep-sequencing were too small, another three RM villous RNA samples (1, 2, 3 V,) and two normal pregnancy villous (C1 V and C8 V) RNA samples, whose integrities were qualified for qRT-PCR analysis (RIN ≥ 6 and 28 s/18 s ≥ 0.7), were also chosen to be used in qRT-PCR analysis. The qRT-PCR results showed that the expression patterns of all eight selected miRNAs were in concordance with the deep-sequencing data, indicating the deep-sequencing data were reliable (Fig. 2).Table 1


Deep-sequencing identification of differentially expressed miRNAs in decidua and villus of recurrent miscarriage patients.

Wang JM, Gu Y, Zhang Y, Yang Q, Zhang X, Yin L, Wang J - Arch. Gynecol. Obstet. (2016)

Expression profile of miRNAs in villus and decidua of RM and normal pregnancy. a Heatmap of differentially expressed miRNAs in decidua. b Log–log scatter plot comparing global miRNAs expression profiles in decidua of RM patients with those of normal pregnancy. c Heatmap of differentially expressed miRNAs in villus. d Log–log scatter plot comparing global miRNAs expression profiles in villus of RM patients with those of normal pregnancy
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829624&req=5

Fig1: Expression profile of miRNAs in villus and decidua of RM and normal pregnancy. a Heatmap of differentially expressed miRNAs in decidua. b Log–log scatter plot comparing global miRNAs expression profiles in decidua of RM patients with those of normal pregnancy. c Heatmap of differentially expressed miRNAs in villus. d Log–log scatter plot comparing global miRNAs expression profiles in villus of RM patients with those of normal pregnancy
Mentions: Human decidua and villus tissues were obtained from 18 RM patients and 15 normal pregnant women. No significant differences were observed in the age and gestational period between the two groups (Table 1). Total RNA product was respectively extracted from each tissue sample. After checking the RNA integrity, all of 18 RM decidua RNA samples and 15 normal pregnancy decidua RNA samples were qualified for subsequent analysis; however, only three RM villus RNA samples (4, 10 and 12 V) and four normal pregnancy villus RNA samples (C2 V, C4 V, C7 V and C10 V) were qualified to construct cDNA libraries for deep sequencing [RNA integrities (RIN) ≥ 7 and 28 s/18 s ≥ 0.7]. Differentially expressed miRNAs were screened out by meeting our designated criteria: P < 0.05 and fold change >2. A total of 32 miRNAs were screened to be significantly up-regulated in decidua of RM patients, whereas a total of nine miRNAs was differentially expressed in villus of RM patients, including four up-regulated and five down-regulated miRNAs (Fig. 1; Table 2). To validate the reliability of the deep-sequencing data, we selected four miRNAs from decidua (hsa-miR-516a-5p, 517a-3p, 519a-3p and 519d) and four miRNAs from villus (hsa-miR-1, 372, 100-5p and 146a-5p) to confirm their differential expressions in decidua or villus between RM and normal pregnancy group by qRT-PCR analysis. As the villous RNA sample sizes used for deep-sequencing were too small, another three RM villous RNA samples (1, 2, 3 V,) and two normal pregnancy villous (C1 V and C8 V) RNA samples, whose integrities were qualified for qRT-PCR analysis (RIN ≥ 6 and 28 s/18 s ≥ 0.7), were also chosen to be used in qRT-PCR analysis. The qRT-PCR results showed that the expression patterns of all eight selected miRNAs were in concordance with the deep-sequencing data, indicating the deep-sequencing data were reliable (Fig. 2).Table 1

Bottom Line: In the decidual of RM patients, expression of hsa-miR-516a-5p, -517a-3p, -519a-3p and -519d were significantly up-regulated compared to normal pregnancy.In the villi of RM patients, expression of hsa-miR-100 and -146a-5p were significantly higher, while hsa-miR-1 and -372 were significantly lower compared to normal pregnancy.These data suggested that the pathogenic process of RM might be associated with the alteration of miRNAs expression profiles in decidua and villus.

View Article: PubMed Central - PubMed

Affiliation: The Second Hospital of Tianjin Medical University, Tianjin, China.

ABSTRACT

Purpose: MicroRNAs (miRNAs) are small non-coding RNA molecules that play critical roles in post-transcriptional gene expression regulation. The aim of this study was to identify differentially expressed miRNAs in decidua and villus of recurrent miscarriage (RM) patients.

Methods: Participants were recruited at the outpatient Department of Gynecology and Obstetrics, The Second Hospital of Tianjin Medical University, China. Decidua and villus tissues were collected by curettage from recruited RM patients and normal pregnant women with their informed consent. MiRNAs expression profiles in decidua or villus were respectively determined by the deep-sequencing analysis. The predicated target genes of these differentially expressed miRNAs were analyzed by miRWalk. The differential expressions of four miRNAs in decidua and four miRNAs in villus between the six pairs of RM patients and normal pregnant women were confirmed by qRT-PCR analysis. The expression patterns of two predicated target genes, Bcl-2 and Pten, in the same six pairs of decidual or villus tissues were detected by Western blotting analysis, respectively.

Results: Totally 18 RM patients and 15 normal pregnant women were recruited. Thirty-two miRNAs in decidua and four miRNAs in villus of RM patients were screened out to be significantly up-regulated compared to that of normal pregnant women, and five miRNAs in villus of RM patients were screened out to be remarkably down-regulated compared to that of normal pregnant women (P value < 0.05 and Fold change >2). These differentially expressed miRNAs were predicted to target a large number of genes that involved in cell apoptosis, p53 signaling pathway, cell cycle and other cellular bio-functions. Differential expressions of hsa-miR-516a-5p, -517a-3p, -519a-3p and -519d in decidua, as well as hsa-miR-1, -372, -100-5p and -146a-5p in villus, were validated by qRT-PCR analysis. In the decidual of RM patients, expression of hsa-miR-516a-5p, -517a-3p, -519a-3p and -519d were significantly up-regulated compared to normal pregnancy. In the villi of RM patients, expression of hsa-miR-100 and -146a-5p were significantly higher, while hsa-miR-1 and -372 were significantly lower compared to normal pregnancy. Furthermore, the expression of Bcl-2 and Pten, a predicated target gene of hsa-miR-1 or hsa-miR-372 respectively, was significantly up-regulated in the villi of RM patients.

Conclusions: These data suggested that the pathogenic process of RM might be associated with the alteration of miRNAs expression profiles in decidua and villus. Especially, the aberrant placental expression of hsa-miR-1 and -372 might be involved in the progression of RM, but need to be further investigated by larger studies in the future.

No MeSH data available.


Related in: MedlinePlus