MicroRNA-30b-5p inhibits autophagy in ovarian granulosa cells in polycystic ovary syndrome rats by targeting Atg5

Abstract

Abstract:

Objective ·To explore the expression of microRNA-30b-5p (miR-30b-5p) in polycystic ovary syndrome (PCOS) rats and the effect of miR-30b-5p overexpression on ovarian granulosa cell (GC) autophagy. Methods ·Dehydroepiandrosterone (DHEA) was performed to establish a PCOS rat model, and real-time fluorescent quantitative PCR (qRT-PCR) and Western blotting were performed to measure the expression of miR-30b-5p and autophagy-associated protein 5 homologue (Atg5) in the ovarian tissues of the normal group and PCOS group. Primary PCOS rat ovarian GCs were isolated and cultured, and divided into control group, miR-NC group, miR-30b-5p overexpression group, miR-30b-5p overexpression+pcDNA3.1-NC group, and miR-30b-5p overexpression+pcDNA3.1-Atg5 group. In addition, the ovarian GC of the normal group was taken as the blank group. MiR-30b-5p mimic, pcDNA3.1-Atg5 and the corresponding negative control were transfected into the cells, and 48 h after transfection, qRT-PCR was performed to measure the expression of miR-30b-5p and Atg5 mRNA of cells in each group to verify the transfection effect. CCK-8 and flow cytometer were performed to measure cell viability and apoptosis rate, respectively; immunofluorescence staining was performed to measure the positive expression of microtubule-associated protein 1 light chain 3 (LC3) in each group. Western blotting was performed to measure the protein expression of autophagy-related proteins Atg5, p62, Beclin-1 and LC3. Results ·The expression level ofmiR-30b-5pin the ovarian tissue of the PCOS group was significantly lower than that of the normal group, and the levels of Atg5 mRNA and protein were significantly higher than those of the normal group (all P=0.000). After transfection, compared with the blank group, the miR-30b-5p level, apoptosis rate, and p62 protein level in the ovarian GC of the control group were significantly reduced, the Atg5 mRNA and protein levels, cell proliferation activity, LC3 positive cell percentage, Beclin-1 protein level and LC3Ⅱ/LC3Ⅰ ratio were significantly increased (all P=0.000). Compared with the control group, the miR-30b-5p level, apoptosis rate, and p62 protein level in the ovarian GC of the miR-30b-5p overexpression group were significantly increased, and the Atg5 mRNA and protein levels, cell proliferation activity, LC3 positive cell percentage, Beclin-1 protein level and LC3Ⅱ/LC3Ⅰ ratio were significantly reduced (all P=0.000). Up-regulation of Atg5 can significantly attenuate the inhibitory effect of miR-30b-5p overexpression on ovarian GC proliferation and autophagy (all P=0.000). Conclusion ·MiR-30b-5p is lowly expressed in PCOS; overexpression of miR-30b-5p can inhibit the proliferation and autophagy of ovarian GC in PCOS rats, and promote cell apoptosis, and its mechanism may be related to the inhibition of Atg5 expression.

Key words: polycystic ovary syndrome, microRNA-30b-5p, autophagy, autophagy-associated protein 5 homologue(Atg5), proliferation, apoptosis

CLC Number:

R711.75

WANG Xuemin, WANG Yanan, NIU Aiqin, YE Ying, LI Fei. MicroRNA-30b-5p inhibits autophagy in ovarian granulosa cells in polycystic ovary syndrome rats by targeting Atg5[J]. Journal of Shanghai Jiao Tong University (Medical Science).

Figures/Tables 8

Tab 1 Primer sequences used for qRT-PCR

Gene	Forward primer （5′-3′）	Reverse primer （5′-3′）	Length/bp
miR-30b-5p	GCG CTG TAA ACA TCC TAC AC	GTG CAG GGT CCG AGG T	245
Atg5	TGA CCA GTT TTG GAC CAT CA	AGG GTA TGC AGC TGT CCA TC	158
β-actin	CTA AGG CCA ACC GTG AAA AG	ACC AGA GGC ATA CAG GGA CA	143
U6	CTC GCT TCG GCA GCA CAT ATA CT	ACG CTT CAG AAT TTG CGT GTC	129

Fig 1 Expression of miR-30b-5p and Atg5 in ovarian tissue of PCOS rats

Fig 2 Identification of primary rat ovarian GC (×200)

Fig 3 Comparison of miR-30b-5p and Atg5 levels in ovarian GC of rats in each group

Fig 4 Proliferative activity of rat ovarian GC in each group

Fig 5 Apoptosis levels of ovarian GC in rats in each group

Fig 6 Positive expression of LC3 in rat ovarian GC in each group

Fig 7 Expression of p62, Beclin-1 and LC3 proteins in ovarian GC of rats in each group

TrendMD

References 16

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