微RNA-30b-5p通过靶向Atg5抑制多囊卵巢综合征大鼠卵巢颗粒细胞自噬

摘要/Abstract

摘要：

目的·探究microRNA-30b-5p（miR-30b-5p）在多囊卵巢综合征（polycystic ovary syndrome，PCOS）大鼠中的表达及miR-30b-5p过表达对卵巢颗粒细胞（granulosa cell，GC）自噬的影响。方法·采用脱氢表雄酮（dehydroepiandrosterone，DHEA）建立PCOS大鼠模型，实时荧光定量PCR（real-time fluorescent quantitative PCR，qRT-PCR）和Western blotting检测正常组和PCOS组大鼠卵巢组织中miR-30b-5p和自噬相关蛋白5同源物（autophagy-associated protein 5 homologue，Atg5）的表达。分离并培养原代PCOS大鼠卵巢GC，分为对照组、miR-NC组、miR-30b-5p过表达组、miR-30b-5p过表达+pcDNA3.1-NC组、miR-30b-5p过表达+pcDNA3.1-Atg5组，另取正常组大鼠卵巢GC为空白组；将miR-30b-5p mimic和pcDNA3.1-Atg5及相应的阴性对照转染到细胞中，转染48 h后，qRT-PCR检测各组细胞中miR-30b-5p和Atg5 mRNA表达，验证转染效果。CCK-8及流式细胞仪分别检测细胞活力和凋亡率；免疫荧光染色检测各组细胞微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3，LC3）阳性表达；Western blotting检测自噬相关蛋白Atg5、p62、Beclin-1和LC3蛋白表达。结果·PCOS组大鼠卵巢组织中miR-30b-5p表达水平显著低于正常组，Atg5 mRNA和蛋白水平显著高于正常组（均P=0.000）。转染后，与空白组相比，对照组卵巢GC中miR-30b-5p水平、细胞凋亡率、p62蛋白水平显著降低，Atg5 mRNA和蛋白水平、细胞增殖活性、LC3阳性细胞百分比、Beclin-1蛋白水平和LC3Ⅱ/LC3Ⅰ比值显著升高（均P=0.000）；与对照组相比，miR-30b-5p过表达组卵巢GC中miR-30b-5p水平、细胞凋亡率、p62蛋白水平显著升高，Atg5 mRNA和蛋白水平、细胞增殖活性、LC3阳性细胞百分比、Beclin-1蛋白水平和LC3Ⅱ/LC3Ⅰ比值显著降低（均P=0.000）。上调Atg5可明显减弱miR-30b-5p过表达对卵巢GC增殖和自噬的抑制作用（均P=0.000）。结论·MiR-30b-5p在PCOS中呈低表达；过表达miR-30b-5p可抑制PCOS大鼠卵巢GC增殖和自噬，促进细胞凋亡，其作用机制可能与抑制Atg5表达有关。

关键词: 多囊卵巢综合征, microRNA-30b-5p, 自噬, 自噬相关蛋白5同源物, 增殖, 凋亡

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

中图分类号:

R711.75

王雪敏, 王亚楠, 牛爱琴, 叶英, 李飞. 微RNA-30b-5p通过靶向Atg5抑制多囊卵巢综合征大鼠卵巢颗粒细胞自噬[J]. 上海交通大学学报（医学版）.

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).

图/表 8

表1 用于qRT-PCR的引物序列

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

图1 miR-30b-5p和Atg5在PCOS大鼠卵巢组织中的表达Note： A. Expression of miR-30b-5p. B. Expression of Atg5 mRNA. C. Expression of Atg5 protein. D. Detection of Atg5 protein in ovarian tissue by Western blotting. ①P=0.000, compared with the normal group.

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

图2 原代大鼠卵巢GC的鉴定(×200)Note： A. Morphology of ovarian GC. B. Identification of ovarian GC. Red fluorescence represents FSHR positive; blue fluorescence represents DAPI-stained nuclei; FSHR can be seen expressed on the cell membrane.

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

图3 各组大鼠卵巢GC中miR-30b-5p和Atg5水平比较Note：A. Relative expression of miR-30b-5p. B. Relative expression of Atg5 mRNA. C. Relative expression of Atg5 protein. D. Western blotting to detect the expression of Atg5 protein in ovarian GC of rats in each group. a—blank group; b—control group; c—miR-NC group; d—miR-30b-5p overexpression group; e—miR-30b-5p overexpression+pcDNA3.1-NC group; f—miR-30b-5p overexpression+pcDNA3.1-Atg5 group. ①P=0.000, compared with the blank group; ②P=0.000, compared with the control group; ③P=0.000, compared with the miR-30b-5p overexpression group.

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

图4 各组大鼠卵巢GC增殖活性Note： a—blank group; b—control group; c—miR-NC group; d—miR-30b-5p overexpression group; e—miR-30b-5p overexpression+pcDNA3.1-NC group; f—miR-30b-5poverexpression+pcDNA3.1-Atg5 group. ①P=0.012, ②P=0.008, compared with the blank group; ③P=0.027, compared with the control group; ④P=0.047, ⑤P=0.044, compared with the miR-30b-5p overexpression group.

Fig 4 Proliferative activity of rat ovarian GC in each group

图5 各组大鼠卵巢GC凋亡水平Note：A. Detection of apoptosis by flow cytometry. B. GC apoptosis rate in each group. a—blank group; b—control group; c—miR-NC group; d—miR-30b-5p overexpression group; e—miR-30b-5p overexpression+pcDNA3.1-NC group; f—miR-30b-5p overexpression+pcDNA3.1-Atg5 group. ①P=0.000, compared with the blank group; ②P=0.000, compared with the control group; ③P=0.000, compared with the miR-30b-5p overexpression group.

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

图6 各组大鼠卵巢GC中LC3阳性表达Note：A. Immunofluorescence LC3 staining (×400). B. LC3 positive cells in each group. a—blank group; b—control group; c—miR-NC group; d—miR-30b-5p overexpression group; e—miR-30b-5p overexpression+pcDNA3.1-NC group; f—miR-30b-5p overexpression+pcDNA3.1-Atg5 group. ①P=0.000, ②P=0.002, compared with the blank group; ③P=0.002, ④P=0.001, compared with the control group; ⑤P=0.010, compared with the miR-30b-5p overexpression group.

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

图7 各组大鼠卵巢GC中p62、Beclin-1和LC3蛋白表达Note：A. Relative expression of p62 protein. B. Relative expression of Beclin-1 protein. C. Relative expression of LC3Ⅱ/LC3Ⅰ. D. Western blotting to detect protein expression in ovarian GC of rats in each group. a—blank group; b—control group; c—miR-NC group; d—miR-30b-5p overexpression group; e—miR-30b-5p overexpression+pcDNA3.1-NC group; f—miR-30b-5p overexpression+pcDNA3.1-Atg5 group. ①P=0.000, compared with the blank group; ②P=0.000, compared with the control group; ③P=0.000, compared with the miR-30b-5p overexpression group.

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

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