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

doi:10.3969/j.issn.1674-8115.2023.01.003

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), 2023, 43(1): 20-28.

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

1	BJEKIĆ-MACUT J, VUKAŠIN T, VELIJA-AŠIMI Z, et al. Polycystic ovary syndrome: a contemporary clinical approach[J]. Curr Pharm Des, 2021, 27(36): 3812-3820.
2	ASADUZZAMAN M, RODGERS R J, YOUNG F M. Quantification of viable granulosa cells in murine ovarian follicles[J]. Biotech Histochem, 2020, 95(7): 540-554.
3	LI X X, QI J, ZHU Q L, et al. The role of androgen in autophagy of granulosa cells from PCOS[J]. Gynecol Endocrinol, 2019, 35(8): 669-672.
4	YEFIMOVA M G, LEFEVRE C, BASHAMBOO A, et al. Granulosa cells provide elimination of apoptotic oocytes through unconventional autophagy-assisted phagocytosis[J]. Hum Reprod, 2020, 35(6): 1346-1362.
5	LUO X D, GONG Y Y, CAI L Y, et al. Chemerin regulates autophagy to participate in polycystic ovary syndrome[J]. J Int Med Res, 2021, 49(11): 3000605211058376.
6	XU X Y, SONG X L, XU X H, et al. Inhibition of sestrin 1 alleviates polycystic ovary syndrome by decreasing autophagy[J]. Aging, 2021, 13(8): 11774-11785.
7	杨潇曼, 王克华, 王晓丹, 等. MiRNA-2861、miRNA-483-3p与多囊卵巢综合征患者内分泌代谢及卵母细胞质量的相关性[J]. 现代妇产科进展, 2021, 30(9): 666-669.
	YANG X M, WANG K H, WANG X D, et al. Correlation of miRNA-2861, miRNA-483-3p with endocrine metabolism and oocyte quality in patients with polycystic ovary syndrome[J]. Progress in Obstetrics and Gynecology, 2021, 30(9):666-669.
8	CHEN Q, LI Z, XU Z, et al. MiR-378d is involved in the regulation of apoptosis and autophagy of and E2 secretion from cultured ovarian granular cells treated by sodium fluoride[J]. Biol Trace Elem Res, 2021, 199(11): 4119-4128.
9	GUO H J, PU M, TAI Y S, et al. Nuclear miR-30b-5p suppresses TFEB-mediated lysosomal biogenesis and autophagy[J]. Cell Death Differ, 2021, 28(1): 320-336.
10	SUN B, OU H, REN F, et al. Propofol protects against cerebral ischemia/reperfusion injury by down-regulating long noncoding RNA SNHG14[J]. ACS Chem Neurosci, 2021, 12(16): 3002-3014.
11	LIU Z Q, WEI X Q, ZHANG A Q, et al. Long non-coding RNA HNF1A-AS1 functioned as an oncogene and autophagy promoter in hepatocellular carcinoma through sponging hsa-miR-30b-5p[J]. Biochem Biophys Res Commun, 2016, 473(4): 1268-1275.
12	邵梅, 王家传. 淫羊藿素对多囊卵巢综合征颗粒细胞凋亡和自噬的影响[J]. 中国临床药理学杂志, 2021, 37(20): 2830-2833.
	SHAO M, WANG J C. Effect of icariin on apoptosis and autophagy of granulosa cells in polycystic ovary syndrome[J]. The Chinese Journal of Clinical Pharmacology, 2021, 37(20): 2830-2833.
13	YE H, LIU X J, HUI Y, et al. Downregulation of microRNA-222 reduces insulin resistance in rats with PCOS by inhibiting activation of the MAPK/ERK pathway via pten[J]. Mol Ther Nucleic Acids, 2020, 22: 733-741.
14	SHEN H R, WANG Y. Activation of TGF-β1/Smad3 signaling pathway inhibits the development of ovarian follicle in polycystic ovary syndrome by promoting apoptosis of granulosa cells[J]. J Cell Physiol, 2019, 234(7): 11976-11985.
15	CAI F Z, LI J W, ZHANG J Y, et al. Knockdown of Circ_CCNB2 sensitizes prostate cancer to radiation through repressing autophagy by the miR-30b-5p/KIF18A axis[J]. Cancer Biother Radiopharm, 2022, 37(6): 480-493.
16	LIU M, ZHU H Q, ZHU Y, et al. Guizhi Fuling Wan reduces autophagy of granulosa cell in rats with polycystic ovary syndrome via restoring the PI3K/AKT/mTOR signaling pathway[J].J Ethnopharmacol, 2021, 270: 113821.

[1]	DING He, CAO Yanglin, HE Yang, WEI Xing, YANG Jianfeng. SMAD7 expression in multiple myeloma and its effect on cell proliferation and drug resistance [J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(7): 858-865.
[2]	HU Zhexuan, ZHANG Xin, WO Lulu, LI Jingchi, WANG Jiao, ZHOU Cixiang, ZHAO Qian. Study on the function of TRMT61A in liver cancer cell and its mechanism [J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(6): 742-750.
[3]	WANG Xinpeng, WANG Junying, CAI Jiayi, FU Wanbing, ZHONG Hua. Effect of low-dose decitabine on the biological behavior of bone marrow mesenchymal stem cells derived from patients with immune thrombocytopenia [J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(6): 758-767.
[4]	XU Feixiang, WANG Sheng, XUE Mingming, TONG Chaoyang, CHEN Yumei. Effect of altered expression of long non-coding RNA-B230352I09 on proliferation and cycle of H9C2 cardiomyocytes [J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(5): 578-582.
[5]	Ming CHEN, Jing ZHANG. Inhibitory effect of sanguinarine on proliferaton and invasion of gastric cancer cells by upregulating m⁶A methyltransferase 14 [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2022, 42(2): 135-141.
[6]	SUN Jinli, SONG Weiwei, XU Ming, LI Jingquan. Oxidative damage and malignant migration of hepatocellular carcinoma cells LM3 induced by 14 weeks exposure to sodium arsenite [J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(12): 1677-1684.
[7]	DU Lin, HE Ping. Effect of Leptospira interrogans 56606v on apoptosis of murine neutrophil [J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(11): 1517-1523.
[8]	YUAN Yongmei, CHENG Xiaodan, SUN Jiaan, CHANG Dongge, HE Yingying, LIU Chang. lncRNA NEAT1 affects the proliferation, invasion and migration of ox-LDL-induced human vascular smooth muscle cells through miR-377-3p/Wnt pathway [J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(11): 1534-1541.
[9]	Jian ZHANG, Fei SONG, Xiqiao WANG. Role of autosis of fibroblasts in hypertrophic scar regression [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2022, 42(1): 44-50.
[10]	Yi LI, Da-wei SUN, De-rong CUI. Research progress in the relationship between endosomal sorting complexes required for transport and autophagy [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(9): 1256-1260.
[11]	Bing-qian ZHOU, Li HAN, Zhe-yi CHEN, Shi-yu CHEN, Ying-xia ZHENG. Expression of protein arginine methyltransferase 5 in lung cancer and its mechanism of promoting lung cancer [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(8): 1009-1016.
[12]	Yan-qi HE, Rui CHI, Meng-ping CHEN, Si CHEN, Chun-liang LIU, Yun-xia LIU, Hai-peng SUN. Function of branched-chain amino acid catabolism in lung cancer cells [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(7): 858-864.
[13]	Xu TONG, Lin-jing SHU. Effect of miR-877-3p on proliferation of bone marrow mesenchymal stem cells in osteoporosis [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(7): 884-890.
[14]	Jing-jing LIU, Hui-jie HU, Zi-kai LIU, Ming-ke SONG. Suppressing effect of the Na⁺/Ca²⁺ exchanger blocker CB-DMB on the growth of human glioblastoma cells [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(6): 710-716.
[15]	Meng-di LIU, Yi-lian PAN, Xiao-na ZHU, Shuo YANG, Qian YANG, Yun YU. Molecular mechanism of apoptosis-inducing factor regulated by FBXO22 and its role in cancer cell apoptosis [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(4): 427-433.