circRAF1调节人卵巢颗粒细胞的增殖与凋亡

doi:10.3969/j.issn.1006-5725.2024.07.006

Abstract

Abstract:

Objective To investigate the expression of circRAF1 in primary ovarian insufficiency （POI） and explore its effect on cell proliferation and apoptosis of human ovarian granulosa cells （GCs） line （KGN cells）. Methods The expression of circRAF1 in GCs and serum of patients with normal ovarian reserve function （n = 50） and patients with POI （n = 50） were detected with RT-qPCR. The correlation of circRAF1 with ovarian reserve function indexes was analyzed. Small interfering RNA （siRNA） targeting circRAF1 was constructed and transfected into KGN cells， with the cell proliferation detected by CCK-8 and EdU assay， and the cell apoptosis detected by JC-1 and Tunel assay. The mRNA and protein levels of genes related to cell proliferation and apoptosis （FSHR， PCNA， Bcl-2， Casp-9， Bax） were detected by RT-qPCR and WB. Results The expression of circRAF1 decreased in GCs and serum of POI patients. The expression of circRAF1 was positively correlated with serum E₂ and AMH levels （P < 0.001）， but negatively correlated with serum FSH and LH levels （P < 0.001）. At the same time， the expression of circRAF1 was positively correlated with AFC （P < 0.001）. Interfering with the expression of circRAF1 could inhibit the proliferation of KGN cells and promote their apoptosis. Conclusion The expression of circRAF1 in the GCs and serum of POI patients is down-regulated， which is correlated with the decline of ovarian reserve function. Interfering with circRAF1 can inhibit the proliferation of GCs and promote their apoptosis.

Key words: circRAF1, POI, GCs, ovarian reserve, cell proliferation and apoptosis

CLC Number:

R711.75

Wenxin LI,Minjun LU,Li LIN,Yueqin LIU,Xiaolan. ZHU. circRAF1 regulates the proliferation and apoptosis of human ovarian granulosa cells[J]. The Journal of Practical Medicine, 2024, 40(7): 910-917.

Figures/Tables 6

Tab.1

Tab. 2

Fig.1

Fig.2

Fig.3

Fig.4

References 38

1	CHON S J， UMAIR Z， YOON M S. Premature Ovarian Insufficiency： Past， Present， and Future［J］.Front Cell Dev Biol， 2021， 9： 672890. doi:10.3389/fcell.2021.672890 doi: 10.3389/fcell.2021.672890
2	ZHANG X， ZHANG R， HAO J， et al. miRNA‐122‐5p in POI ovarian‐derived exosomes promotes granulosa cell apoptosis by regulating BCL9［J］. Cancer Med， 2022， 11（12）： 2414-2426. doi:10.1002/cam4.4615 doi: 10.1002/cam4.4615
3	HOSSEINISADAT R， FARSI NEJAD A， MOHAMMADI F. Intra-ovarian infusion of autologous platelet-rich plasma in women with poor ovarian reserve： A before and after study［J］. Eur J Obstet Gynecol Reprod Biol， 2023， 280： 60-63. doi:10.1016/j.ejogrb.2022.11.001 doi: 10.1016/j.ejogrb.2022.11.001
4	DING C， QIAN C， HOU S， et al. Exosomal miRNA-320a Is Released from hAMSCs and Regulates SIRT4 to Prevent Reactive Oxygen Species Generation in POI［J］. Mol Ther Nucleic Acids， 2020， 21： 37-50. doi:10.1016/j.omtn.2020.05.013 doi: 10.1016/j.omtn.2020.05.013
5	LIU M， ZHANG D， ZHOU X， et al. Cell-free fat extract improves ovarian function and fertility in mice with premature ovarian insufficiency［J］. Stem Cell Res Ther， 2022， 13： 320. doi:10.1186/s13287-022-03012-w doi: 10.1186/s13287-022-03012-w
6	WANG C， SUN H， DAVIS J S， et al. FHL2 deficiency impairs follicular development and fertility by attenuating EGF/EGFR/YAP signaling in ovarian granulosa cells［J］. Cell Death Dis， 2023， 14（4）： 239. doi:10.1038/s41419-023-05759-3 doi: 10.1038/s41419-023-05759-3
7	ZHOU J， PENG X， MEI S. Autophagy in Ovarian Follicular Development and Atresia［J］. Int J Biol Sci， 2019， 15（4）： 726-737. doi:10.7150/ijbs.30369 doi: 10.7150/ijbs.30369
8	杨延周，陈敏慧，裴承斌. miR-23a-3p通过调控靶基因Gja1参与调控大鼠卵泡闭锁及颗粒细胞凋亡［J］. 生殖医学杂志， 2021， 30（11）： 1486-1493. doi:10.3969/j.issn.1004-3845.2021.11.014 doi: 10.3969/j.issn.1004-3845.2021.11.014
9	KHAN A， KHAN M Z， DOU J， et al. RNAi-Mediated Silencing of Catalase Gene Promotes Apoptosis and Impairs Proliferation of Bovine Granulosa Cells under Heat Stress［J］.Animals （Basel）， 2020， 10（6）： 1060. doi:10.3390/ani10061060 doi: 10.3390/ani10061060
10	HUANG A， ZHENG H， WU Z， et al. Circular RNA-protein interactions： functions， mechanisms， and identification［J］. Theranostics， 2020， 10（8）： 3503-3517. doi:10.7150/thno.42174 doi: 10.7150/thno.42174
11	JU X， TANG Y， QU R， et al. The Emerging Role of Circ-SHPRH in Cancer［J］. Onco Targets Ther， 2021， 14： 4177-4188. doi:10.2147/ott.s317403 doi: 10.2147/ott.s317403
12	ZHAO C， LV Y， DUAN Y， et al. Circulating Non-coding RNAs and Cardiovascular Diseases［J］. Adv Exp Med Biol， 2020， 1229： 357-367. doi:10.1007/978-981-15-1671-9_22 doi: 10.1007/978-981-15-1671-9_22
13	DONG J， ZENG Z， HUANG Y， et al. Challenges and opportunities for circRNA identification and delivery［J］. Crit Rev Biochem Mol Biol， 2023， 58（1）： 19-35. doi:10.1080/10409238.2023.2185764 doi: 10.1080/10409238.2023.2185764
14	KRISTENSEN L S， JAKOBSEN T， HAGER H， et al. The emerging roles of circRNAs in cancer and oncology［J］. Nat Rev Clin Oncol， 2022， 19（3）： 188-206. doi:10.1038/s41571-021-00585-y doi: 10.1038/s41571-021-00585-y
15	YU C Y， KUO H C. The emerging roles and functions of circular RNAs and their generation［J］. J Biomed Sci， 2019， 26： 29. doi:10.1186/s12929-019-0523-z doi: 10.1186/s12929-019-0523-z
16	熊英，徐克惠. 早发性卵巢功能不全的早期诊断和预防［J］. 中国实用妇科与产科杂志， 2023， 39（9）： 906-909.
17	HOLESH J E， BASS A N， Physiology LORD M.， Ovulation［M］//StatPearls. Treasure Island （FL）： StatPearls Publishing， 2023.
18	HE Y， CHEN Q， DAI J， et al. Single‐cell RNA‐Seq reveals a highly coordinated transcriptional program in mouse germ cells during primordial follicle formation［J］. Aging Cell， 2021， 20（7）： e13424. doi:10.1111/acel.13424 doi: 10.1111/acel.13424
19	LI D， WANG X， LI G， et al. LncRNA ZNF674-AS1 regulates granulosa cell glycolysis and proliferation by interacting with ALDOA［J］. Cell Death Discov， 2021， 7（1）： 107. doi:10.1038/s41420-021-00493-1 doi: 10.1038/s41420-021-00493-1
20	徐昊，方梦蝶，李超，等. 新型肿瘤靶标环状RNA的研究进展［J］. 中国医学科学院学报， 2021， 43（3）： 435-444.
21	HUANG Y， ZHANG Y， JIA L， et al. Circular RNA ABCB10 promotes tumor progression and correlates with pejorative prognosis in clear cell renal cell carcinoma［J］. Int J Biol Markers， 2019， 34（2）： 176-183. doi:10.1177/1724600819842279 doi: 10.1177/1724600819842279
22	SHAO Y， TAO X， LU R， et al. Hsa_circ_0065149 is an Indicator for Early Gastric Cancer Screening and Prognosis Prediction［J］. Pathol Oncol Res， 2020， 26（3）： 1475-1482. doi:10.1007/s12253-019-00716-y doi: 10.1007/s12253-019-00716-y
23	LI J， HAN T， LI Z， et al. A Novel circRNA hsa_circRNA_002178 as a Diagnostic Marker in Hepatocellular Carcinoma Enhances Cell Proliferation， Invasion， and Tumor Growth by Stabilizing SRSF1 Expression［J］. J Oncol， 2022， 2022： 4184034. doi:10.1155/2022/4184034 doi: 10.1155/2022/4184034
24	WANG Y， PEI L， YUE Z， et al. The Potential of Serum Exosomal hsa_circ_0028861 as the Novel Diagnostic Biomarker of HBV-Derived Hepatocellular Cancer［J］. Front Genet， 2021， 12： 703205. doi:10.3389/fgene.2021.703205 doi: 10.3389/fgene.2021.703205
25	李文强，刘慧颖，任卫东. 干扰Hsa＿circ＿0023642调控miR-653对甲状腺癌细胞生物行为的影响［J］. 实用医学杂志， 2021， 37（21）： 2727-2732. doi:10.3969/j.issn.1006-5725.2021.21.005 doi: 10.3969/j.issn.1006-5725.2021.21.005
26	WANG L P， PENG X Y， LV X Q， et al. High throughput circRNAs sequencing profile of follicle fluid exosomes of polycystic ovary syndrome patients［J］. J Cell Physiol， 2019， 234（9）： 15537-15547. doi:10.1002/jcp.28201 doi: 10.1002/jcp.28201
27	MA Z， ZHAO H， ZHANG Y， et al. Novel circular RNA expression in the cumulus cells of patients with polycystic ovary syndrome［J］. Arch Gynecol Obstet， 2019， 299（6）： 1715-1725. doi:10.1007/s00404-019-05122-y doi: 10.1007/s00404-019-05122-y
28	XING J， ZHANG M， ZHAO S， et al. EIF4A3-Induced Exosomal circLRRC8A Alleviates Granulosa Cells Senescence Via the miR-125a-3p/NFE2L1 axis［J］. Stem Cell Rev Rep， 2023， 19（6）： 1994-2012. doi:10.1007/s12015-023-10564-8 doi: 10.1007/s12015-023-10564-8
29	SUN X， LUO L， GAO Y. Circular RNA PVT1 enhances cell proliferation but inhibits apoptosis through sponging microRNA-149 in epithelial ovarian cancer［J］. J Obstet Gynaecol Res， 2020， 46（4）： 625-635. doi:10.1111/jog.14190 doi: 10.1111/jog.14190
30	LUO X， LIU H， GUO H， et al. RAF1 mediates the FSH signaling pathway as a downstream molecule to stimulate estradiol synthesis and secretion in mouse ovarian granulosa cells［J］. Ann Transl Med， 2022， 10（6）： 314. doi:10.21037/atm-22-393 doi: 10.21037/atm-22-393
31	LI Y， PAN M， LU T， et al. RAF1 promotes lymphatic metastasis of hypopharyngeal carcinoma via regulating LAGE1： an experimental research［J］. J Transl Med， 2022， 20（1）： 255. doi:10.1186/s12967-022-03468-7 doi: 10.1186/s12967-022-03468-7
32	HUANG X， WU B， CHEN M， et al. Depletion of exosomal circLDLR in follicle fluid derepresses miR-1294 function and inhibits estradiol production via CYP19A1 in polycystic ovary syndrome［J］. Aging， 2020， 12（15）： 15414-15435. doi:10.18632/aging.103602 doi: 10.18632/aging.103602
33	JIA C， WANG S， YIN C， et al. Loss of hsa_circ_0118530 inhibits human granulosa-like tumor cell line KGN cell injury by sponging miR-136［J］. Gene， 2020， 744： 144591. doi:10.1016/j.gene.2020.144591 doi: 10.1016/j.gene.2020.144591
34	ZHAO C， ZHOU Y， SHEN X， et al. Circular RNA expression profiling in the fetal side of placenta from maternal polycystic ovary syndrome and circ_0023942 inhibits the proliferation of human ovarian granulosa cell［J］. Arch Gynecol Obstet， 2020， 301（4）： 963-971. doi:10.1007/s00404-020-05495-5 doi: 10.1007/s00404-020-05495-5
35	CHEN Y， MIAO J， LOU G. Knockdown of circ-FURIN suppresses the proliferation and induces apoptosis of granular cells in polycystic ovary syndrome via miR-195-5p/BCL2 axis［J］. J Ovarian Res， 2021， 14（1）： 156. doi:10.1186/s13048-021-00891-0 doi: 10.1186/s13048-021-00891-0
36	PEREIRA A L， MAGALHÃES L， PANTOJA R P， et al. The Biological Role of Sponge Circular RNAs in Gastric Cancer： Main Players or Coadjuvants？［J］. Cancers， 2020， 12（7）： 1982. doi:10.3390/cancers12071982 doi: 10.3390/cancers12071982
37	吴田方，戴东，王常元. circ-0008583结合EIF4A3蛋白促进肝癌细胞增殖并抑制其凋亡［J］. 实用医学杂志， 2022， 38（9）： 1088-1093. doi:10.3969/j.issn.1006-5725.2022.09.009 doi: 10.3969/j.issn.1006-5725.2022.09.009
38	LEI M， ZHENG G， NING Q， et al. Translation and functional roles of circular RNAs in human cancer［J］. Mol Cancer， 2020， 19（1）： 30. doi:10.1186/s12943-020-1135-7 doi: 10.1186/s12943-020-1135-7

项目	NC组（n = 50）	POI组（n = 50）	P值
年龄（岁）	31.50 ± 5.50	33.00 ± 5.00	0.458
BMI（kg/m²）	21.10 ± 2.84	21.53 ± 2.57	0.513
AMH（ng/mL）	4.94 ± 3.90	0.50 ± 0.49	0.001
E₂（pmol/L）	181.00 ± 91.00	80.50 ± 67.50	0.001
FSH（IU/L）	6.26 ± 3.68	32.19 ± 7.11	0.001
LH（IU/L）	6.58 ± 4.33	22.55 ± 11.52	0.001
AFC（个）	12.50 ± 5.50	5.00 ± 4.00	0.001

基因	上游引物	下游引物
circRAF1	5′-CAGAGGCAGAGGTCGACATC-3′	5′-AAGGTAGTGCTGGGACTCCA-3′
发散引物	5′-TGAAGGTTCCCTCTCCCAGA-3′	5′-AAGGTGAAGGCGTGAGGTGT-3′
聚合引物	5′-GGTGATAGTGGAGTCCCAGCA-3′	5′-GTGGTGCTGACCATGTGGA-3′
RAF1	5′-GTGCTGACCATGTGGACATTAGG-3′	5′-GATATTCTACACCTCACGCCTTCAC-3′
PCNA	5′-TGATGAGGTCCTTGAGTG-3′	5′-GAGTGGTCGTTGTCTTTC-3′
FSHR	5′-GGCCATGCTCATCTTCACTG-3′	5′-ATAGAGGAAGGGGTTGGCAC-3′
Bcl-2	5′-GCCTTCTTTGAGTTCGGTGGG-3′	5′-TGCCGGTTCAGGTACTCAGTCATC-3′
Bax	5′-TCCACAGAGCGATGTTGTCC-3′	5′-TCGCGACTTTGCAGAGATGT-3′
Casp-3	5′-CGTGTCATAAAATACCAGTGGA-3′	5′-AAATTCTGTTGCCACCTTTCG-3′
GAPDH	5′-CAGGAGGCATTGCTGATGAT-3′	5′-GAAGGCIGGGGCICATTT-3′
U6	5′-AGAGAAGATTAGCATGGCCCCTG-3	5′-ATCCAGTGCAGGGICCGAGG-3′

[1]	Yuxuan DING,Lining GUO,Jiayi SHEN,Lijun. WANG. Safety and efficacy of radiotherapy and PD⁃1/PD⁃L1 inhibitor + TKI for MSS/pMMR colorectal cancer with liver metastases [J]. The Journal of Practical Medicine, 2024, 40(9): 1293-1297.
[2]	Xiaona MENG,Xu SUN,Huaimin LIU. Advances in the study of immune checkpoint inhibitors⁃related colitis [J]. The Journal of Practical Medicine, 2024, 40(9): 1314-1319.
[3]	Rong SHI,Xianfang ZHENG,Qun TAO,Junmo CHEN,Xiaohua GE. Effect of laparoscopic salpingectomy on ovarian reserve function and serum neuropeptide Y and cortisol in patients with ectopic pregnancy [J]. The Journal of Practical Medicine, 2024, 40(6): 801-806.
[4]	Jiang SHAO,Lin LI,Yansong GUO,Chengyuan SUN,Xichao WEN,Kebin ZHENG,Yanfang SHI. Research progress of CD73/NT5E in glioblastoma [J]. The Journal of Practical Medicine, 2024, 40(3): 428-431.
[5]	Kun PENG,Baozhong YAO,Hongcun CHEN,Jun ZHANG,Wenzhong BAO,Wenbo LI,Weitao SONG,Sailong SANG,Li LIN,Zhixing JIA,Liang LI. Efficacy of autofluorescence point⁃spectral analysis combined with the immune colloidal gold technique for the detection of ectopic microscopic parathyroid glands to guide surgery for secondary hyperparathyroidism [J]. The Journal of Practical Medicine, 2024, 40(20): 2905-2912.
[6]	Peiqian LAI,Pei HU,Hongshen WANG,Yanhua ZHENG,Meiyan LAN,Shaohua CHEN. Effect of acupoint sticking therapy along meridians on gastrointestinal function recovery in patients after lumbar internal fixation surgery [J]. The Journal of Practical Medicine, 2024, 40(2): 267-271.
[7]	Yuqiao ZHANG,Weijian MEI. Landmark Achievements in Treating Solid Tumors with Immune Checkpoint Inhibitors [J]. The Journal of Practical Medicine, 2024, 40(2): 272-277.
[8]	Guojun CHEN,Tianen ZHOU,Hongfeng LIU,Li′na PENG,Jun JIANG,Chunming. XIE. Diagnostic value of POCT⁃cTnT for acute myocardial infarction based on symptom onset time [J]. The Journal of Practical Medicine, 2024, 40(16): 2326-2332.
[9]	Shengfang YUAN,Bu WANG,Baoli XIANG,Jianqing ZHAO,Jingjing SHEN,Zhihua. ZHANG. Prediction of immune therapy efficacy and prognosis for advanced non-small cell lung cancer using peripheral blood circulation tumor DNA [J]. The Journal of Practical Medicine, 2024, 40(15): 2110-2115.
[10]	Xuejian MA,Dongzhe ZHANG,Chaoyang NIU,Shuaigang DU,Wuyang WANG,Shilong CHENG,Yingyuan XIAO,Xuechang. WANG. A clinical study on the treatment of refractory tennis elbow with arc-edge needle therapy based on zaodian theory [J]. The Journal of Practical Medicine, 2024, 40(15): 2161-2165.
[11]	Yapeng LIANG,Chaopu ZHANG,Hao ZHANG,Zhongqun. WANG. The relationship between the expression of serum ANGPTL8 and KLF2 and the degree of coronary artery disease and the occurrence of major adverse cardiac events in patients with acute myocardial infarction [J]. The Journal of Practical Medicine, 2024, 40(13): 1827-1832.
[12]	Huinan ZHOU,Kewei QIN,Lijun. ZHOU. Current research status and clinical application progress of the immune checkpoint LAG⁃3 and its targeting drugs [J]. The Journal of Practical Medicine, 2024, 40(11): 1607-1612.
[13]	Zijuan FU,Qian LI,Lin LU,Yongqiu. LI. Relationship of serum ANGPTL3 and NFATc1 levels with the severity and prognosis in cerebral infarction patients [J]. The Journal of Practical Medicine, 2024, 40(10): 1407-1411.
[14]	Dan LIANG,Ying YI,Weifu HUANG,Xiansheng. NONG. A retrospective study on multi⁃targeted small molecule tyrosine kinase inhibitors combined with immune checkpoint inhibitors for 21 patients with advanced solid tumors after standard treatment failure [J]. The Journal of Practical Medicine, 2024, 40(1): 102-107.
[15]	PENG Yingnan, BIAN Zhilei, CAO Weijie, LI Li, ZHANG Suping, WAN Dingming.. Efficacy and safety study of posaconazole and voriconazole for the prevention of invasive fungal infections after allogeneic hematopoietic stem cell transplantation [J]. The Journal of Practical Medicine, 2023, 39(6): 742-746.

circRAF1 regulates the proliferation and apoptosis of human ovarian granulosa cells

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 6

References 38

Related Articles 15

Recommended Articles

Metrics

Comments