Effect of the transcription factor SOX3 on ovarian granulosa cell proliferation and estradiol secretion
10.19405/j.cnki.issn1000-1492.2024.03.001
- VernacularTitle:转录因子SOX3对卵巢颗粒细胞增殖和雌二醇分泌的影响
- Author:
Rui Cai
1
;
Hao Zhang
1
;
Zhuang Liu
1
;
Yuanhua Chen
1
;
Fenfen Xie
1
;
Qiang Hong
1
Author Information
1. Dept of Histology and Embryology, School of Basic Medical Sciences, Anhui Medical University, He fei 230032
- Publication Type:Journal Article
- Keywords:
SOX3;
ovarian granulosa cell;
lentiviral vector;
proliferation;
estradiol;
green fluorescent protein
- From:
Acta Universitatis Medicinalis Anhui
2024;59(3):371-376, 383
- CountryChina
- Language:Chinese
-
Abstract:
Objective :To study the effect of sex-determining region Y-frame protein 3 (SOX3) on proliferation and estradiol secretion in human ovarian granulosa cells (KGN cell line) .
Methods:The gene sequence of human SOX3 (NM_005634. 3) was searched in Gene-Bank , an NCBI database , and the target gene SOX3 was amplified by PCR , which was cloned into lentiviral vector pLV-EF1a-GFP-2A-Puro to obtain the overexpression lentiviral re- combinant plasmid pLV-EF1a-GFP-2A-Puro- SOX3 ; the correctly sequenced overexpressed lentiviral recombinant plasmid as well as packaging plasmids ( pGag/Pol , pRev , pVSV-G) were co-transfected into human embryonic kidney cell line ( HEK 293T) cells ( pLV-SOX3 group) , and pLV-EF1a-GFP-2A-Puro and packaging plasmids (pGag/Pol , pRev , pVSV- G) were co-transfected into HEK 293T cells (pLV-NC group) , the lentiviral particles of both groups were collected and the titers of the viruses were measured after 48 h of transfection , the lentiviruses of the two groups were infected into KGN cells , and the stably expressed cell lines were obtained after puromycin screening for 2 weeks; real-time fluorescence quantitative PCR (RT-qPCR) and Western blot were used to detect the SOX3 mRNA and protein levels in the two groups; CCK-8 assay was used to detect the proliferative ability of the cells in the two groups; ELISA was used to determine the concentration of estradiol in the two groups .
Results:The identification of PCR products and sequencing results showed that the SOX3 gene fragment was amplified successfully , and the enzyme digestion and sequencing results indicated that the construction of overexpression lentiviral recombinant plasmid was completed; green fluorescence could be detected after lentiviral infection of HEK 293T cells , which indicated that lentiviral packaging was successful; the lentivirus was screened by puromycin after lentiviral infection of KGN cells , and the cells were observed to express green fluorescence under the fluorescence microscope; RT- qPCR and Western blot assays both showed that the expression level of SOX3 in the pLV-SOX3 group was significantly higher than that in the pLV-NC group ( P < 0. 05) . CCK-8 assay results showed that the proliferation ability of the cells in the pLV-SOX3 group significantly increased compared with that in the pLV-NC
group (P < 0. 01) . ELISA results showed that estradiol concentration was elevated in the pLV-SOX3 group com- pared with the pLV-NC group (P < 0. 05) .
Conclusion:Overexpression of the transcription factor SOX3 can pro- mote the proliferation and estradiol secretion of human ovarian granulosa cells KGN .
- Full text:2024061819301771983转录因子SOX3对卵巢颗粒细胞增殖和雌二醇分泌的影响_蔡睿.pdf
