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. 2020 Aug 15;12(8):4659-4668.
eCollection 2020.

Effect of rNA interference on Oatp3a1 gene expression on biological characteristics and immune factors of ovarian granulosa cells in rats with PCOS

Weimin Yi  1 , Xue Li  1 , Ke Chen  1 , Jianjun Li  1 , Kefang Chen  1 , Aizhen Pan  1
Affiliations

Effect of rNA interference on Oatp3a1 gene expression on biological characteristics and immune factors of ovarian granulosa cells in rats with PCOS

Weimin Yi et al. Am J Transl Res. .

Abstract

Polycystic ovary syndrome (PCOS) is a common endocrinal metabolic disease, and its pathogenesis has not yet been thoroughly studied. The purpose of this experiment was to investigate the effect of RNA interference on Oatp3a1 gene expression on the biological viability and immune factors of ovarian granulosa cells in rats with PCOS. First, rats were intragastrically administered 1 mg/kg letrozole to successfully construct PCOS model. Western blot, qRT-PCR, CCK8 and flow cytometry were used to detect the gene expression, immune factor protein expression, cell proliferation and apoptosis in ovarian granulosa cells transfected with siRNA Oatp3a1 in rats with PCOS, respectively. The results showed that follicle stimulating hormone receptor (FSHR) was located on the cell membrane of rat ovarian granulosa cells, and letrozole successfully induced PCOS rat model. In PCOS rat ovarian granulosa cells, the mRNA expression level of Oapta1 was higher than that in normal rat ovarian granulosa cells. At the same time, compared with the sham group, the protein expression of NF-κB, TGF-β1 and VEGF in si-Oatp3a1 group was significantly down-regulated (P < 0.05), and the cell proliferation rate was significantly decreased in si-Oatp3a1 group (P < 0.05) in comparison with the sham group. The apoptotic rate was increased obviously (P < 0.05), which was about 2.5 times that of the sham group. This indicates that in the ovarian granulosa cells of rats with PCOS, the interference of Oatp3a1 gene expression can significantly inhibit cell proliferation and promote apoptosis, while inhibiting the expression of immune factors TGF-β1 and VEGF can reduce the expression of NF-κB protein, thereby suppressing the activation of the NF-κB signaling pathway.

Keywords: NF-κB; Oatp3a1; ovarian granulosa cells in rats; polycystic ovary syndrome.

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Conflict of interest statement

None.

Figures

Figure 1
Figure 1
Ovarian volume and body weight of rats in PCOS group, control group and normal group. A. Ovarian volume of rats in PCOS group, control group and normal group. B. Body weight of rats in PCOS group, control group and normal group. *P < 0.05, ***P < 0.001.
Figure 2
Figure 2
Changes of hormone level in PCOS group, control group and normal group. After treatment with letrozole for 21 days, serum levels of (A) progesterone (P), (B) estradiol (E2), (C) follicle stimulating hormone (FSH), (D) testosterone (T), (E) luteinizing hormone (LH) were detected in PCOS group, control group and normal group. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3
Figure 3
Purity identification of primary rat ovarian granulosa cells. A. Immunofluorescence was used to detect granulosa cells. Blue: DAPI nuclear staining. Red: FSHR antibody staining. Merge: nucleus and FSHR overlap. B. Morphological observation of normal-GC group and PCOS-GC group.
Figure 4
Figure 4
Oatp3a1 mRNA levels are up-regulated in PCOS rat ovarian granulosa cells. A. RT-qPCR was used to detect the mRNA expression of Oatp3a1 in normal-GC group and PCOS-GC group. B. Validation of the transfection efficiency of Oatp3a1 siRNA. C. The transfection efficiency of si-Oatp3a was detected by RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5
si-Oatp3a1 inhibits proliferation of rat ovarian granulosa cells. A. Rat ovarian granulosa cells were transfected with Oatp3a1 interfering RNA, and the OD values from 0 to 3 days of cells in each group were detected by CCK-8. B. The appreciation rate was calculated from the OD values of ovarian granulosa cells in each group from 0 to 3 days. The data represents mean ± SD and each set of experiments was repeated at least three times. *P < 0.05, **P < 0.01.
Figure 6
Figure 6
si-Oatp3a1 promotes the cell apoptosis of rat ovarian granulosa cells. The data represents mean ± SD and each set of experiments was repeated at least three times. **P < 0.01, ***P < 0.001.
Figure 7
Figure 7
Effects of Oatp3a1 on the expression of NF-κB, VEGF and TGF-β1. Western blot was used to detect the protein expression of NF-κB, VEGF and TGF-β1 in rat ovarian granulosa cells transfected with Oatp3a1 siRNA (si-Oatp3a1), siRNA NC (si-NC). The protein expression level is calculated as the gray value of each protein band, and the data represents mean ± SD. Each set of experiments was repeated at least three times. **P < 0.01.
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