GnIH/RFRP-3通过受体GPR147抑制PI3K/AKT/mTOR通路诱导雌激素受体阳性人乳腺癌MCF-7细胞凋亡的研究

doi:10.3969/j.issn.1006-6233.2021.11.01

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Abstract Objective: To investigate the effects of gonadotropin inhibitory hormone (GnIH) on proliferation, apoptosis and PI3K/Akt/mTOR signaling pathway of human breast cancer MCF-7 cells based on GPR147 receptor. Methods: ① Human breast cancer cell line MCF-7 was cultured in vitro and divided into blank control group, control group and GnIH experimental group (0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL, 10000ng/mL). CCK-8 method was used to detect the cell proliferation; flow cytometry was used to detect the apoptosis rate; real time fluorescent quantitative PCR (QRT-PCR) and Western blot wereused to detect the effect of GnIH on apoptosis related proteins (Bcl-2, Bax, cytochrome C) at mRNA and protein levels . The expression of PI3K, Akt/p-Akt and mTOR proteins in Caspase-3, and p53 and PI3K/Akt/mTOR signaling pathways were detected. Western blot was used to detect the expression of GPR147 in MCF-7 cell line.② The cells were divided into PBS control group, DMSO control group, GnIH group and GnIH+RF9 inhibitor group for 24 hours. PBS 15 μL, DMSO 3μL, GnIH 10000 ng/mL and GnIH 10000 ng/mL+RF9 10μmol were added to the cells for 24 hours. The expression of Akt/p-Akt protein was detected by Western blot. Results: CCK-8 results showed that the GnIH experimental group 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL cells compared with the control group, the difference was not statistically significant (P>0.05); Compared with the control group, GnIH10000ng/mL in the experimental group has a statistically significant difference (P<0.05). The apoptotic rates of control group, 1000ng/mL and 10000ng/mL were (7.76±1.57)% and (16.14±3.001)%, respectively, with significant difference (F=6.164, P=0.0351). The expressions of Bax, cytochrome c, caspase-3 and p53 were significantly up-regulated (P<0.05), while the expressions of Bcl-2 were significantly down regulated (P<0.05). In PI3K/Akt/mTOR signaling pathway, the protein expression of PI3K, Akt/pAkt and mTOR was significantly down regulated (P<0.01). GPR147 was found in MCF-7 cell line. When the drug concentration was 10000ng/mL, the receptor was activated compared with the control group (P<0.01). The expression of GPR147 in the experimental group was significantly higher than that in the control group (P<0.01). After the intervention, the expression of Akt/p-Akt protein was significantly higher in the group of GnIH +RF9 than in the group of GnIH (P<0.05). Conclusion: GnIH receptor GPR147 exists in breast cancer cell line MCF-7. GnIH may inhibit the proliferation and induce apoptosis of MCF-7 cells by binding to gpr147 receptor, and the occurrence of apoptosis may be related to the inhibition of PI3K/Akt/mTOR signaling pathway.

Key words： GnIH Human breast cancer MCF-7 cells Apoptosis GPR147

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http://www.hbyxzzs.cn/EN/10.3969/j.issn.1006-6233.2021.11.01 OR http://www.hbyxzzs.cn/EN/Y2021/V27/I11/1761

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