【摘要】 目的:基于G蛋白耦联受体147(GPR147)受体探讨促性腺激素抑制激素(gonadotropin inhibitory hormone,GnIH)对人乳腺癌MCF-7细胞增殖、凋亡和PI3K/AKT/mTOR信号通路的影响及分子机制。方法:(1)体外培养人乳腺癌细胞系MCF-7,设空白调零组、对照组和GnIH实验组(0.1ng/mL、1ng/mL、10ng/mL、100ng/mL、1000ng/mL、10000ng/mL)。应用CCK-8法检测细胞增殖能力;用流式细胞仪检测凋亡率;通过实时荧光定量PCR(qRT-PCR)和蛋白免疫印记分析(Western Blot),在mRNA和蛋白质水平上检测GnIH对凋亡相关蛋白(Bcl-2、Bax、cytochrome C、Caspase-3、P53)和PI3K/AKT/mTOR信号通路上PI3K、AKT/p-AKT、mTOR蛋白表达情况。Western Blot法检测GnIH的受体GPR147在MCF-7细胞系上的表达情况。(2)将细胞分为PBS对照组、DMSO对照组、GnIH组和GnIH+RF9抑制剂组培养24h,分别加入PBS15μL、DMSO3μL、GnIH10000ng/mL和Gn IH10000ng/mL+RF910μmoL进行干预,继续培养24h。Western Blot检测AKT/p-AKT蛋白表达情况。结果:(1)CCK-8结果显示:Gn IH实验组0.1ng/mL、1ng/mL、10ng/mL、100ng/mL、1000ng/mL的细胞与对照组相比,差异无统计学意义(P>0.05); GnIH10000ng/mL实验组与对照组相比,差异有统计学意义(P <0.05)。流式细胞仪检测结果:对照组和GnIH实验组10000ng/mL时的细胞凋亡率分别为(7.76±1.57)%和(16.14±3.001)%,差异有统计学意义(F=6.164,P=0.0351)。凋亡相关蛋白Bax、cytochrome C、Caspase-3蛋白表达(P<0.01)和P53蛋白表达显著上调(P <0.05),Bcl-2蛋白表达显著下调(P <0.01)。PI3K/AKT/mTOR信号通路中,PI3K、AKT/p AKT、mTOR蛋白表达显著下调(P <0.05)。MCF-7细胞系上存在GnIH的受体GRP147,当药物浓度为10000ng/mL时,与对照组比较受体激活(P<0.01)。抑制剂干预后,与GnIH组相比,GnIH+抑制剂RF9组细胞的AKT/p-AKT蛋白表达水平显著升高(P<0.05)。结论:乳腺癌细胞系MCF-7上存在GnIH受体GPR147。GnIH可能通过与GPR147受体结合抑制乳腺癌细胞MCF-7的增殖和诱导凋亡,并且凋亡的发生可能与抑制PI3K/Akt/mTOR信号通路有关。更多还原

【Abstract】 Objective: To investigate the effects of gonadotropin inhibitory hormone( Gn IH) 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. 1 ng/mL,1 ng/mL,10 ng/mL,100 ng/mL,1000 ng/mL,10000 ng/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,Gn IH group and Gn IH+RF9 inhibitor group for 24 hours. PBS 15 μL,DMSO 3μL,GnIH 10000 ng/mL and Gn IH 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 0.1 ng/mL,1 ng/mL,10 ng/mL,100 ng/mL,1000 ng/mL cells compared with the control group,the difference was not statistically significant( P >0.05); Compared with the control group,GnIH10000 ng/mL in the experimental group has a statistically significant difference( P < 0. 05). The apoptotic rates of control group,1000 ng/mL and 10000 ng/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 10000 ng/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.更多还原