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  

GnIH/RFRP-3 Inhibits PI3K/Akt/MTOR Pathway Through GPR147 and Induces Apoptosis of Estrogen Receptor Positive Human Breast Cancer MCF-7 Cells

作　　者：王凤霞 魏萌[1] 司丽娜[1] 杨松鹤[1] 程露阳[1] 乔跃兵[1] 马秀艳[2] WANG Fengxia;WEI Meng;SI Lina(Chengde Medical University, Hebei Chengde 067000, China)

机构地区：[1]承德医学院,河北承德067000 [2]承德医学院附属医院,河北承德067000

出　　处：《河北医学》2021年第11期1761-1766,共6页Hebei Medicine

基　　金：国家自然科学基金项目,(编号:81602318);河北省自然科学基金资助,(编号:H2013406115);河北省科学技术研究与发展计划,(编号:08276101D-20);河北省高等学校科学研究,(编号:QN2015121);河北省高校人体解剖学与组织胚胎学重点发展学科建设项目资助,(编号:冀教高[2013]4号)。

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GnIH10000ng/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:①CCK-8结果显示:GnIH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/pAKT、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�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:①Human 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.②The cells were divided into PBS control group,DMSO control group,GnIH group and GnIH+RF9 inhibitor group for 24 hours.PBS 15μL,DMSO 3μL,GnIH 10000 ng/mL and GnIH 10000 ng/mL+RF910μmol 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

关 键 词：促性腺激素抑制激素 人乳腺癌MCF-7细胞 细胞凋亡 G蛋白耦联受体147 

分 类 号：R73[医药卫生—肿瘤]