hsa-miRNA-424-5p靶向GPR30对宫颈癌恶性生物学行为的影响  

Effect of hsa-miRNA-424-5p targeting GPR30 on malignant biological behavior of cervical cancer

作　　者：王佳佳 丁佳 常永霞[2] 王新生[2] 张斌[2] 刘训涛 徐丹丹 张凡[2] WANG Jiajia;DING Jia;CHANG Yongxia;WANG Xinsheng;ZHANG Bin;LIU Xuntao;XU Dandan;ZHANG Fan(Life Science Research Center,Hebei North University,Zhangjiakou 075000,China;不详)

机构地区：[1]河北北方学院生命科学研究中心,河北张家口075000 [2]河北北方学院附属第一医院病理科

出　　处：《山东医药》2021年第28期42-46,共5页Shandong Medical Journal

基　　金：河北省财政厅专科能力建设和专科带头人培养项目(361009)。

摘　　要：目的探讨hsa-miRNA-424-5p(miR-424-5p)对宫颈癌恶性生物学行为的影响及机制。方法选取12例正常宫颈组织和33例宫颈癌组织;将宫颈癌HeLa细胞随机分为NCmimics组、mimics组、NCinhibitor组、inhibitor组,分别转染miR-424-5p模拟物阴性对照、模拟物、抑制物阴性对照、抑制物序列。采用实时荧光定量PCR检测宫颈癌组织中miR-424-5p及新型雌激素受体G蛋白偶联受体30(GPR30)mRNA表达及宫颈癌细胞转染效率;平板克隆形成实验检测克隆形成能力;Transwell小室测定细胞侵袭能力;AnnexinV-PE/7-AAD双染色法测算细胞凋亡率;双荧光素酶实验初步验证miR-424-5p与GPR30的靶向关系;Westernblotting法进一步证实miR-424-5p和GPR30的靶向关系并检测PI3K/AKT/mTOR通路相关蛋白。结果实时荧光定量PCR实验结果显示,宫颈癌组织较正常宫颈组织中miR-424-5p相对表达量减少,而GPR30 mRNA表达增加(P均<0.05)。转染24 h后,NCmimics组、mimics组、NCinhibitor组、inhibitor组miR-424-5p相对表达量分别为1.00±0.06、10.91±0.42、1.00±0.03、0.01±0.00,细胞克隆率分别为(36.87±0.35)%、(17.43±1.40)%、(14.67±0.65)%、(21.23±0.68)%,细胞侵袭数分别为(179±9)、(71±5)、(133±5)、(221±11)个,细胞凋亡率分别为(12.73±0.31)%、(18.00±1.65)%、(22.37±2.17)%、(15.77±1.88)%,GPR30蛋白表达分别为1.00±0.03、0.72±0.03、1.00±0.11、2.55±0.13,p-AKT/AKT分别为1.00±0.07、0.84±0.02、1.00±0.05、1.17±0.08,p-mTOR/mTOR分别为1.00±0.08、0.65±0.14、1.00±0.05、1.43±0.25,mimics组与NCmimics组相比,inhibitor组与NCinhibitor组相比,P均<0.01。双荧光素酶证实GPR30为miR-424-5p的直接靶基因。结论miR-424-5p在宫颈癌中发挥抑癌基因的作用,过表达miR-424-5p能够抑制宫颈癌细胞的克隆、侵袭,并促进细胞凋亡;miR-424-5p可能通过GPR30作用于PI3K/AKT/mTOR通路从而抑制宫颈癌的恶性生物学行为。Objective To investigate the effect of hsa-miRNA-424-5p(miR-424-5p)on the malignant biological behavior of cervical cancer and its mechanism.Methods Twelve cases of normal tissues and 33 cases of cervical cancer tissues were selected as the control group and cancer group,respectively.The cervical cancer HeLa cells were randomly divided into four groups:NC mimics group,mimics group,NC inhibitor group,and inhibitor group,which were transfected with miR-424-5p mimics negative control,miR-424-5p mimics,miR-424-5p inhibitor negative control and miR-424-5p inhibitor,respectively.The qRT-PCR was used to verify the expression of miR-424-5p and G protein-coupled estrogen receptor 30(GPR30)mRNA in the cervical cancer tissues and transfection efficiency of cervical cancer cells.The cloning ability was detected by plate colony formation experiment.The invasion ability was detected by Transwell invasion assay.Besides,Annexin V-PE/7-AAD double staining was used to observe the apoptosis rate.Dual-luciferase assay was used to preliminarily identify the targeted relationship between miR-424-5p and GPR30.Western blotting was used to further identify the targeted relationship between miR-424-5p and GPR30,and detect the protein expression of PI3K/AKT/mTOR signaling pathway.Results The expression level of miR-424-5p in the cancer group was significantly lower than that in the control group,whereas the GPR30 mRNA expression was higher.After 24-hour transfection,the expression levels of miR-424-5p in the NC mimics group,mimics group,NC inhibitor group,and inhibitor group were 1.00±0.06,10.91±0.42,1.00±0.03,and 0.01±0.00,respectively;the clonal formation rates in the four groups were 36.87%±0.35%,17.43%±1.40%,14.67%±0.65%,and 21.23%±0.68%,respectively;the number of cells penetrating into matrigel were 179±9,71±5,133±5,and 221±11,respectively;the apoptosis rates were 12.73%±0.31%,18.00%±1.65%,22.37%±2.17%,and 15.77%±1.88%,respectively;the expression levels of GPR30 protein were 1.00±0.03,0.72±0.03,1.00±0.11,2.55±0.13,

关 键 词：宫颈癌 微小RNA-424-5p 细胞克隆 细胞侵袭 细胞凋亡 

分 类 号：R711.74[医药卫生—妇产科学]