Hsa_circ_0000392靶向miR-145-5p/CRKL轴调控细胞外调节蛋白激酶通路影响宫颈癌的辐射敏感性

Hsa_circ_0000392 affects the radiation sensitivity of cervical cancer by targeting the miR-145-5p/CRKL/MAPK pathway

目的探讨hsa_circ_0000392(circ_0000392)对宫颈癌细胞放疗敏感性的影响及其潜在作用机制。方法收集2016—2019年于河南大学淮河医院首次经病理诊断明确的42例宫颈癌患者的宫颈癌组织和癌旁正常组织,根据患者对放射治疗的反应,将癌组织分为放疗敏感和放疗耐受。采用实时荧光定量聚合酶链反应和Western blot法检测宫颈癌放疗敏感和放疗耐受肿瘤组织及宫颈癌Hela、SiHa细胞中circ_0000392、miR-145-5p、CT10激酶调节子样蛋白(CRKL)的表达。将siRNA circ_0000392、miR-145-5p mimic、miR-145-5p inhibitor、pcDNA 3.1-CRKL及其阴性对照分别转染至Hela和SiHa细胞,细胞经辐射诱导后,采用克隆形成实验检测细胞存活能力,流式细胞术检测细胞凋亡情况,Western blot检测Bax和Bcl-2、磷酸化细胞外调节蛋白激酶(p-ERK)1/2和细胞外调节蛋白激酶(ERK)1/2的表达。双荧光素酶报告基因实验验证circ_0000392、miR-145-5p和CRKL之间的靶向关系。裸鼠成瘤实验观察circ_0000392对宫颈癌体内放疗敏感性的影响。结果circ_0000392和CRKL在宫颈癌放疗耐受组织和癌细胞中均呈高表达,miR-145-5p在宫颈癌放疗耐受组织和癌细胞中呈低表达(均P<0.05)。si-circ_0000392#1+6 Gy组Hela和SiHa细胞的克隆形成数分别为(78.67±10.97)个和(71.00±9.54)个,均低于si-Ctrl+6 Gy组[分别为(176.00±22.27)个和(158.33±17.56)个,均P<0.05],细胞凋亡率分别为(41.55±3.40)%和(31.41±3.29)%,均高于si-Ctrl+6 Gy组[分别为(15.91±1.37)%和(13.70±1.89)%,均P<0.05],si-circ_0000392#1+6 Gy组细胞中Bax蛋白表达水平高于si-Ctrl+6 Gy组,Bcl2蛋白表达水平低于si-Ctrl+6 Gy组(均P<0.05)。si-circ_0000392#1+miR-145-5p inhibitor+6 Gy组Hela和SiHa细胞的克隆形成数分别为(171.33±25.01)个和(137.00±21.66)个,均高于si-circ_0000392#1+inhibitor NC+6 Gy组[分别为(84.67±17.79)个和(71.00±11.00)个,均P<0.05],细胞凋亡率分别为(17.41±2.58)%和(15.96±1.25)%,均低于si-circ_0000392#1+inhibitor NC+6 Gy组[分别为(40.29±2.92)%和(30.82±2.34)%,均P<0.05],si-circ_0000392#1+miR-145-5p inhibitor+6 Gy组细胞中Bax蛋白表达水平低于si-circ_0000392#1+inhibitor NC+6 Gy组,Bcl2蛋白表达水平高于si-circ_0000392#1+inhibitor NC+6 Gy组(均P<0.05)。circ_0000392能够与miR-145-5p靶向结合,而CRKL是miR-145-5p的下游靶基因。miR-145-5p mimic+6 Gy组Hela和SiHa细胞的克隆形成数分别为(74.33±10.02)个和(66.00±12.17)个,均低于mimic NC+6 Gy组[分别为(197.67±17.21)个和(157.67±11.59)个,均P<0.05],细胞凋亡率分别为(45.58±2.16)%和(32.10±3.55)%,均高于mimic NC+6 Gy组[分别为(15.85±2.45)%和(13.99±1.69)%,均P<0.05],miR-145-5p mimic+6 Gy组细胞中Bax蛋白表达水平高于mimic NC+6 Gy组,Bcl2蛋白表达水平低于mimic NC+6 Gy组(均P<0.05)。miR-145-5p mimic+pcDNA-CRKL+6 Gy组Hela和SiHa细胞的克隆形成数分别为(158.00±15.88)个和(122.33±13.65)个,均高于miR-145-5p mimic+pcDNA组+6 Gy组[分别为(71.33±8.02)个和(65.67±12.22)个,均P<0.05],细胞凋亡率分别为(19.50±3.45)%和(17.04±0.94)%,均低于miR-145-5p mimic+pcDNA组+6 Gy组[分别为(44.33±2.36)%和(32.05±2.76)%,均P<0.05],miR-145-5p mimic+pcDNA-CRKL+6 Gy组细胞中Bax蛋白表达水平低于miR-145-5p mimic+pcDNA组+6 Gy组,Bcl2蛋白表达水平高于miR-145-5p mimic+pcDNA组+6 Gy组(均P<0.05)。sh-circ_0000392组小鼠肿瘤体积较小,肿瘤重量降低(均P<0.05)。小鼠移植瘤组织中circ_0000392、miR-145-5p、CRKL mRNA相对表达水平降低减少,CRKL、Bcl-2和p-ERK1/2蛋白相对表达水平降低,Bax蛋白相对表达水平升高(均P<0.05)。结论circ_0000392可增强宫颈癌细胞的放疗敏感性,其作用机制可能与靶向miR-145-5p调控CRKL/ERK信号通路有关。

ObjectiveTo investigate the effect of hsa_circ_0000392(circ_0000392)on the radiosensitivity of cervical cancer cells and explore its potential mechanism.MethodsCervical cancer tissues and adjacent normal tissues of 42 patients with cervical cancer who were confirmed pathologically for the first time in Huaihe Hospital of Henan University from 2016 to 2019 were collected.According to the patients′response to radiotherapy,the cancer tissues were divided into radio-sensitive tissues and radio-resistant tissues.The expressions of circ_0000392,miR-145-5p,and CRKL in radiation-sensitive,radiation-resistant cervical cancer tissues and Hela,SiHa cells were detected by reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR)and western blot.SiRNA circ_0000392,miR-145-5p mimic,miR-145-5p inhibitor,pcDNA 3.1-CRKL and its negative control were transfected into HeLa and Siha cells,respectively.After radiation induction,the survival fraction of cells was detected by clone formation assay,apoptosis was detected by flow cytometry,and the expressions of apoptosis-related proteins Bax and Bcl-2 and ERK pathway protein p-ERK1/2 and ERK1/2 were detected by western blot.The targeting relationship between circ_0000392,miR-145-5p and CRKL was verified by dual luciferase reporter gene assay.The effect of circ_0000392 on radiotherapy sensitivity of cervical cancer in vivo was observed in the tumor formation experiment in nude mice.Resultscirc_0000392 and CRKL were upregulated in radiation-resistant tissues and cancer cells of cervical cancer,while miR-145-5p was downregulated.The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+6 Gy group were(78.67±10.97)and(71.00±9.54),respectively,which were lower than those in si-Ctrl+6 Gy group[(176.00±22.27)and(158.33±17.56),respectively].The apoptosis rates were(41.55±3.40)%and(31.41±3.29)%,respectively,which were higher than those in si-Ctrl+6 Gy group[(15.91±1.37)%and(13.70±1.89)%,P<0.05].The protein expression of Bax was higher than that of si-Ctrl+6 Gy group,and the protein expressions of Bcl2 was lower than those of si-Ctrl+6 Gy group.The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+miR-145-5p inhibitor+6 Gy group were(171.33±25.01)and(137.00±21.66),higher than those in si-circ_0000392#1+inhibitor NC+6 Gy group[(84.67±17.79)vs(71.00±11.00),P<0.05].The apoptosis rates were(17.41±2.58)%and(15.96±1.25)%,lower than those of si-circ_0000392#1+inhibitor NC+6 Gy[(40.29±2.92)%and(30.82±2.34)%,respectively,P<0.05].The expression of Bax protein was lower than that of si-circ_0000392#1+inhibitor NC+6 Gy group,and the expressions of Bcl2 protein were higher than those of si-circ_0000392#1+inhibitor NC+6 Gy group.Circ_0000392 can target miR-145-5p,and CRKL is the downstream target gene of miR-145-5p.The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+6 Gy group were(74.33±10.02)and(66.00±12.17),respectively,which were lower than those of mimic NC+6 Gy group[(197.67±17.21)vs(157.67±11.59),respectively,P<0.05].The apoptosis rates were(45.58±2.16)%and(32.10±3.55)%,higher than those of mimic NC+6 Gy group[(15.85±2.45)%and(13.99±1.69)%,respectively,P<0.05].The expression of Bax protein was higher than that of the mimic NC+6 Gy mimic group,and the expression of Bcl2 protein was lower than that of the mimic NC+6 Gy group.The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+pcDNA-CRKL+6 Gy group were(158.00±15.88)and(122.33±13.65),respectively,which were higher than those of miR-145-5p mimic+pcDNA+6 Gy group[(71.33±8.02)vs(65.67±12.22),P<0.05].The apoptosis rates were(19.50±3.45)%and(17.04±0.94)%,respectively,which were lower than those of miR-145-5p mimic+pcDNA+6 Gy group[(44.33±2.36)%and(32.05±2.76)%,respectively,P<0.05].The expression of Bax protein was lower than that of miR-145-5p mimic+pcDNA group+6 Gy group,and the expression of Bcl2 protein was higher than that of miR-145-5p mimic+pcDNA+6 Gy group.Sh-circ_0000392 group had smaller tumor volume and decreased tumor weight(P<0.05).The relative mRNA expression levels of circ_0000392,miR-145-5p and CRKL and the relative protein expression levels of CRKL,Bcl-2 and p-ERK1/2 were decreased,while the relative expression level of Bax protein was increased(P<0.05).ConclusionCirc_0000392 could enhance the radiosensitivity of cervical cancer cells,and its mechanism may be related to the regulation of CRKL/ERK signaling pathway by targeting miR-145-5p,which provides a new reference for enhancing the radiosensitivity of cervical cancer cells.