微小RNA-148a-3p靶向核心1β13-半乳糖基转移酶1增强肺腺癌细胞的放疗敏感性的研究

MicroRNA-148a-3p enhances sensitivity of radiotherapy of lung adenocarcinoma cells by targeting core 1β13-galactosyltransferase 1

目的探讨微小RNA-148a-3p(miR-148a-3p)在肺腺癌中的表达及临床意义,分析miR-148a-3p靶向蛋白核心1β13-半乳糖基转移酶1(C1GALT1)对肺腺癌细胞放疗敏感性的影响及机制。方法选取肺腺癌组织芯片中76例患者肿瘤组织及肺腺癌A549细胞株为研究对象。对组织芯片分别进行miR-148a-3p原位杂交(ISH)染色和C1GALT1免疫组织化学染色,分析76例肺腺癌患者肿瘤组织中miR-148a-3p的表达与临床病理、预后及C1GALT1表达的关系。采用细胞转染技术对A549细胞进行miR-148a-3p过表达质粒的转染;转染细胞接受2 Gy放疗后采用克隆形成实验检测细胞的放疗敏感性;采用蛋白质印迹法检测细胞C1GALT1蛋白表达水平;采用双荧光素酶报告基因实验验证miR-148a-3p与C1GALT1的靶向调控关系;采用共转染技术分析miR-148a-3p调控A549细胞放疗敏感性的机制。结果76例肺腺癌组织中低表达miR-148a-3p与患者淋巴结转移显著相关(P=0.012);miR-148a-3p高表达者预后显著优于miR-148a-3p低表达者(P=0.005);肺腺癌组织中miR-148a-3p与C1GALT1的表达呈显著负相关(P=0.023)。多因素分析显示,miR-148a-3p低表达、T分期及淋巴结转移是预后的独立危险因素。克隆形成实验显示,过表达miR-148a-3p组克隆形成数显著低于对照组(P<0.001);Western Blot结果显示,miR-148a-3p过表达可以显著下调细胞中C1GALT1蛋白水平(P<0.001)。双荧光素酶报告基因实验显示miR-148a-3p通过结合C1GALT1的3′UTR来调控C1GALT1的表达。功能拯救试验显示C1GALT1能够部分抵消miR-148a-3p的放疗增敏效应。结论肺腺癌中miR-148a-3p低表达与淋巴结转移、C1GALT1高表达及预后不良显著相关,miR-148a-3p通过负调控C1GALT1的表达增强肺腺癌细胞的放疗敏感性。

Objective To investigate the expression and clinical significance of microRNA-148a-3p(miR-148a-3p)in lung adenocarcinoma and analyze the effect and mechanism of miR-148a-3p on radiotherapy sensitivity of lung adenocarcinoma cells by targeting the protein core 1β13-galactosyltransferase 1(C1GALT1).Methods Seventy-six patients′tumor tissues from lung adenocarcinoma tissue microarrays and lung adenocarcinoma A549 cell line were selected for the study.The miR-148a-3p in situ hybridizations(ISH)and C1GALT1 immunohistochemical staining were performed on the tissue microarrays to analyze the correlations of miR-148a-3p expression with clinical pathology,prognosis and C1GALT1 expression in the tumor tissues of the 76 patients with lung adenocarcinoma.A549 cells were transfected with miR-148a-3p overexpression plasmid by using cell transfection technique;the clone formation assay was used to detect the sensitivity of the transfected cells for radiotherapy after receiving 2 Gy radiotherapy;the protein expression level of cellular C1GALT1 was detected by western blot;the targeted regulatory relationship between miR-148a-3p and C1GALT1 was verified by dual-luciferase radiotherapy was analyzed by co-transfection technique.Results Low expression of miR-148a-3p in 76 cases of lung adenocarcinoma tissues was significantly associated with lymph node metastasis(P=0.012);the prognosis of patients with high expression of miR-148a-3p was significantly better than that of patients with low expression of miR-148a-3p(P=0.005);there was a significant negative correlation between the expression of miR-148a-3p and C1GALT1 in lung adenocarcinoma tissues(P=0.023).Multivariate analysis showed that low expression of miR-148a-3p,T staging and lymph node metastasis were the independent risk factors for prognosis.Clone formation experiment showed that the number of clone formation in the miR-148a-3p overexpression group was significantly lower than that in the control group(P<0.001);western blot result showed that overexpression of miR-148a-3p was able to significantly down-regulate the level of C1GALT1 protein in cells(P<0.001).Dual luciferase reporter gene experiment showed that miR-148a-3p was able to regulate the expression of C1GALT1 by binding to the 3′UTR of C1GALT1.Functional rescue experiment showed that C1GALT1 could partially offset the radiosensitization effect of miR-148a-3p.Conclusion Low expression of miR-148a-3p in lung adenocarcinoma is significantly associated with lymph node metastasis,high expression of C1GALT1 and poor prognosis,and miR-148a-3p can enhance the radiosensitivity of lung adenocarcinoma cells by negatively regulating the expression of C1GALT1.