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lncRNA_MEG8
通过
促进
细胞
肺癌
肿瘤
进展
林燕明
基金项目:湛江市科技计划项目(2022B01089)作者简介:林燕明,男,1986 09 生,硕士,主治医师,E-mail:mlzsac163 com收稿日期:2022 10 11lncNA MEG8 通过调控 mi-363-3p/PAX6 轴促进非小细胞肺癌的肿瘤进展林燕明,陈玉婷,林慕文,李姝君,王永存*(广东医科大学附属医院肺部肿瘤专科,湛江524000;*通讯作者,E-mail:wycdiyi163 com)摘要:目的探讨长非编码 NA 母系表达基因8(lncNA MEG8)通过调控 mi-363-3p/人类配对盒基因6(PAX6)轴促进非小细胞肺癌(NSCLC)的肿瘤进展。方法选取 116 例经确诊为 NSCLC 患者的肿瘤组织与癌旁正常组织标本,常规培养人NSCLC 细胞株 A549、H1299,采用 T-qPC 法检测组织和细胞中 lncNA MEG8、mi-363-3p 和 PAX6 mNA 的表达。将A549、H1299 细胞分别分为对照组(control 组,空白培养基处理)、pcDNA 组(转染 pcDNA)、pcDNA-MEG8 组(转染 pcDNA-MEG8)、pcDNA-MEG8+mi-NC 组(pcDNA-MEG8 与 mi-NC 共转染)、pcDNA-MEG8+mi-363-3p mimic 组(pcDNA-MEG8 与mi-363-3p mimic 共转染)。CCK-8 法检测培养 24,48,72 h 各组细胞增殖能力;细胞划痕实验检测细胞迁移能力;流式细胞术检测细胞凋亡;采用 Western blot 检测凋亡相关蛋白 Bax、Bcl-2、Caspase-3 和 PAX6 蛋白的表达。双荧光素酶报告基因实验分别验证 MEG8 和 mi-363-3p、mi-363-3p 和 PAX6 的关系。NA 结合蛋白免疫沉淀(IP)实验检测 lncNA MEG8、mi-363-3p和 PAX6 之间的结合。结果与正常组织相比,肿瘤组织中 lncNA MEG8、PAX6 mNA 高表达,mi-363-3p 呈现低表达(均 P0 05)。与 control 组和 pcDNA 组相比,pcDNA-MEG8 组培养 48,72 h A549 和 H1299 细胞增殖能力、细胞迁移率、PAX6、Bcl-2 蛋白表达显著升高(P 0 05),细胞凋亡率和 Bax、Caspase-3 蛋白显著降低(P 0 05)。与 pcDNA-MEG8 组和pcDNA-MEG8+mi-NC 组相比,pcDNA-MEG8+mi-363-3 mimic 组培养 48,72 h A549 和 H1299 细胞增殖能力、细胞迁移率、PAX6、Bcl-2 蛋白表达显著降低(P0 05),mi-363-3p 表达、细胞凋亡率、Bax、Caspase-3 蛋白表达显著升高(P 0 05)。双荧光素酶报告基因实验结果显示,与 mi-NC+MEG8-WT 共转染组相比,mi-363-3p mimic+MEG8-WT 共转染组荧光素酶活性显著降低(P0 05);与 mi-NC+MEG8-MUT 共转染组相比,mi-363-3p mimic+MEG8-MUT 共转染组荧光素酶活性无显著差异(P0 05)。与 mi-NC+PAX6-WT 共转染组相比,mi-363-3p mimic+PAX6-WT 共转染组荧光素酶活性显著降低(P 0 05);与 mi-NC+PAX6-MUT 共转染组相比,mi-363-3p mimic+PAX6-MUT 共转染组荧光素酶活性无显著性差异(P 0 05)。IP 实验结果显示,与 IgG 处相比,lncNA MEG8 和 mi-363-3p、mi-363-3p 和 PAX6 均主要富集在 Ago2 处,IgG 处与Ago2 处的 lncNA MEG8、mi-363-3p 和 PAX6NA 相对表达水平均具有统计学差异(P 0 05),提示 lncNA MEG8 和 mi-363-3p、mi-363-3p 和 PAX6 能靶向结合。结论过表达 lncNA MEG8 可能通过下调 mi-363-3p 并促进 PAX6 蛋白的表达,进而促进 NSCLC 的进展。关键词:母系表达基因 8;mi-363-3p/人类配对盒基因 6;非小细胞肺癌;细胞凋亡;细胞增殖;细胞迁移中图分类号:734 2文献标志码:A文章编号:1007 6611(2023)04 0416 09DOI:1013753/j issn1007 6611202304002LncNA MEG8 promotes tumor progression in non-small cell lung cancer by regulating mi-363-3p/PAX6 axisLIN Yanming,CHEN Yuting,LIN Muwen,LI Shujun,WANG Yongcun*(Department of Lung Cancer,Affiliated Hospital of Guang-dong Medical University,Zhanjiang 524000,China;*Corresponding author,E-mail:wycdiyi163 com)Abstract:ObjectiveTo investigate the effect of long non-coding NA maternally expressed gene 8(lncNA MEG8)on tumor pro-gression of non-small cell lung cancer(NSCLC)by regulating the mi-363-3p/human paired box gene 6(PAX6)axisMethodsTumor tissue and adjacent normal tissue specimens from 116 patients diagnosed with NSCLC were collected,and human NSCLC celllines A549 and H1299 were routinely cultured,and then T-qPC was used to detect the expression of lncNA MEG8,mi-363-3pand PAX6 mNA in tissues and cells A549 and H1299 cells were divided into control group(blank medium treatment),pcDNA group(transfected with pcDNA),pcDNA-MEG8 group(transfected with pcDNA-MEG8),pcDNA-MEG8+mi-NC group(co-transfectedwith pcDNA-MEG8 and mi-NC),and pcDNA-MEG8+mi-363-3p mimic group(co-transfected with pcDNA-MEG8 and mi-363-3pmimic)CCK-8 method was applied to detect the cell proliferation ability at 24,48,72 h;cell scratch assay was applied to detect thecell migration ability;flow cytometry was applied to detect the apoptosis;Western blot was applied to detect the expression of apoptosis-related proteins Bax,Bcl-2,Caspase-3 and PAX6 Dual-luciferase reporter assays were applied to verify the relationship betweenlncNA MEG8 and mi-363-3p,mi-363-3p and PAX6,respectively NA binding protein immunoprecipitation(IP)assay wasused to detect the binding between lncNA MEG8,mi-363-3p and PAX6esultsCompared with normal tissues,lncNA MEG8614J Shanxi Med Univ,Apr 2023,Vol 54 No 4and PAX6 mNA expression levels were increased in tumor tissues,and mi-363-3p expression was decreased(P0 05)Comparedwith control group and pcDNA group,the cell proliferation ability at 48 h and 72 h,the cell migration rate,and the protein expressionof PAX6,Bcl-2 in A549 and H1299 cells were significantly increased in pcDNA-MEG8 group(P 0 05),while the apoptosis rate,and Bax and Caspase-3 protein levels were significantly decreased(P0 05)Compared with pcDNA-MEG8 group and pcDNA-MEG8+mi-NC group,the proliferative capacity at 48 h and 72 h,the cell migration rate,PAX6 and Bcl-2 protein expression levels inA549 and H1299 cells were significantly decreased in pcDNA-MEG8+mi-363-3 mimic group(P 0 05),while the expression ofmi-363-3p,the apoptosis rate,Bax and Caspase-3 protein expression levels were significantly increased(P 0 05)The results ofdouble luciferase reporter gene experiment showed that the luciferase activity in mi-363-3p mimic+MEG8-WT co-transfection groupwas significantly lower than that in mi-NC+MEG8-WT co-transfection group(P 0 05)Compared with mi-NC+MEG8-MUT co-transfection group,the luciferase activity had no significant difference in mi-363-3p mimic+MEG8-MUT co-transfection group(P 005)Compared with mi-NC+PAX6-WT co-transfection group,the luciferase activity in mi-363-3p mimic+PAX6-WT co-transfectiong