肿瘤研究与临床2024，Vol.36Issue(3) ：191-198.DOI:10.3760/cma.j.cn115355-20230719-00012

长链非编码RNA RP13-349O20.2在子宫颈癌组织中的表达及其对子宫颈癌细胞迁移、侵袭能力和化疗敏感性影响的实验研究

Expression of long non-coding RNA RP13-349020.2 in cervical cancer tissues and experimental study of its effect on migration,invasion abilities and chemosensitivity of cervical cancer cells

柴守辉 吴然然 殷宪明 徐行丽
肿瘤研究与临床2024，Vol.36Issue(3) ：191-198.DOI:10.3760/cma.j.cn115355-20230719-00012
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长链非编码RNA RP13-349O20.2在子宫颈癌组织中的表达及其对子宫颈癌细胞迁移、侵袭能力和化疗敏感性影响的实验研究

Expression of long non-coding RNA RP13-349020.2 in cervical cancer tissues and experimental study of its effect on migration,invasion abilities and chemosensitivity of cervical cancer cells

柴守辉 1吴然然 1殷宪明 1徐行丽1
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作者信息

  • 1. 徐州医科大学附属滕州市中心人民医院妇科,滕州 277500
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摘要

目的 探讨长链非编码RNA(lncRNA)RP 13-349O20.2在子宫颈癌组织中的表达水平,及其体外对子宫颈癌细胞迁移、侵袭能力和化疗敏感性的影响及可能机制.方法 利用GEPIA.CANCER网站(数据更新时间2023年6月)分析RP13-349O20.2表达水平与253例子宫颈癌患者总生存的关系.回顾性收集2020年1月至2022年8月徐州医科大学附属滕州市中心人民医院40例子宫颈癌患者癌组织和癌旁组织(距离肿瘤边缘＞2 cm),采用人正常子宫颈上皮细胞株H8和人子宫颈癌细胞株HCC94、C33A、Hela、HCC1106、SiHa进行体外细胞实验.采用实时荧光定量聚合酶链反应(qRT-PCR)检测子宫颈癌组织和癌旁组织、各细胞株中RP13-349O20.2的相对表达量.向RP13-349O20.2相对表达水平最高的C33A细胞分别转染RP13-349O20.2的小干扰RNA(siRNA)及其阴性对照序列的siRNA,分别为si-RP13-349O20.2组和si-Con组.采用划痕愈合实验检测两组C33A细胞的迁移能力,Transwell实验检测C33A细胞侵袭能力,CCK-8法检测C33A细胞对5-氟尿嘧啶的敏感性(以吸光度值表示细胞增殖能力,吸光度值越低,增殖能力越弱,则对药物敏感性越强).双荧光素酶报告基因实验验证RP13-349O20.2和 miRNA-493-5p(miR-493-5p)、miR-493-5p和 Nectin-4 的靶向关系.采用 qRT-PCR检测两组 C33A 细胞miR-493-5p和Nectin-4 mRNA的相对表达量,蛋白质印迹法检测两组细胞Nectin-4蛋白和PI3K-AKT信号通路相关蛋白的表达情况.结果 根据GEPIA.CANCER网站数据分析显示,RP 13-349O20.2低表达患者的总生存较高表达患者好(P＜0.01).40例子宫颈癌患者癌组织和癌旁组织中RP13-349O20.2相对表达量分别为4.04±0.32和1.18±0.14,差异有统计学意义(t=8.29,P＜0.01).与H8细胞比较,人子宫颈癌细胞株 HCC94、C33A、Hela、HCC1106、SiHa 细胞中 RP13-349O20.2 表达水平均高(均 P＜0.01).si-Con组和 si-RP13-349O20.2 组 C33A 细胞中 RP 13-349O20.2 相对表达量分别为 7.30±0.30 和 1.01±0.27,差异有统计学意义(t=15.62,P＜0.01).si-Con组和si-RP13-349O20.2组C33A细胞划痕抑制率分别为(32±9)％和(75±6)％(t=3.97,P＜0.01),侵袭细胞数分别为(106±12)个和(36±8)个(t=4.79,P＜0.01).在5、10、20、40、80 μmol/L 5-氟尿嘧啶作用24 h后,si-RP13-349O20.2组C33A细胞吸光度值均低于si-Con组.双荧光素酶报告基因实验证实P13-349O20.2与miR-493-5p存在靶向关系(P＜0.01),miR-493-5p与 Nectin-4存在靶向关系(P＜0.01).si-Con组和 si-RP13-349O20.2组 C33A 细胞miR-493-5p的相对表达量分别为1.02±0.13和5.48±0.85(t=5.21,P＜0.01),Nectin-4 mRNA的相对表达量分别为5.65±0.33 和 0.99±0.34(t=9.87,P＜0.01).si-RP13-349O20.2组 C33A 细胞中 Nectin-4 蛋白表达水平较si-Con组低(t=9.21,P=0.001),PI3K-AKT信号通路蛋白 p-STAT3、p-PI3K、p-AKT、p-mTOR表达水平均低于si-Con组(均P＜0.01).结论 子宫颈癌组织中RP 13-349O20.2水平较高,且其高表达可能预示患者预后不良.体外干扰RP13-349O20.2表达能够抑制子宫颈癌细胞迁移、侵袭能力,促进子宫颈癌细胞对5-氟尿嘧啶的敏感性,其机制可能与miR-493-5p/Nectin-4信号通路和PI3K-AKT信号通路有关.

Abstract

Objective To explore the expression of long non-coding RNA(lncRNA)RP13-349O20.2 in cervical cancer tissues and its impact on the migration,invasion abilities and chemotherapy sensitivity of cervical cancer cells in vitro and the possible mechanisms.Methods The GEPIA.CANCER website(the data was updated in June 2023)was used to analyze the relationship between the expression level of RP13-349O20.2 and the overall survival of 253 cervical cancer patients.From January 2020 to August 2022,cancer tissues and paracancerous tissues(＞2 cm from the tumor edge)from 40 cervical cancer patients in the Affiliated Tengzhou Central People's Hospital of Xuzhou Medical University were retrospectively collected.Human normal cervical epithelial cells H8 and human cervical cancer cell lines HCC94,C33A,Hela,HCC1106 and SiHa were used for cell experiments in vitro.Real-time fluorescence polymerase chain reaction(qRT-PCR)was used to detect the relative expression of RP13-349O20.2 in cervical cancer tissues,paracancerous tissues and each cell line.The C33A cells with the highest relative expression level of RP13-349O20.2 were transfected with small interfering RNA(siRNA)of RP13-349O20.2 and siRNA of its negative control sequence,and they were si-RP13-349O20.2 group and si-Con group,respectively.The scratch healing assay was used to detect the migration ability of C33A cells in the two groups,the Transwell assay was used to detect the invasion ability of C33A cells,and the CCK-8 method was used to detect the sensitivity of C33A cells to 5-fluorouracil.The absorbance value indicated the cell proliferation ability,the lower the absorbance value,the weaker the proliferation ability,the more sensitive to the drug.Dual-luciferase reporter gene assay was used to verify the targeting relationship between RP13-349O20.2 and miRNA-493-5p(miR-493-5p),miR-493-5p and Nectin-4.qRT-PCR was used to detect the relative expression of miR-493-5p and Nectin-4 mRNA in two groups of C33A cells,and Western blotting was used to detect the expressions of Nectin-4 protein and PI3K-AKT signaling pathway proteins in two groups of cells.Results Analysis based on data from GEPIA.CANCER website shows that patients with low expression of RP13-349O20.2 had better overall survival than patients with high expression(P＜0.01).The relative expression levels of RP13-349O20.2 in cervical cancer tissues and paracancerous tissues of 40 patients were 4.04±0.32 and 1.18±0.14,and the difference was statistically significant(t=8.29,P＜0.01).Compared with H8 cells,the expressions of RP13-349O20.2 in human cervical cancer cell lines HCC94,C33A,Hela,HCC1106 and SiHa were higher(all P＜0.01).The relative expression levels of RP13-349O20.2 in C33A cells in the si-Con group and si-RP13-349O20.2 group were 7.30±0.30 and 1.01±0.27,and the difference was statistically significant(t=15.62,P＜0.01).The scratch healing rates of C33A cells in the si-Con group and si-RP13-349O20.2 group were(32±9)％and(75±6)％(t=3.97,P＜0.01),and the numbers of invasive cells were(106±12)cells and(36±8)cells(t=4.79,P＜0.01).After the action of 5,10,20,40 and 80 μmol/L 5-fluorouracil for 24 h,the absorbance value of C33A cells in the si-RP13-349O20.2 group was lower than that in the si-Con group.Dual-luciferase reporter gene assay confirmed that there was a targeting relationship between P13-349O20.2 and miR-493-5p(P＜0.01),and there was a targeting relationship between miR-493-5p and Nectin-4(P＜0.01).The relative expression levels of miR-493-5p in C33A cells in the si-Con group and si-RP13-349O20.2 group was 1.02±0.13 and 5.48±0.85(t=5.21,P＜0.01).The relative expression levels of Nectin-4 mRNA were 5.65±0.33 and 0.99±0.34(t=9.87,P＜0.01).The expression of Nectin-4 protein in C33A cells in the si-RP13-349O20.2 group was lower than that in the si-Con group(t=9.21,P=0.001),and the expressions of PI3K-AKT signaling pathway proteins p-STAT3,p-PI3K,p-AKT and p-mTOR were lower than those in the si-Con group(all P＜0.01).Conclusions The level of RP13-349O20.2 in cervical cancer tissues is high,and its high expression may indicate the poor prognosis of patients.Interfering with the expression of RP13-349O20.2 in vitro can inhibit the migration and invasion abilities of cervical cancer cells and promote the sensitivity of cervical cancer cells to 5-fluorouracil.The mechanism may be related to the miR-493-5p/Nectin-4 signaling pathway and the PI3K-AKT signaling pathway.

关键词

宫颈肿瘤/RNA,长链非编码/细胞增殖/细胞运动/抗药性

Key words

Uterine cervical neoplasms/RNA,long non-coding/Cell proliferation/Cell movement/Drug resistance

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基金项目

徐州医科大学附属医院发展基金(优秀人才基金)(XYFZ2020053)

出版年

2024
肿瘤研究与临床
中华医学会,山西省肿瘤研究所,山西省肿瘤医院

肿瘤研究与临床

CSTPCD
影响因子：0.705
ISSN：1006-9801
参考文献量25
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