hsa-miR-103a-3p影响食管鳞状细胞癌细胞化疗耐药性的机制研究

Mechanism of hsa-miR-103a-3p affecting chemoresistance of esophageal squamous cell carcinoma

目的:探讨hsa-miR-103a-3p影响食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)细胞对奥沙利铂(oxaliplatin,OXA)耐药的机制。方法:采用RT-qPCR验证测序结果中hsa-miR-103a-3p在不同ESCC细胞系中的表达;转染hsa-miR-103a-3p mimics或inhibitor,验证hsa-miR-103a-3p是否可以逆转ESCC细胞系的耐药表型。应用双萤光素酶报告基因实验和RT-qPCR验证hsa-miR-103a-3p与RASSF8(Ras association domain family member 8)的靶向调控关系;挽救实验验证改变RASSF8的表达是否可以逆转由hsa-miR-103a-3p引起的耐药表型。PCR-array实验检测hsa-miR-103a-3p参与ESCC的耐药机制;Kaplan-Meier生存分析预测hsa-miR-103a-3p高表达与患者生存时间的关系。结果:hsa-miR-103a-3p在OXA敏感ESCC细胞系中高表达(P<0.05);hsa-miR-103a-3p的表达影响ESCC细胞对OXA的敏感性(P<0.05);与对照组相比,过表达hsa-miR-103a-3p能够降低RASSF83’端非翻译区萤光素酶报告基因质粒的活性(P<0.05);挽救实验结果显示,改变RASSF8的表达可有效挽救由hsa-miR-103a-3p引起的耐药表型(P<0.05)。PCR-array实验结果显示,RASSF8高表达能够促进XPA(xeroderma pigmentosum complementation group A)和XPC(xeroderma pigmentosum complementation group C)表达。配对t检验分析结果显示,hsamiR-103a-3p在肿瘤组织中高表达;Kaplan-Meier生存分析显示,与hsa-miR-103a-3p低表达的患者相比,hsa-miR-103a-3p高表达的患者生存时间显著缩短(P<0.05)。结论:hsa-miR-103a-3p低表达通过靶向上调RASSF8的表达促进ESCC细胞对OXA耐药。

AIM:To investigate the mechanism of hsa-miR-103a-3p affecting resistance of esophageal squamous cell carcinoma(ESCC)to oxaliplatin(OXA).METHODS:The expression of hsa-miR-103a-3p was detected in different cell lines by RT-qPCR. The eversal effect of varying hsa-miR-103a-3p expression on drug-resistant phenotype of ESCC cells was confirmed by transfection of hsa-miR-103a-3p mimics or inhibitor. Regulation relationship between hsamiR-103a-3p and Ras association domain family member 8(RASSF8) was verified by luciferase reporter assay and RTqPCR. The reversal effect of RASSF8 on drug-resistant phenotype caused by hsa-miR-103a-3p was verified by rescue experiment. The mechanism of hsa-miR-103a-3p affecting the resistance to OXA was investigated by PCR-array assay. The relationship between high expression of hsa-miR-103a-3p and prognosis of ESCC patients was predicted by Kaplan-Meier survival analysis.RESULTS:hsa-miR-103a-3p was highly expressed in OXA-sensitive ESCC cell line(P<0. 05). Overexpression or knockdown of hsa-miR-103a-3p affected the drug-resistant phenotype of ESCC cells(P<0. 05). Results of luciferase reporter assay showed that the activity of RASSF8 3’-untranslated region reporter was reduced in hsa-miR-103a-3p overexpression group compared with control group(P<0. 05). The chemoresistance caused by hsa-miR-103a-3p was reversed through the expression of RASSF8(P<0. 05). hsa-miR-103a-3p could affect the expression of xeroderma pigmentosum complementation group A(XPA) and xeroderma pigmentosum complementation group C(XPC) via regulating RASSF8. Paired t-test showed that hsa-miR-103a-3p was highly expressed in tumor tissue. Kaplan-Meier survival analysis showed that the patients with high expression of hsa-miR-103a-3p had short survival time(P<0. 05).CONCLUSION:Low expression of hsa-miR-103a-3p promoted the resistance of ESCC cells to OXA by targeting up-regulation of RASSF8 expression.