微小RNA hsa-miR-93靶向STAT3提高鼻咽癌对放疗敏感性的机制研究

目的 研究hsa-miR-93在鼻咽癌放疗抗拒过程中的作用及分子机制。方法 以人鼻咽癌细胞株CNE-1、CNE-2、CNE-2R、HONE1、C666.1以及鼻咽上皮永生细胞NP460为研究对象,采用实时荧光定量PCR(qRT-PCR)法检测各组细胞经放疗后hsa-miR-93的表达;采用Western blot法检测各组细胞经放疗后STAT3蛋白的表达;通过miRWalk软件和RNAHybrid软件预测hsa-miR-93和STAT3的结合作用及结合位点;通过双荧光素酶报告基因法检测hsa-miR-93对STAT3靶向结合情况;瞬时转染小分子模拟物或抑制剂调控hsa-miR-93的表达后,采用qRT-PCR和Western blot法检测STAT3的表达;转染siSTAT3沉默STAT3的表达后,通过细胞集落形成实验检测鼻咽癌细胞经放疗后的集落形成能力。结果 与CNE-1、CNE-2细胞(相对放疗敏感)相比,hsa-miR-93在HONE1、CNE-2R细胞(相对放疗抗拒)中表达降低(P＜0.001),且各组鼻咽癌细胞经放疗后hsa-miR-93的表达呈剂量及时间依赖性降低(P＜0.05);与CNE-1、CNE-2细胞相比,STAT3在HONE1、CNE-2R细胞中表达升高,且各组鼻咽癌细胞经放疗后表达呈剂量及时间依赖性升高;经预测,hsa-miR-93可直接靶向结合STAT3。过表达hsa-miR-93可以在mRNA以及蛋白水平上显著抑制STAT3的表达(P＜0.05),而抑制hsa-miR-93的表达可以显著上调STAT3的表达(P＜0.05);过表达hsa-miR-93或沉默STAT3均使鼻咽癌细胞的集落形成显著减少(P＜0.001);抑制hsa-miR-93减弱了鼻咽癌对放疗的敏感性,而同时抑制hsa-miR-93和STAT3后,可提高鼻咽癌对放疗的敏感性。结论 hsa-miR-93可能通过靶向STAT3提高鼻咽癌对放疗的敏感性。

Research on the mechanism of hsa-miR-93 targeting STAT3 to improve the radiosensitivity of nasopharyngeal carcinoma

Objective The Objective of this study was to study the role and molecular mechanism of hsa-miR-93 in radiotherapy resistance of nasopharyngeal carcinoma(NPC). Methods Human NPC cell lines CNE-1,CNE-2,CNE-2R,HONE1,C666.1 and nasopharyngeal epithelial immortalized NP460 cell line were used as the research objects.The expression of hsa-miR-93 after radiotherapy in each group of cells was detected by qRT-PCR;The expression of STAT3 protein after radiotherapy in each group of cells was detected by Western blot;MiRwalk software and RNAHybrid software were used to predict the function and binding site of the combination of hsa-miR-93 and STAT3;The binding sites of hsa-miR-93 to STAT3 was detected by dual luciferase reporter gene assay;The expression of STAT3 was detected by qRT-PCR and Western blot;After the expression of STAT3 was silenced by transfection with siSTAT3,the clonogenic ability of NPC cells after radiotherapy was detected by cell colony formation assay. Results Compared with CNE-1 cells and CNE-2 cells(relative radiotherapy sensitivity),the expression of hsa-miR-93 in HONE1 cells and CNE-2R cells(relative radiotherapy resistance)decreased(P＜0.001),and the expression of hsa-miR-93 in each group was decreased in a dose- and time- dependent manner after radiotherapy(P＜0.05).Compared with CNE-1 cells and CNE-2 cells,the expression of STAT3 increased in HONE1 cells and CNE-2R cells,and the expression of STAT3 in NPC cells of each group after radiotherapy increased in a dose- and time-dependent manner.It was predicted that hsa-miR-93 could directly target STAT3.Overexpression of hsa-miR-93 could significantly inhibit the expression of STAT3 at mRNA and protein levels(P＜0.05),while inhibition of hsa-miR-93 could significantly upregulate the expression of STAT3(P＜0.05).Overexpression of hsa-miR-93 or silencing of STAT3 significantly reduced the number of NPC cell colony(P＜0.001).Inhibition of hsa-miR-93 attenuated the sensitivity of NPC to radiotherapy,while simultaneous inhibition of hsa-miR-93 and STAT3 could increase the sensitivity of NPC to radiotherapy. Conclusion hsa-miR-93 increases the sensitivity of NPC to radiotherapy by targeting STAT3.