可编辑DNA纳米花抑制LLC细胞增殖、迁移及侵袭的实验研究

An experimental study of editable DNA nanoflowers inhibiting the proliferation，migration and invasion of Lewis lung carcinoma cells

摘要

目的 探究载有阿霉素（doxorubicin，DOX）和缺氧诱导因子-1α（hypoxia inducible factor-1α，HIF-1α）反义寡核苷酸的DNA纳米花（DNA nanoflower，DF）对小鼠肺癌（lewis lung carcinoma，LLC）细胞增殖、迁移及侵袭能力的影响。 方法 通过滚环扩增反应（rolling circle amplification，RCA）合成含有HIF-1α反义寡核苷酸的DHA-DF，并检测其物理特性；负载DOX后获得载DOX纳米花（DOX-loaded DNA nanoflower，DDF）。通过细胞计数试剂（cell counting kit-8，CCK-8）评价DHA-DDF对LLC细胞活性的影响；通过划痕实验和Transwell小室实验检测DHA-DDF对LLC细胞迁移及侵袭能力的影响；通过蛋白质印记实验和定量聚合酶链反应检测LLC细胞HIF-1α的表达水平。 结果 DHA-DF为多孔花瓣状球形颗粒，粒径为（421.26±7.90） nm；与游离DOX和DA-DDF相比，经DHA-DDF处理后的LLC细胞HIF-1α蛋白及mRNA的表达量明显下降（均P<0.05），且其细胞活性、划痕愈合面积及侵袭细胞数目明显减少（均P<0.05）。 结论 DHA-DDF能有效降低HIF-1α的表达并抑制LLC细胞的增殖、迁移和侵袭，有望为临床肿瘤治疗提供新的思路。

Abstract

Objective To investigate the effects on the proliferation，migration and invasion of Lewis lung carcinoma（LLC） cells induced by DNA nanoflowers（DFs） carrying doxorubicin（DOX） and hypoxia inducible factor-1α（HIF-1α） antisense oligonucleotides. Methods DFs containing HIF-1α antisense oligonucleotides（DHA-DF） were synthesized by rolling circle amplification，and the physical properties of DHA-DF were examined. DOX was loaded into DFs to form DDF.We evaluated the cell viability of LLC cells treated with DHA-DDF by using Cell Counting Kit-8； assessed the effects of DHA-DDF on the migration and invasion of LLC cells by using scratch assay and Transwell assay； and measured the expression level of HIF-1α in LLC cells by Western blot and quantitative polymerase chain reaction. Results DHA-DF appeared flower-like porous spherical particles with a size of （421.26±7.90） nm. Compare with free DOX and DA-DDF，DHA-DDF significantly decreased the mRNA and protein expression of HIF-1α in LLC cells，and significantly reduced cell viability，the area of wound healed and the number of invasive cells（all P<0.05）. Conclusion DHA-DDF could effectively reduce the expression of HIF-1α，and inhibit the proliferation，migration and invasion of LLC cells，providing new ideas for clinical treatment of tumors.

关键词

DNA纳米花 / 缺氧 / 肺癌 / 反义寡核苷酸

Key words

DNA nanoflower / hypoxia / lung cancer / antisense oligonucleotide

基金资助

重庆市技术创新与应用发展专项面上资助项目(cstc2019jscx-msxmX0255)