ShRNA 介导的基质金属蛋白酶-2 基因沉默可保护正常细胞并增强癌细胞对电离辐射诱导损伤的敏感性。

ShRNA-mediated matrix metalloproteinase-2 gene silencing protects normal cells and sensitizes cancer cells against ionizing-radiation induced damage.

机构信息

Cancer Biology Lab, Department of Biochemistry, GIS, GITAM (Deemed to be University), Vishakhapatnam, India.

Department of Biotechnology, GIS, GITAM (Deemed to be University), Vishakhapatnam, India.

出版信息

J Cell Biochem. 2020 Feb;121(2):1332-1352. doi: 10.1002/jcb.29369. Epub 2019 Sep 6.

DOI:10.1002/jcb.29369

PMID:31489968

Abstract

INTRODUCTION

Ionizing radiation (IR) affects healthy tissues during the treatment of cancer radiation therapy and other nuclear and radiological accidents. Some natural compounds showed nonspecific radioprotective activity with severe side effects. The present study is aimed to develop potent and specific radioprotective short hairpin RNA (shRNA), which selectively protects normal cells from IR by specifically targeting matrix metalloproteinases (MMP-2).

RESULTS

IR reduced the viability of human normal dermal fibroblasts (HDFs) in a dose-response manner. It enhanced the expression of MMP-2 at 10 Gy. Plasmid MMP-2shRNA (pMMP-2) reduced the IR (10 Gy) induced cytotoxicity analyzed by lactate dehydrogenase (LDH) assay, normalized IR induced cellular and morphological changes with enhanced the clonogenicity in 48 hours at 2 µg/mL. It reduced the ROS generation, released HDFs from G /M arrest and rescued from apoptosis analyzed by DCFDA dye, cell cycle analysis by PI stain and annexin V assay, respectively. pMMP-2 also modulates the expression of EGFR and reduced IR induced expression of DNA damage response protein, ATM and increased the expression of repair proteins, KU70/KU80, and RAD51. In addition, decreased the expression of cell cycle regulatory proteins cyclin-dependent kinases (CDK1) and Cyclin B as well as proapoptotic proteins BAX, caspase-3, and Cytochrome-C and increased the expression of survival protein, Bcl-2. In contrary pMMP-2 decreased the LDH activity, survival fraction and blocked G /M phase of cell cycle and increased apoptosis in MCF-7 cells. In addition, decreased the expression of EGFR, proapoptotic BAX and DNA repair proteins ATM, KU70/80 and RAD51, increased expression of cyclinB as well as CDK1.

CONCLUSION

Results conclude that pMMP-2 protected HDFs from IR and sensitized the MCF-7 cells. Therefore, pMMP-2 can be employed for better treatment of radiation accidents and during the treatment of radiotherapy.

摘要

简介

电离辐射（IR）在癌症放射治疗和其他核与放射事故的治疗过程中会影响健康组织。一些天然化合物表现出非特异性的放射保护活性，但伴有严重的副作用。本研究旨在开发有效的、特异性的放射保护短发夹 RNA（shRNA），通过特异性靶向基质金属蛋白酶（MMP-2），选择性地保护正常细胞免受 IR 的影响。

结果

IR 以剂量反应的方式降低人正常皮肤成纤维细胞（HDF）的活力。它在 10Gy 时增强了 MMP-2 的表达。质粒 MMP-2shRNA（pMMP-2）降低了乳酸脱氢酶（LDH）分析的 IR（10Gy）诱导的细胞毒性，在 2μg/mL 时在 48 小时内使 IR 诱导的细胞和形态变化正常化，并增强了集落形成能力。它降低了 ROS 的产生，通过 DCFDA 染料分析使 HDF 从 G/M 期阻滞中释放出来，并通过细胞周期分析用 PI 染色和 annexin V 分析分别从凋亡中得到挽救。pMMP-2 还调节 EGFR 的表达，并降低 IR 诱导的 DNA 损伤反应蛋白 ATM 的表达，增加修复蛋白 KU70/KU80 和 RAD51 的表达。此外，降低细胞周期调节蛋白周期蛋白依赖性激酶（CDK1）和细胞周期蛋白 B 的表达以及促凋亡蛋白 BAX、caspase-3 和细胞色素-C 的表达，并增加生存蛋白 Bcl-2 的表达。相反，pMMP-2 降低了 LDH 活性、生存分数并阻断了 MCF-7 细胞的 G/M 期，增加了细胞凋亡。此外，降低了 EGFR、促凋亡 BAX 和 DNA 修复蛋白 ATM、KU70/80 和 RAD51 的表达，增加了细胞周期蛋白 B 以及 CDK1 的表达。