Zhang Jun, Chou Xubin, Zhuang Ming, Zhu Chenlei, Hu Yong, Cheng Dong, Liu Zhiwei
Department of Spine Surgery, The Third Affiliated Hospital of Soochow University, Changzhou 213001, P.R. China.
Exp Ther Med. 2020 Nov;20(5):65. doi: 10.3892/etm.2020.9193. Epub 2020 Sep 9.
Circular RNAs (circRNAs) have been demonstrated to be involved in osteosarcoma (OS) development; however, the underlying mechanism of circKMT2D in OS progression remains unclear. The present study aimed to elucidate how circKMT2D could affect hydrogen peroxide (HO)-induced OS progression. HO (100 µmol/l) was used to treat MG63 and U2OS cells. The cell viability, invasive ability, apoptosis and circKMT2D expression were detected using Cell Counting Kit-8 assay, Transwell assay, flow cytometry and reverse transcription-quantitative PCR, respectively. Furthermore, MG63 and U2OS cells transfected with circKMT2D short hairpin RNA and negative control were treated with HO, and circKMT2D expression and cell phenotype were determined. Dual-luciferase reporter assay was conducted to determine the association between circKMT2D and miR-210 expression level. Rescue experiments were conducted to examine the mechanisms through which circKMT2D and miR-210 could affect HO-treated MG63 cells. In addition, the effects of miR-210 on the expression of the autophagy-related proteins Beclin1 and p62 in HO-treated MG63 cells were detected by western blotting. An autophagy inhibitor was used to treat the MG63 cells, and whether miR-210 could affect the HO-treated MG63 cell phenotype through autophagy was investigated. The results demonstrated that HO treatment promoted cell apoptosis and decreased cell viability, invasive ability and circKMT2D expression in MG63 and U2OS cells. Furthermore, circKMT2D knockdown decreased the cell viability and invasive ability and enhanced the apoptosis of HO-treated MG63 and U2OS cells. circKMT2D possessed binding sites for miR-210 and inhibited miR-210 expression. In HO-treated MG63 cells, miR-210 silencing partially reversed the circKMT2D knockdown-induced cell viability inhibition and apoptosis promotion. In addition, miR-210 elevated Beclin1 expression and decreased p62 expression in HO-treated MG63 cells. The use of the autophagy inhibitor partially reversed the miR-210 overexpression-induced promotion of apoptosis and inhibition of the viability and invasive ability of HO-treated MG63 cells. Taken together, these findings indicated that circKMT2D knockdown may contribute to the inhibition of HO-attenuated OS progression via miR-210/autophagy pathway.
环状RNA（circRNAs）已被证明参与骨肉瘤（OS）的发展；然而，circKMT2D在OS进展中的潜在机制仍不清楚。本研究旨在阐明circKMT2D如何影响过氧化氢（HO）诱导的OS进展。使用HO（100µmol/l）处理MG63和U2OS细胞。分别采用细胞计数试剂盒-8法、Transwell法、流式细胞术和逆转录定量PCR检测细胞活力、侵袭能力、凋亡和circKMT2D表达。此外，用circKMT2D短发夹RNA和阴性对照转染的MG63和U2OS细胞用HO处理，并测定circKMT2D表达和细胞表型。进行双荧光素酶报告基因检测以确定circKMT2D与miR-210表达水平之间的关联。进行挽救实验以研究circKMT2D和miR-210影响HO处理的MG63细胞的机制。此外，通过蛋白质印迹法检测miR-210对HO处理的MG63细胞中自噬相关蛋白Beclin1和p62表达的影响。使用自噬抑制剂处理MG63细胞，并研究miR-210是否可以通过自噬影响HO处理的MG63细胞表型。结果表明，HO处理促进了MG63和U2OS细胞的凋亡，降低了细胞活力、侵袭能力和circKMT2D表达。此外，circKMT2D敲低降低了HO处理的MG63和U2OS细胞的活力和侵袭能力，并增强了其凋亡。circKMT2D具有miR-210的结合位点并抑制miR-210表达。在HO处理的MG63细胞中，miR-210沉默部分逆转了circKMT2D敲低诱导的细胞活力抑制和凋亡促进。此外，miR-210提高了HO处理的MG63细胞中Beclin1的表达并降低了p62的表达。使用自噬抑制剂部分逆转了miR-210过表达诱导的HO处理的MG63细胞凋亡促进以及活力和侵袭能力抑制。综上所述，这些发现表明，circKMT2D敲低可能通过miR-210/自噬途径抑制HO减弱的OS进展。
