Department of Orthopedics, Bozhou People's Hospital, Bozhou, China.
Eur Rev Med Pharmacol Sci. 2021 Jan;25(2):678-686. doi: 10.26355/eurrev_202101_24629.
The aim of this study was to explore the effect of long non-coding ribonucleic acid (lncRNA) FOXD2-adjacent opposite strand RNA 1 (FOXD2-AS1) on the sensitivity of osteosarcoma cells to cisplatin and its possible underlying mechanism. Our findings might help to provide a certain reference for clinically preventing the drug resistance of osteosarcoma cells.
Cisplatin with a certain concentration gradient was used to induce the stable acquired resistance of human osteosarcoma U2-OS cell line. Subsequently, the expression level of lncRNA FOXD2-AS1 was determined in osteosarcoma cells in non-resistance group (Control group) and Cisplatin-resistance group (Cisplatin-RES group), respectively. Next, the cell line with stable lncRNA FOXD2-AS1 knockdown was constructed in Cisplatin-RES group using small interfering RNA (siRNA). The effects of stable knockdown of lncRNA FOXD2-AS1 on the proliferation of human osteosarcoma cells and the half-maximal inhibitory concentration (IC50) of cisplatin were detected by Cell Counting Kit-8 (CCK-8) assay. 5-ethynyl-2'-deoxyuridine (EdU) staining was performed to measure deoxyribonucleic acid (DNA) replication level in each group of cells. The protein expression levels of apoptosis-associated genes B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax) in each group of cells were measured via Western blotting. The migration and invasion abilities of cells in each group were determined using wound-healing assay and transwell assay. In addition, the expression of micro RNA (miR)-143 in each group of cells was detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR).
Compared with Control group, the expression level of lncRNA FOXD2-AS1 rose significantly in cells in Cisplatin-RES group (p<0.05). Knockdown of FOXD2-AS1 evidently decreased the IC50 of cisplatin in human osteosarcoma cells (p<0.05). According to EdU staining results, the knockdown of FOXD2-AS1 distinctly inhibited the proliferation of osteosarcoma cells (p<0.05). Western blotting results demonstrated that the knockdown of FOXD2-AS1 remarkably upregulated the expression of pro-apoptotic protein Bax and repressed that of anti-apoptotic protein Bcl-2 in drug-resistant human osteosarcoma cells (p<0.05). Moreover, the knockdown of FOXD2-AS1 significantly weakened the migration and invasion abilities of drug-resistant human osteosarcoma cells (p<0.05). Finally, it was found that the expression level of miR-143 was distinctly elevated in drug-resistant human osteosarcoma cells after knockdown of FOXD2-AS1 (p<0.05).
LncRNA FOXD2-AS1 knockdown inhibits the resistance of human osteosarcoma cells to cisplatin, promotes their apoptosis and weakens their invasion and migration abilities. The possible underlying mechanism may be related to the inhibition of miR-143 expression by lncRNA FOXD2-AS1 in drug-resistant cell lines.
本研究旨在探讨长链非编码 RNA（lncRNA）FOXD2-相邻反义 RNA 1（FOXD2-AS1）对骨肉瘤细胞对顺铂敏感性的影响及其可能的作用机制。我们的研究结果可能有助于为临床上预防骨肉瘤细胞耐药性提供一定的参考。
采用一定浓度梯度的顺铂诱导人骨肉瘤 U2-OS 细胞系获得稳定的获得性耐药。然后，分别检测非耐药组（对照组）和顺铂耐药组（顺铂-RES 组）骨肉瘤细胞中 lncRNA FOXD2-AS1 的表达水平。接下来，在顺铂-RES 组中使用小干扰 RNA（siRNA）构建稳定敲低 lncRNA FOXD2-AS1 的细胞系。通过细胞计数试剂盒-8（CCK-8）检测稳定敲低 lncRNA FOXD2-AS1 对人骨肉瘤细胞增殖和半抑制浓度（IC50）的影响。通过 5-乙炔基-2'-脱氧尿苷（EdU）染色检测各组细胞的脱氧核糖核酸（DNA）复制水平。通过 Western blot 检测各组细胞中凋亡相关基因 B 细胞淋巴瘤 2（Bcl-2）和 Bcl-2 相关 X 蛋白（Bax）的蛋白表达水平。通过划痕愈合实验和 Transwell 实验检测各组细胞的迁移和侵袭能力。此外，通过逆转录-聚合酶链反应（RT-PCR）检测各组细胞中 micro RNA（miR）-143 的表达。
与对照组相比，顺铂-RES 组细胞中 lncRNA FOXD2-AS1 的表达水平显著升高（p<0.05）。FOXD2-AS1 敲低明显降低了人骨肉瘤细胞中顺铂的 IC50（p<0.05）。EdU 染色结果表明，FOXD2-AS1 敲低明显抑制了骨肉瘤细胞的增殖（p<0.05）。Western blot 结果表明，FOXD2-AS1 敲低显著上调耐药人骨肉瘤细胞中促凋亡蛋白 Bax 的表达，下调抗凋亡蛋白 Bcl-2 的表达（p<0.05）。此外，FOXD2-AS1 敲低显著削弱了耐药人骨肉瘤细胞的迁移和侵袭能力（p<0.05）。最后，发现 FOXD2-AS1 敲低后耐药人骨肉瘤细胞中 miR-143 的表达水平明显升高（p<0.05）。
lncRNA FOXD2-AS1 敲低抑制人骨肉瘤细胞对顺铂的耐药性，促进其凋亡，削弱其侵袭和迁移能力。其可能的作用机制与 lncRNA FOXD2-AS1 抑制耐药细胞系中 miR-143 的表达有关。
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