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LncRNAMALAT1对骨肉瘤细胞阿霉素耐药性的影响机制研究
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| 篇名： | LncRNAMALAT1对骨肉瘤细胞阿霉素耐药性的影响机制研究 |
| TITLE： | Effect mechanism of LncRNA MALAT1 on doxorubicin resistance in osteosarcoma cells |
| 摘要： | 目的 探讨长链非编码RNA（LncRNA）肺腺癌转移相关转录本1（MALAT1）与骨肉瘤（OS）细胞阿霉素（DOX）耐药性的关系及作用机制。方法将MG-63和MG-63/DOX细胞用不同浓度DOX（0、0.01、0.05、0.1、1μmol/L）处理后，以CCK-8法检测其存活率和半数抑制浓度（IC50）；采用实时荧光定量聚合酶链反应法检测MG-63和MG-63/DOX细胞中LncRNAMALAT1表达。将MG-63/DOX细胞分为Control（对照）组、敲低LncRNAMALAT1的阴性对照（sh-NC）组、sh-MALAT1组、sh-MALAT1+抑制（anti）-NC组、sh-MALAT1+anti-miR-154-5p组。检测各组MG-63/DOX细胞中LncRNAMALAT1、miR-154-5p、细胞周期素D1（CCND1）mRNA相对表达量，采用CCK-8法、划痕实验、Transwell实验、流式细胞仪分别检测敲低LncRNAMALAT1对MG-63/DOX细胞增殖、迁移、侵袭、凋亡的影响；采用Westernblot法检测MG-63/DOX细胞中增殖细胞核抗原（PCNA）、CCND1蛋白的表达；采用双荧光素酶报告基因实验检测LncRNAMALAT1与miR-154-5p、miR-154-5p与CCND1之间的相互作用。结果与0μmol/LDOX比较，0.01、0.05、0.1、1μmol/LDOX均能降低MG-63和MG-63/DOX细胞（0.01μmol/LDOX除外）的存活率（P＜0.05），IC50分别为0.07、0.13μmol/L。sh-MALAT1组MG-63/DOX细胞存活率、迁移数、侵袭数、划痕愈合率、LncRNAMALAT1mRNA表达、CCND1mRNA和蛋白表达、PCNA蛋白表达均显著低于sh-NC组、Control组，细胞凋亡率和miR-154-5p表达显著高于sh-NC组、Control组（P＜0.05）；相较于sh-MALAT1组，sh-MALAT1+anti-miR-154-5p组的上述生物学作用均被逆转（P＜0.05）。在转染MALAT1-野生型（WT）和CCND1-WT的MG-63/DOX细胞中，miR-154-5p模拟物（mimic）组的荧光素酶活性均显著低于其阴性对照组（P＜0.05）。结论敲低LncRNAMALAT1可以抑制OS细胞的DOX耐药性，其机制可能是通过靶向miR-154-5p/CCND1轴实现的。 |
| ABSTRACT： | OBJECTIVE To investigate the relationship of long non-coding RNA （LncRNA） metastasis-associated lung adenocarcinoma transcript 1 （MALAT1） and doxorubicin （DOX） resistance in osteosarcoma （OS） cells. METHODS MG-63 and MG-63/DOX cells were treated with different concentrations of DOX （0， 0.01， 0.05， 0.1， 1 μmol/L）， and survival rates and half maximal inhibitory concentration were determined using CCK-8 assay. The expressions of LncRNA MALAT1 in MG-63 and MG-63/ DOX cells were detected by real-time quantitative fluorescence PCR. MG-63/DOX cells were divided into Control group， knocking down LncRNA MALAT1 negative control （sh-NC） group， sh-MALAT1 group， sh-MALAT1+anti-NC group， and sh-MALAT1+ anti-miR-154-5p group. The expressions of LncRNA MALAT1， miR-154-5p and cyclin D1 （CCND1） mRNA in MG-63/DOX cells of each group were detected. The effects of knocking down LncRNA MALAT1 on the proliferation， migration， invasion， and apoptosis of MG-63/DOX cells were detected by CCK-8 assay， scratch test， Transwell experiment and flow cytometry， respectively. The expression of proliferating cell nuclear protein （PCNA） and CCND1 protein in MG-63/DOX cells was detected by Western blot assay. Interactions between LncRNA MALAT1 and miR-154-5p， miR-154-5p and CCND1 were detected by dual luciferase reporter gene experiment. RESULTS Compared with 0 μmol/L DOX， 0.01， 0.05， 0.1 and 1 μmol/L DOX could reduce the survival rates of MG-63 and MG-63/DOX cells （except for 0.01 μmol/L DOX） （P＜0.05）， IC50 were 0.07 and 0.13 μmol/L， respectively. The survival rate， cell migration number and invasion number of MG-63/DOX cells， scratch closure rate， mRNA expressions of LncRNA MALAT1， mRNA and protein expressions of CCND1， and PCNA protein expression in sh-MALAT1 group were significantly lower than sh-NC group and Control group； the apoptosis rate and miR-154-5p expression were significantly higher than sh-NC group and Control group （P＜0.05）. sh-MALAT1+anti-miR-154-5p group was able to reverse the aforementioned biological effects in sh-MALAT1 group （P＜0.05）. In MG-63/DOX cells transfected with both MALAT1-wild type （WT） and CCND1-WT， the luciferase activity in the miR-154-5p mimic group was significantly lower than mimic negative control group （P＜ 0.05）. CONCLUSIONS Knocking down LncRNA MALAT1 can inhibit the DOX resistance of OS cells， and its mechanism may be targeting the miR-154-5p/CCND1 axis. |
| 期刊： | 2025年第36卷第06期 |
| 作者： | 梁福东；狄淑芳；罗伟；齐江华；刘利兵 |
| AUTHORS： | LIANG Fudong，DI Shufang，LUO Wei，QI Jianghua，LIU Libing |
| 关键字： | 长链非编码RNA；肺腺癌转移相关转录本1；微小RNA-154-5p；细胞周期蛋白D1；骨肉瘤；阿霉素；耐药性 |
| KEYWORDS： | long non-coding RNA； metastasis-associated |
| 阅读数： | 4 次 |
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