| 高晓丹,王丽洁,王书梦,苏洁.miR-103通过NF-κB通路上调MGMT表达诱导胃癌细胞紫杉醇耐药[J].浙江中西医结合杂志,2022,32(10): |
| miR-103通过NF-κB通路上调MGMT表达诱导胃癌细胞紫杉醇耐药 |
| miR-103 up-regulates the expression of MGMT through the NF-κB pathway to induce paclitaxel resistance in gastric cancer cells |
| 投稿时间：2022-04-21 修订日期：2022-09-15 |
| DOI： |
| 中文关键词: 微小RNA-103 核转录因子-κB抑制蛋白α 核转录因子-κB O6-甲基鸟嘌呤-DNA甲基转移酶 紫杉醇 胃癌 耐药 |
| 英文关键词:microRNA-103 IKBα nuclear factor κB O6-methylguanine-DNA methyltransferase paclitaxel gastric cancer drug resistance |
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| 中文摘要: |
| 目的 观察miR-103在胃癌细胞紫杉醇(PTX)耐药中的作用,并对其机制进行探讨。方法 构建PTX耐药胃癌NCI-N87细胞(N87/PTX)；N87/PTX 细胞转染miR-103 inhibitor和阴性对照,分别设为miR-103 inhibitor组和阴性对照组(NC),同时以未转染的N87/PTX细胞作为空白对照组(Control)。四甲基偶氮唑盐比色法(MTT)检测细胞活力；实时荧光定量PCR(RT-qPCR)检测细胞miR-103表达水平；生物信息学分析miR-103的靶基因；结晶紫染色法检测细胞贴壁存活率；免疫荧光法检测细胞NF-κB p65定位；蛋白印记法检测p-NF-κB p65、IKBα、MGMT蛋白表达变化。结果 N87/PTX耐药指数为26.4。IKBα蛋白的编码基因NFKBIA与miR-103存在互补结合位点。与正常N87细胞相比,N87/PTX细胞miR-103、p-NF-κBp65和MGMT表达显著升高,IKBα表达明显下调(P＜0.01)。miR-103 inhibitor可显著降低PTX对N87/PTX细胞的IC50(P＜0.01)。miR-103 inhibitor显著增强PTX(3 μmol.L-1)诱导的N87/PTX细胞贴壁存活率降低(P＜0.01)。NF-κB p65在NC组N87/PTX细胞中表达于细胞质和细胞核,而在miR-103 inhibitor组中主要表达于细胞质。miR-103 inhibitor显著下调N87/PTX细胞p-NF-κB p65和MGMT蛋白表达,上调IKBα蛋白表达(P＜0.01)。结论 miR-103可能通过降解IKBα诱导NF-κB p65入核,NF-κB p65增强MGMT转录表达从而介导胃癌细胞对PTX耐药。 |
| 英文摘要: |
| Objective Observe the role of miR-103 in paclitaxel (PTX) resistant gastric cancer cells, and explore its mechanism. Methods Construction of PTX-resistant gastric cancer NCI-N87 cells (N87/PTX). N87/PTX cells were transfected with miR-103 inhibitor and negative control, which were set as miR-103 inhibitor group and negative control group (NC), while untransfected cells were used as blank control group (Control). Tetramethylazolium salt colorimetric method (MTT) was used to detect cell viability. Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expression level of miR-103 in cells. Bioinformatics analysis of miR-103 target genes. Crystal violet staining method was used to detect cell viability. Immunofluorescence method was used to detect the localization of NF-κB p65 in cells. Western blotting was used to detect p-NF-κB p65, IKBα, and MGMT protein expression changes. Results The resistance index of N87/PTX was 26.4. IKBα encoding gene NFKBIA and miR-103 have complementary binding sites. Compared with normal N87 cells, the expressions of miR-103, p-NF-κBp65 and MGMT in N87/PTX cells were significantly increased, and the expression of IKBα was significantly down-regulated (P<0.01). miR-103 inhibitor can significantly reduce the IC50 of PTX on N87/PTX cells (P<0.01). miR-103 inhibitor significantly enhanced PTX (3 μmol.L-1)-induced reduction in the survival rate of N87/PTX cells (P<0.01). NF-κB p65 was expressed in the cytoplasm and nucleus in N87/PTX cells in the NC group, while it was mainly expressed in the cytoplasm in the miR-103 inhibitor group. miR-103 inhibitor significantly down-regulated p-NF-κB p65 and MGMT protein expression in N87/PTX cells, and significantly up-regulated IKBα protein expression (P<0.01). Conclusion miR-103 may induce NF-κB p65 to enter the nucleus by degrading IKBα to enhance the transcriptional expression of MGMT, thereby inducing gastric cancer cells to be resistant to PTX. |
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