miR-145过表达对宫颈癌细胞辐射敏感性的影响

Effect of miR-145 over-expression on radiosensitivity of cervical cancer cells

目的:探讨微小RNA(miR)-145过表达增强宫颈癌细胞放疗敏感性的分子机制。方法:运用Lipofectamine 2000将miR-145-mimic转染4株宫颈癌细胞系He La、Ca Ski、C33A和Si Ha,以NC-mimic为阴性对照,并以real-time PCR检测子宫内膜基质细胞(ESC)和4株宫颈癌细胞中miR-145的表达水平;转染的细胞经电离辐射照射后于不同时点运用MTT法和Annexin V-FITC/PI染色法联合流式细胞术分别测定细胞活力和细胞凋亡率;运用免疫荧光检测组蛋白γH2AX的表达水平;Western blot检测细胞中螺旋酶样转录因子(HLTF)的蛋白表达水平。结果:miR-145在ESC中高表达,而在4株宫颈癌细胞中均呈低表达,转染miR-145-mimic后4株宫项癌细胞中miR-145的表达水平显著高于NC-mimic组细胞(P<0.05)。相同条件下,辐照后miR-145过表达的宫颈癌细胞的活力显著低于NC-mimic组细胞,72 h的细胞凋亡率明显增加(P<0.05)。免疫荧光观察结果显示miR-145过表达显著促进电离辐射诱导的γH2AX激活;Western blot结果显示,miR-145过表达显著抑制HLTF的表达,与NC-mimic比较差异有统计学意义(P<0.05)。结论:miR-145在正常子宫内膜上皮细胞中高表达,而在宫颈癌细胞系中低表达;miR-145过表达可显著抑制宫颈癌细胞的活力,促进电离辐射诱导的细胞凋亡。miR-145过表达增强宫颈癌细胞的辐射敏感性,其机制可能与下调HLTF表达、抑制DNA损伤修复、促进细胞凋亡有关。

AIM:To investigate the molecular mechanism of microRNA(miR)-145 enhancing radiosensitivity of cervical cancer cells.METHODS:Cervical cancer lines HeLa,CaSki,C33A and SiHa were transfected with miR-145-mimic and NC-mimic using Lipofectamine 2000 kit,and the expression levels of miR-145 in the cervical cancer lines and endometrial stromal cells(ESC)were detected by real-time PCR.The transfected cells were exposed to X-ray radiation.At different time points,the cell viability and apoptotic rate were measured by MTT assay and flow cytometry with AnnexinV-FITC/PI staining,respectively.The protein levels ofγH2AX and helicase-like transcription factor(HLTF)were monitored by immunofluorescence and Western blot,respectively.RESULTS:High miR-145 levels was found in ESC,while low miR-145 levels were found in HeLa,SiHa,C33A and CaSki cells.The level of miR-145 in the cells transfected with miR-145-mimic was significantly higher than that in the negative control cells(P<0.05).The viability of cervical cancer cells with miR-145 over-expression was notably lower than that of the negative control cells,and the apoptotic rate at 72 h was remarkablely increased(P<0.05)under the same condition.The results of immunofluorescence labeling indicated that the protein level ofγH2AX significantly increased in the cervical cancer cells with miR-145 over-expression exposed to radiation compared with the negative control cells(P<0.05).The results of Western blot indicated that the protein level of HLTF significantly decreased in the cervical cancer cells with miR-145 over-expression compared with the negative control cells(P<0.05).CONCLUSION:miR-145 was highly expressed in normal endometrial epithelial cells,while low expressed in cervical cancer cells.miR-145 over-expression inhibits growth and significantly enhances radiosensitivity of cervical cancer cells.The molecular mechanism is possiblely related to downregulating HLTF expression and inhibiting DNA damage repair,thus resulting in apoptosis.