光动力学处理对宫颈癌细胞的miRNA的影响

Photodynamic Treatment on miRNA in cancer cells

目的研究光动力学(PDT)处理对宫颈癌细胞的miRNA的影响。方法将He La细胞接种到黑色透明底的96孔微Optilux板(1×104细胞/孔)。24 h后,当细胞附着在培养板上,除去培养基,并将培养物在磷酸盐缓冲盐溶液(PBS,p H 7.4)中洗涤3次,进行不同浓度的光敏剂PDT处理。评价PDT对He La细胞活力影响,并进行细胞计数,以评估其对PDT灵敏度。激光照射1 h后,所有He La细胞的总细胞RNA经Quick Gene RNA细胞培养试剂盒提取后,进行DNA酶处理,使用大容量c DNA逆转录试剂盒进行逆转录(RT)。实时PCR定量测定凋亡相关的miRNA(MIR-7,miR-148a,miR-204,miR-210,miR-216和miR-296的表达水平。结果 PDT处理1 h后,与未做任何干预处理的对照组比较,He La细胞存活率显著降低至78%。PDT处理24 h后,He La细胞全部死亡。此外,PDT处理1 h后,He La细胞内聚半胱天冬酶活性,显著增高。另外,PDT处理1h后,miR-210和miR-296的表达水平显著高于对照组。然而,其他miRNA的表达水平包括miR-7,miR-148a,miR-204和miR-216与对照组无差异。结论 PDT处理宫颈癌细胞后,miRNA表达水平不同,miR-210和miR-296的表达水平最高,可以作为宫颈癌PDT疗效的标记物。

Objective To study the photodynamic（ PDT） effect on ervical cancer cells,miRNA expression. Methods He La cells were seeded into a black with clear bottom 96-well micro Optilux plates（ BD Biosciences,CA）（ 1 × 104 cells / well）. 24 h later,when cells were attached to the culture plate,the medium was removed,and the cultures were washed 3 times in phosphate buffered saline（ PBS,p H 7. 4）,PDT treatment with different concentrations of photosensitizer. He La cell viability evaluation PDT on impact,and cell count to assess their sensitivity for PDT. After laser irradiation 1 h,total cellular RNA of all He La cells Use Quick Gene RNA extraction cell culture kit. All DNA samples were enzymatically treated in order to avoid contamination of DNA,followed by the use of high capacity c DNA reverse transcription kit reverse transcription（ RT）. Apoptosis-related miRNA（ miR-7,miR-148 a,miR-204,miR-210,miR-216 and miR-296 expression levels were determined by quantitative real-time PCR. Results After PDT treatment 1h,compared with the control group without making any intervention treatment,He La cell viability was significantly reduced to 78%. 24 hours after PDT treatment,He La cells all died. In addition,PDT poly caspase activity in He La cells within one hour after treatment,was significantly increased. In addition,the PDT treatment 1 h,miR-210 and miR-296 expression levels were significantly higher.However,the expression levels of other miRNA include miR-7,miR-148 a,miR-204 and miR-216 no difference with the control group.Conclusions Talaporfin sodium PDT photosensitizers treated as cervical cancer cells,distinct miRNA expression levels,the highest miR-210 and miR-296 expression levels can be used as markers of cervical cancer PDT efficacy thereof.